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Conversion to Greenhouse Cultivation from Continuous Corn Production Decreases Soil Bacterial Diversity and Alters Community Structure

Agronomy 2024, 14(9), 2144; https://doi.org/10.3390/agronomy14092144

by Yaqiong Fan^1,2, Yamin Jia¹, Xinyang Zhang¹, Guoqiang Geng¹, Ronghao Liu¹, Lixia Shen¹, Jingjuan Hu¹ and Xinmei Hao^2,3,*

Reviewer 1: Anonymous

Reviewer 2:

Helvi Heinonen-Tanski

Reviewer 3: Anonymous

Agronomy 2024, 14(9), 2144; https://doi.org/10.3390/agronomy14092144

Submission received: 4 July 2024 / Revised: 10 September 2024 / Accepted: 18 September 2024 / Published: 20 September 2024

(This article belongs to the Section Innovative Cropping Systems)

Round 1

Reviewer 1 Report

Comments and Suggestions for Authors

My commends:

The paper titled "Continue Planting Decreases Soil Bacterial Diversity and Alters Community Structure in Greenhouse Tomato Cultivation" aims to explore how long-term greenhouse tomato cultivation affects soil microbial communities. The topic is both interesting and significant. However, the study suffers from several methodological issues that need to be addressed. The use of a maize field as a control is unjustified and does not provide an appropriate baseline for comparison, which undermines the validity of the results. Additionally, the study fails to adequately control for confounding variables such as soil type, climate, and management practices. This lack of control makes it difficult to attribute observed changes in microbial communities solely to the duration of tomato cultivation. Furthermore, the absence of detailed methodological descriptions for soil sampling and microbial analysis compromises the study’s reproducibility and scientific rigor. These critical flaws prevent the paper from offering reliable insights into the effects of continuous cultivation on soil bacterial diversity. It is recommended that the authors thoroughly address these issues before resubmission.

Specific commends:

Line 13-15: It is not clear why you want to conduct this research. Please clarify your motivation.

L23: "was smaller" - Does it have a significant effect? Please verify.

L24: What do you mean by “Similarity”? Please clarify.

Lines 34-46: The first paragraph should emphasize the importance of soil, soil microbes, bacterial diversity, and community structure.

L35: Does “4 million ha” refer to the entire globe?

L46: Since the research indicated tomatoes, it is better to provide specific citations and data regarding the decline in tomato production.

L50: What do you mean by “Soil community”? Please clarify.

L53-56: It is not sufficient to use only one research result to support your implementation. You should provide more citations and references to explain and support the claim that continuous planting results in changes in the soil microbial community, structure, and composition, as well as changes in biodiversity.

L75: “In contrast” - What pattern does this oppose? Please clarify.

L90-92: You mentioned the importance of the research, but it is not clear why you want to conduct this research. There is a lack of logical reasoning and explanation of the gap that needs to be filled.

L98-99: The detailed method should not be described here.

L101: Replace “The aim of the study” with “The objectives of the study were”

L103-104: “Limit your effect” - What effect are you referring to? Please describe it in the previous paragraph of the “Introduction”.

L106-108: Citations are needed here.

L114: Repeat sentence

L120: It is necessary to add subtitle of “Experimental Design” section to explain and provide more details about the experiment. It is not clear.

L122-124: The description 'These solar greenhouses were rebuilt from adjacent maize fields, thus the surrounding corn fields served as the control (CK) with a planting duration of 0 years' is not clear. I am confused about it.

L127-129: More details are needed to describe the “solar greenhouse.” Does the solar greenhouse have a sealed structure? How are temperature, moisture, and solar radiation controlled? What are the control group conditions? Are the maize crops in the control group also planted in the solar greenhouse?"

L136-138: The soil was also tilled in the controlled (CK) plots?

L139-149: The soil in the greenhouse differed in terms of fertilizer and irrigation compared to the control plots. These differences could affect the results due to the variation in controlled variables.

L172-174: Please provide the methods for assessing soil physicochemical indicators such as carbon-nitrogen ratio, NH4+-N content, NO3--N content, organic matter, total nitrogen, available phosphorus, available potassium, organic carbon, bulk density, electrical conductivity, and pH in the Methods section. I cannot find any results or discussion regarding these soil physicochemical indicators. The term 'environmental factors' is not correctly used in this manuscript.

Results are not clearly described in terms of treatment effects and the results of ANOVA.

L179-184: It appears that the only difference is between the control and all treatments (over a 3-year period), with no differences among y5, y9, and y13 in Table 1. Additionally, there is no table legend indicating the meaning of the lowercase letters.

L185-186: “A statistically significant difference (p < 0.05) was detected between the Y13 and CK treatments for the most dominant phyla (Figure 1a), but this is not clear from Figure 1a.” However, significant differences are observed between the CK and all other treatments in Table 1 (species).

Incomplete Information:

What is the soil type, and what relevant information can you provide regarding the soil?

L 25-27: Missing information in the Results section, Discussion, and Methods: “In terms of soil characteristics, the carbon-nitrogen ratio, organic carbon, and pH were identified as the main negative factors influencing soil bacterial composition, while ammonium nitrogen was the dominant positive factor,” as mentioned in the Abstract.

L165-175: There is no mention of the specific statistical tests used to determine the significance of differences (e.g., ANOVA, t-test).

L179-181: The sentence "Notably, a significant difference was observed between Y5 and Y13 at the order taxonomic level" lacks details on the nature and implications of the difference observed.

Figure 1: The y-axis of Figure 1 is not clearly labeled or described, and the ANOVA results are also unclear.

L229: The text incorrectly refers to "Veen diagrams" instead of "Venn diagrams. refers to "Veen diagrams" instead of "Venn diagrams.

L229-236: The current presentation lacks a clear comparative analysis with the control treatment (CK), which is vital for understanding the impact of different treatment durations. While specific overlaps between treatments and CK are mentioned, the implications of these overlaps are not fully explored.

The percentages of shared and unique ASVs presented in the text appear to be inaccurately calculated or inconsistently reported. The calculation of 1.15% for 207 common ASVs, in particular, seems mathematically implausible given the reported total ASVs for each treatment.

Discussion:

After the results are substantially revised based on the statistical analysis, the discussion section should be modified accordingly to reflect the updated findings.

Overall suggestion:

1. Title:

Suggest to replace the “Reduces” into “decreases” in title “Continue planting decreases soil bacterial diversity and alters community structure in a greenhouse tomato cultivation

2. Abstract

Include specific percentages or fold-changes for changes in bacterial abundance.

Provide specific values or ranges for diversity indices (Chao1, Observed species, Pielou_e.g).

Add specific examples of functional predictions and metabolic pathways.

3. Introduction

General Comments:

Some sentences are overly complex and could be simplified for better readability.

Consider breaking long paragraphs into smaller sections to improve readability and emphasize key points. Some phrases are vague and could be more precise. For example, "Establishing a theoretical foundation for effectively overcoming the challenges associated with long-term planting is very important" could be rephrased for clarity.

Ensure consistent use of terminology. For instance, "facility vegetable planting" and "greenhouse vegetable production" should be standardized.

Specific Comments:

Line 34-35: "The method of facility vegetable planting has been rapidly evolving, resulting in the cultivation of over 4 million hectares of land in 2021[1]."

Suggestion: "The method of greenhouse vegetable planting has rapidly evolved, resulting in over 4 million hectares being cultivated in 2021[1]."

Line 39-40: "Tomato, a globally popular vegetable is commonly grown under greenhouse conditions in northern China [2,3]."

Suggestion: "Tomatoes, a globally popular vegetable, are commonly grown in greenhouses in northern China [2,3]."

Line 40-41: "In practical production setting, continuous planting of greenhouse tomatoes is a conventional scenario due to cost consideration and the desire to maximize soil utilization."

Suggestion: "In practical production, continuous planting of greenhouse tomatoes is common due to cost considerations and the desire to maximize soil utilization."

Line 42-43: "However, this practice often leads to obstacles such as the accumulation of autotoxic substances, degradation of soil physiochemical properties, disruption of native soil microbial communities, and the buildup of soil-borne pathogens [4,5]."

Suggestion: "However, this practice often results in the accumulation of autotoxic substances, degradation of soil physiochemical properties, disruption of native soil microbial communities, and the buildup of soil-borne pathogens [4,5]."

Line 47-49: "Consequently, many studies indicated that mono-cropped or intensively managed greenhouse production systems resulted in the risk of soil acidification and salinization, and prolonged cultivation in such systems can lead to great changes in the structure and abundance of soil community[9]."

Suggestion: "Many studies indicate that monocropped or intensively managed greenhouse production systems risk soil acidification and salinization, and prolonged cultivation can significantly change the structure and abundance of soil communities [ 9]."

Line 50-51: "Soil microorganisms play a pivotal role in nutrient cycles and energy flow within the soil, serving as indicators of soil structure, fertility, stability, and sustainable utilization."

Suggestion: "Soil microorganisms play a pivotal role in nutrient cycles and energy flow, serving as indicators of soil structure, fertility, stability, and sustainable use."

Line 61-64: "The composition of these bacteria is influenced by nitrogen levels or types, copiotrophic groups (e.g., Alphaproteobacteria and Acidobacteria), which grow rapidly, are more prone to proliferate in nutrient-rich environments, whereas oligotrophic groups (e.g., Actinobacteria), which are considered K-strategists that have a slower growth rate, would likely decline[15]."

Suggestion: "The composition of these bacteria is influenced by nitrogen levels or types. Copiotrophic groups (e.g., Alphaproteobacteria and Acidobacteria), which grow rapidly, proliferate in nutrient-rich environments, while oligotrophic groups (e.g., Actinobacteria), considered K-strategists, likely decline [15]."

Clearly articulate the study's aim and hypotheses towards the end of the introduction.

Line 97-98: "The present study investigated the impact of the planting years on the structure and diversity of the soil microbial community in greenhouse tomatoes using real-time PCR."

Suggestion: "This study investigates the impact of planting duration on the structure and diversity of soil microbial communities in greenhouse tomatoes using real-time PCR."

Comments on the Quality of English Language

The English language used throughout the paper is generally clear.

Author Response

Dear Reviewer

We are very grateful and moved by your detailed comments on this paper, which will greatly help to improve the quality of the manuscript. We hope that the current version of the manuscript can be considered for further review. We have carefully examined the comments and made the necessary revisions accordingly, combining some related comments and providing a combined response. The following outlines our responses to the comments.

Comments 1: The paper titled "Continue Planting Decreases Soil Bacterial Diversity and Alters Community Structure in Greenhouse Tomato Cultivation" aims to explore how long-term greenhouse tomato cultivation affects soil microbial communities. The topic is both interesting and significant. However, the study suffers from several methodological issues that need to be addressed. The use of a maize field as a control is unjustified and does not provide an appropriate baseline for comparison, which undermines the validity of the results. Additionally, the study fails to adequately control for confounding variables such as soil type, climate, and management practices. This lack of control makes it difficult to attribute observed changes in microbial communities solely to the duration of tomato cultivation. Furthermore, the absence of detailed methodological descriptions for soil sampling and microbial analysis compromises the study’s reproducibility and scientific rigor. These critical flaws prevent the paper from offering reliable insights into the effects of continuous cultivation on soil bacterial diversity. It is recommended that the authors thoroughly address these issues before resubmission.

Response 1: We understand the concern the reviewer raised regarding using the maize field as the control for studying the effects of long-term greenhouse cultivation on soil bacteria diversity. Ideally, soil samples from one field prior to greenhouse cultivation, and different years into greenhouse cultivation should be used for comparisons, which requires a long-term experimental field with consistent management practices. Without such an experimental setup, some alternative ones could be used to gain information on how greenhouse cultivation affects soil properties. Our experimental setup was based on assumptions that soil properties were similar for the fields prior to the conversion to greenhouse cultivation. We believe the assumption is valid, given that the fields have been growing corn for quite a long time, > 20 years for all fields. With such long monocultural history, soil bacterial compositions and quantities of the corn filed become stable over the years, and consequently could be used as the control. The fields are located within the arid region of northwest China, with similar climate and management practices both for the corn and greenhouse vegetables. Previous studies revealed that cropping is the main cause of changes in the structure of the soil bacterial community, long-term cotton cultivation and the associated farming methods gradually stabilizes after 10 years of repeated fluctuations(Wei and Yu, 2018). Also, cluster analysis showed that the greatest similarities in bacterial communities were between 15 and 20 years of continued cultivation(Yang et al., 2018). Many meta-analyses and reviews of crop growth under the same management mode for a long time also show that soil properties (e.g. soil pH、soil nitrogen、soil organic carbon) are the main factors affecting soil microorganisms.

Considering that the comparisons made in the study were soil bacteria properties between soils from inside the greenhouse of different years and nearby corn fields, we changed the title, objectives, and conclusions accordingly to better reflex the experimental setup. Specifically, the title has been changed to “Conversion to Greenhouse Cultivation from Continuous Corn Production Decreases Soil Bacterial Diversity and Alters Community Structure”, and the objective has been changed to “(1) to investigate the shifts in soil bacterial communities after converting corn fields to greenhouse tomato cultivation; (2) to explore the soil bacterial diversity and composition under different planting years, as well as the associated predicted functions of different bacterial groups; (3) to determine whether there is a “limit year effect” in greenhouse tomato continuous cropping”. Also, detailed descriptions of soil sampling and analysis procedures including the company used are also included in the revision.

References:

Wei, Z., Yu, D. Analysis of the succession of structure of the bacteria community in soil from long-term continuous cotton cropping in Xinjiang using high-throughput sequencing. Arch Microbiol. 2018, 200, 653-662.

Yang, L., Tan, L., Zhang, F., Gale, W. J., Cheng, Z., and Sang, W. Duration of continuous cropping with straw return affects the composition and structure of soil bacterial communities in cotton fields. Can J Microbiol. 2018, 64, 167-181.

Comments 2: Line 13-15: It is not clear why you want to conduct this research. Please clarify your motivation.

Response 2: The sentences have been revised as “Changes in crop types and long-term monoculture substantially impact soil microbial communities. Exploring these changes and their influencing factors is of great significance for addressing the challenges of continuous cropping”.

Comments 3: L23: "was smaller" - Does it have a significant effect? Please verify.

Response 3: The sentence has been revised as “The Beta diversity analysis revealed a pronounced variation in soil bacterial community structure across planting years, with the divergence from CK intensifying”.

Comments 4: L24: What do you mean by “Similarity”? Please clarify.

Response 4: The sentence has been revised as ”In comparison to Y5 vs. CK, Y9 and Y13 exhibit marked differences from CK across a broader and same metabolic pathways, suggesting a potential convergence of microbial activities over time”

Comments 5:

Lines 34-46: The first paragraph should emphasize the importance of soil, soil microbes, bacterial diversity, and community structure.

L35: Does “4 million ha” refer to the entire globe?

L46: Since the research indicated tomatoes, it is better to provide specific citations and data regarding the decline in tomato production.

L50: What do you mean by “Soil community”? Please clarify.

L75: “In contrast” - What pattern does this oppose? Please clarify.

L98-99: The detailed method should not be described here.

L101: Replace “The aim of the study” with “The objectives of the study were”

L103-104: “Limit your effect” - What effect are you referring to? Please describe it in the previous paragraph of the “Introduction”.

L106-108: Citations are needed here.

Response 5: According to the above comments, the introduction section has been fully revised, the logical structure between paragraphs has been adjusted, the writing of the language has been improved, and the citations of relevant references have been supplemented. The following is the revised introduction:

Soil microorganisms play a pivotal role in nutrient cycles and energy flow within the soil, serving as indicators of soil structure, fertility, stability, and sustainable use. They play an indispensable role in regulating the intricate feedback mechanisms between plants and soil, thereby fostering a harmonious and productive environment. Both the alteration in crop cultivation practices and the implementation of continuous planting strategies serve as pivotal measures in adapting to shifts in human dietary preferences. Delving into their respective impacts on microbial communities holds immense significance, as it facilitates a deeper comprehension of the theory underpinning soil microbial community evolution.

To meet the growing demand for vegetables, some fields for food crop cultivation (e.g. corn) have undergone meticulous planning to transition smoothly toward vegetable cultivation. The method of greenhouse vegetable planting has rapidly evolved in China, resulting in the cultivation of over 4 million hectares being cultivated in 2021[1]. Tomato, a globally popular vegetable is commonly grown under greenhouse conditions in northern China[2,3]. A previous study found that the diversity and composition of soil microbiome showed a great alternation with continuously planted American ginseng compared to traditional crops[4]. Oh et al. [5]indicated that soil microbial community composition was affected by vegetation type and Mahnert et al. [6]also revealed that plant community structure is partly responsible for the changes to soil microbial communities. Therefore, it can be reasonably inferred that converting conventional fields to greenhouse tomato cultivation will initially result in a substantial difference in soil microorganisms, primarily due to changes in plant species.

In practical production, continuous planting of greenhouse tomatoes is a conventional scenario due to cost consideration and the desire to maximize soil utilization. Numerous studies have reported that long-term mono-cropping patterns severely deteriorate soil quality and lead to a sharp decline (2-38%) in tomato yields[7-9]. On the other hand, this practice often results in the accumulation of autotoxic substances, degradation of soil physiochemical properties, disruption of native soil microbial communities, and the buildup of soil-borne pathogens[10,11]. Many studies indicated that monocropped or intensively managed greenhouse production systems resulted in the risk of soil acidification and salinization, and prolonged cultivation in such systems can lead to great changes in the structure and abundance of soil community[12.13].

The structure and activity of microbial communities vary significantly across cultivation years due to soil property changes[14]. Dominant bacterial abundance and diversity correlate with soil carbon, nitrogen, and phosphorus levels[15-17]. Zhang et al. [18] discovered that the relative abundance of the Acidobacteria phylum significantly increased with an increase in soil nitrogen input, whereas the Actinobacteria phylum showed contrasting results, indicating a decrease in its relative abundance under similar conditions. Furthermore, factors such as higher soil organic content and varying carbon-to-nitrogen ratios can significantly affect soil microbial diversity[19] and the relative abundance of Proteobacteria, Bacteroidetes, and Acidobacteria[20,21]. Specifically, the relative abundance of the Acidobacteria phylum was observed to be diminished in soils enriched with high concentrations of organic carbon. In contrast, b-Proteobacteria and Bacteroidetes, both exhibiting copiotrophic characteristics, demonstrated the highest relative abundances in soils with high availability of carbon[20].

With long-term cultivation, Xiong et al. [22] revealed that continuous cropping of black pepper led to a decrease in soil bacterial abundance and alterations in soil microbial community composition and structure, which were related to black pepper growth. Tong et al. [23] reported that an increase in cultivation years of P. ginseng in farmland and deforestation fields significantly altered the diversity of soil microbial communities. Similarly, soil bacterial and fungal richness decreased with continuous coffee cropping [24]. However, several studies have illuminated a nonlinear pattern in soil bacterial composition and diversity, featuring a pivotal point we designate as the "limit year effect". Li et al. [1] uncovered a notable decrease in the Shannon and Simpson indices within the first 9 years, followed by an increase in year 13 in greenhouse vegetable fields. Analogous trends were observed in a rice-cherry tomato rotation system, where the alpha diversity of soil microbial communities peaked at year 5, subsequently declining gradually until year 10, with certain beneficial microorganisms experiencing a decline after year 5 or 7 [13]. These discoveries underscore the intricate response of soil microbiome indices to continuous agricultural practices, initially escalating and then declining, or exhibiting a reversal in trend.

Research on temporal variations in soil microbial community diversity across planting cycles uncovers varied patterns. These patterns frequently reflect the unique soil conditions and distinct cultivation practices of different regions. However, studies examining soil bacterial community shifts in response to crop alteration and continued greenhouse cultivation in the arid northwest of China are still limited, resulting in uncertainties about whether soil bacterial composition and diversity undergo a gradual transformation over successive planting years or experience an abrupt change (known as the 'limit year effect'). The findings of this study will be of great significance in understanding the mechanisms that underlie the obstacles to continuous greenhouse tomato cultivation from the perspective of soil bacterial community dynamics. Furthermore, these results are crucial for devising effective management strategies aimed at optimizing soil health and tomato production, thereby contributing to ensuring the long-term sustainability of greenhouse tomato cultivation.

In this study, soil from greenhouse tomatoes for 0 years (corn field), five years (Y5), nine years (Y9), and thirteen years (Y13) were selected as study objects. The objectives of the study were (1) to investigate the shifts in soil bacterial communities after converting corn fields to greenhouse tomato cultivation; (2) to explore the soil bacterial diversity and composition under different planting years, as well as the associated predicted functions of different bacterial groups; (3) to determine whether there is a “limit year effect” in greenhouse tomato continuous cropping. We hypothesize that converting corn fields to greenhouse tomatoes will significantly alter soil microbial structure, and based on our prior findings regarding soil physical and chemical properties[25]. We speculate that nine years of continuous cropping represents a tipping point for changes in soil microbial characteristics.

Comments 6: L114: Repeat sentence

Response 6: The sentence “The region belongs to the temperate continental climate” has been deleted.

Comments 7: L120: It is necessary to add subtitle of “Experimental Design” section to explain and provide more details about the experiment. It is not clear.

Response 7: The section of "Experimental design" has been added and modified as required.

Comments 8: L122-124: The description 'These solar greenhouses were rebuilt from adjacent maize fields, thus the surrounding corn fields served as the control (CK) with a planting duration of 0 years' is not clear. I am confused about it.

Response 8: The sentences have been revised as “Notably, corn cultivation has a rich history here, spanning over two decades. Conversely, the greenhouse tomato sites were recently established, having been restructured from portions of these established corn fields in recent years. Consequently, the adjacent corn fields naturally served as the control (CK), with a planting duration of zero years, providing a valuable benchmark for comparison”.

Comments 9: L127-129: More details are needed to describe the “solar greenhouse.” Does the solar greenhouse have a sealed structure? How are temperature, moisture, and solar radiation controlled? What are the control group conditions? Are the maize crops in the control group also planted in the solar greenhouse?"

Response 9: Detailed information has been added. As previously elucidated, soil microorganisms generally exhibit stability under long-term mono-cropping regimes. Recognizing the potential differences in field management practices between open-field and greenhouse settings, it is logically justified to adopt the microbial status of adjacent field-grown crops as a baseline or initial reference point for evaluating the tomato planting system.

Comments 10: L136-138: The soil was also tilled in the controlled (CK) plots?

Response 10: The sentences have been revised as ” Furthermore, before each transplanting, the shallow soil (0-20 cm) was manually tilled, and after a decade of harvesting, the deeper soil (>40 cm) underwent mechanical turning in greenhouse cultivation. Before planting corn every year, the soil is mechanically rotary-plowed to a maximum depth of no more than 40 cm”.

Comments 11: L139-149: The soil in the greenhouse differed in terms of fertilizer and irrigation compared to the control plots. These differences could affect the results due to the variation in controlled variables.

Response 11: Different irrigation and fertilization systems in tomato and corn cultivation are objectively necessary, as they will lead to changes in soil physical and chemical properties, thereby affecting the variation of soil microorganisms. Therefore, the discussion section of this study also explains that crop changes and continuous planting cause differences in soil bacterial composition and diversity by influencing soil physical and chemical properties.

According to the above comments, the section of experimental design has been revised as follows:

2.2. Experimental Design

The study selected centralized greenhouse tomato planting and corn planting as sampling sites. Notably, corn cultivation has a rich history here, spanning over two decades. Conversely, the greenhouse tomato sites were recently established, having been restructured from portions of these established corn fields in recent years. Consequently, the adjacent corn fields naturally served as the control (CK), with a planting duration of zero years, providing a valuable benchmark for comparison. Additionally, tomato greenhouses that had undergone continuous monocropping for 5, 9, and 13 years were chosen in the vicinity (designated as Y5, Y9, and Y13, respectively). To ensure reproducibility, three greenhouses/corn fields were included for each study year.

The type of solar greenhouse features an earth brick structure, with a planting area of approximately 500 m² (the external length × width × height is about 80 m × 70 m × 3.5 m). The soil in the corn field and greenhouse tomato is primarily composed of sandy loam. The corn field is an open corn field, usually planted in mid-April and harvested in early October. Tomatoes are cultivated twice annually, with growth cycles extending from early April to late August and then from early October until the end of February the subsequent year. Sampling occurred in late August 2021, when a few greenhouses were preparing to replant with ginseng fruits in less than two weeks. Consequently, any potential differences stemming from diverse crops were disregarded in this study. The greenhouse lacked heating and was equipped with a ventilation system that automatically opened a 0.5-meter-wide vent at its apex whenever temperatures surpassed 30°C. Typically, this ventilation system remained operational from late April onwards, with additional ventilation achieved by rolling up the lower 70 centimeters of the greenhouse walls from late May until the harvest in August. During winter, straw mats were placed atop the greenhouse to ensure interior temperatures remained above 5°C, and the ventilation was manually opened to approximately 20 centimeters for roughly 5 hours, starting from midday.

Furthermore, before each transplanting, the shallow soil (0-20 cm) was manually tilled, and after a decade of harvesting, the deeper soil (>40 cm) underwent mechanical turning in greenhouse cultivation. Before planting corn every year, the soil is mechanically rotary-plowed to a maximum depth of no more than 40 cm. The irrigation method adopted in the solar greenhouse involves trench irrigation, with approximately seventeen irrigations over the growth period, totaling 350 mm. The corn field in the control group was irrigated on the surface, with an irrigation interval of around 30 to 50 days, and a total irrigation volume of approximately 480 mm. Prior to transplantation, micronutrient fertilizer preceded vegetable transplantation, while a nitrogen, phosphorus, and potassium compound fertilizer was used during growth. For corn cultivation, only nitrogen, phosphorus, and potassium fertilizer were applied. All greenhouses adhered to a uniform and standardized management protocol. The specific geographical location, management measures, etc., of 12 plots were listed in Supplementary Materials Table S1.

Comments 12: L172-174: Please provide the methods for assessing soil physicochemical indicators such as carbon-nitrogen ratio, NH4+-N content, NO3--N content, organic matter, total nitrogen, available phosphorus, available potassium, organic carbon, bulk density, electrical conductivity, and pH in the Methods section. I cannot find any results or discussion regarding these soil physicochemical indicators. The term 'environmental factors' is not correctly used in this manuscript.

Response 12: The methods utilized for measuring these factors have been comprehensively supplemented, and the exhaustive outcomes of these factors in soil depth of 0-100 cm have been duly published in a Chinese-language paper. To prevent redundancy and ensure efficient use of data, the results of soil physicochemical indicators in soil depth of 0-20 cm have been added in the revised manuscript. The section has been revised as follows:

2.3. Soil sample collection and determination of soil physicochemical and microbial properties

Mixed samples were collected from the surface layer (0 - 20 cm) of each corn field and greenhouse, utilizing the plum blossom distribution method. Therefore, there were a total of 12 soil samples in this study. Part of the soil was naturally air-dried to determine the physical and chemical properties of the soil, while the other part was securely stored at -80 ℃ to facilitate soil DNA extraction. Soil bulk density (ρ) was determined using the cutting ring method, while soil pH was determined by a glass electrode pH meter with a soil-to-water ratio of 1:5. Soil electrical conductivity (EC) was determined by a conductivity meter in a 1:5 soil water (w/v) suspension. Soil NH₄⁺-N and NO₃^--N content were determined using a flow analyzer, following extraction with a KCl solution (50 mL KCl for 10 g soil). Soil available phosphorus (AP) was determined by following extraction with sodium bicarbonate solution. Soil available potassium (AK) content was determined by a flame photometry, following extraction with a CH₃COONH₄ solution. Soil total nitrogen (TN) content was analyzed by the Kjeldahl method. Soil total organic carbon (OC) content was determined using the Total Organic Carbon Analyzer.

DNA was extracted from 0.5 g of soil using the Fast DNA Spin Kit for Soil (MP Biomedicals, CA, USA) and subsequently purified with the PowerClean DNA Clean-up Kit (Mobio, CA, USA), adhering strictly to the manufacturer's protocols. The quality and concentration of the extracted DNA were assessed through gel electrophoresis (0.8% agarose) and a NanoDrop NC2000 spectrophotometer (Thermo Fisher Scientific, Waltham, MA, USA), and the purified DNA was then stored at -20°C.

The V3-V4 regions of bacterial 16S rRNA genes were amplified using primers 338F (ACTCCTACGGGAGGCAGCA) and 806R (GGACTACHVGGGTWTCTAAT). PCR amplicons were purified with Vazyme VAHTSTM DNA Clean Beads (Vazyme, Nanjing, China) and quantified utilizing the Quant-iT PicoGreen dsDNA Assay Kit (Invitrogen, Carlsbad, CA, USA). After individual quantification, amplicons were pooled in equal molar concentrations, and pair-end 2×250 bp sequencing was carried out on the Illumina NovaSeq platform with the NovaSeq 6000 SP Reagent Kit (500 cycles) at Shanghai Personal Biotechnology Co., Ltd. (Shanghai, China).

Sequence data analyses were primarily conducted using QIIME2 and R packages (version 3.2.0). The obtained sequences underwent amplicon sequence variant (ASV) clustering, denoising, and dereplication to generate a comprehensive ASV species abundance profile (Table S2). The soil bacterial taxonomy in the study was assigned using the Silva 138.1 database.

Comments 13: Results are not clearly described in terms of treatment effects and the results of ANOVA.

Response 13: The relevant results have been modified in the revised manuscript.

Comments 14: L179-184: It appears that the only difference is between the control and all treatments (over a 3-year period), with no differences among y5, y9, and y13 in Table 1. Additionally, there is no table legend indicating the meaning of the lowercase letters.

Response 14: The paragraph has been revised as follows:

Regarding the four treatments, the denoised sequence count of high quality falls within a range of 40793 to 51922 (Table S2). There was no significant variation in the abundance of bacterial ASVs across different treatments at the phyla, family, and genus taxonomic levels. However, a notable significant difference was observed specifically compared Y9 and Y13 to Y5 at the class taxonomic level, and Y9 and Y13 to CK at the order taxonomic level. Furthermore, when compared to the CK treatment, Y5, Y9, and Y13 all demonstrated a significant increase of 60.5%, 67.4%, and 51.2% respectively in ASV counts at the species taxonomic level (Table 2).

The bacteria belonging to Actinobacteria emerged as the most abundant bacterial phylum across all four treatments, accounting for an average of 24.4% of all taxa, followed by Pseudomonadota (23.7%), Chloroflexi (15.8%), Firmicutes (9.6%). A significantly higher abundance of Pseudomonadota, and Firmicute were found in Y13 than in CK while the opposite results were found in an abundance of Actinobacteria, Acidobacteria, and Gemmatimonadetes (p<0.05) (Figure 1a). At the class taxonomic level, a significantly higher relative abundance of Actinobacteria was found in CK than in Y9 and Y13 (p<0.05), while Y9 and Y13 resulted in a significantly higher relative abundance of Anaerolineae and Gammaproteobacteria, respectively than CK and Y5 treatments (p<0.05) (Figure 1b).

The note of the table has been added to explain the meanings of the lowercase letters.

Comments 15: What is the soil type, and what relevant information can you provide regarding the soil?

Response 15: the soil in the study area is primarily composed of sandy loam. The determination methods of soil physical and chemical indexes, the result analysis, and relevant discussion have been supplemented in the corresponding sections.

Comments 16: L165-175: There is no mention of the specific statistical tests used to determine the significance of differences (e.g., ANOVA, t-test).

Response 16: The sentence has been revised as “The soil bacterial diversity indices (ASVs, the relative abundance of bacterial phyla and classes, alpha diversity indexes, and relative abundance of metabolic pathways) in different treatments were compared by one-way ANOVA. Significant effects were further determined using Fisher’s least significant difference (LSD) test”.

Comments 17: L179-181: The sentence "Notably, a significant difference was observed between Y5 and Y13 at the order taxonomic level" lacks details on the nature and implications of the difference observed.

Response 17: The sentences have been revised as “Regarding the four treatments, the denoised sequence count of high quality falls within a range of 40793 to 51922 (Table S2). There was no significant variation in the abundance of bacterial ASVs across different treatments at the phyla, family, and genus taxonomic levels. However, a notable significant difference was observed specifically compared Y9 and Y13 to Y5 at the class taxonomic level, and Y9 and Y13 to CK at the order taxonomic level. Furthermore, when compared to the CK treatment, Y5, Y9, and Y13 all demonstrated a significant increase of 60.5%, 67.4%, and 51.2% respectively in ASV counts at the species taxonomic level (Table 2)”.

Comments 18: Figure 1: The y-axis of Figure 1 is not clearly labeled or described, and the ANOVA results are also unclear.

Response 18: In the submitted paper, this issue may have arisen due to the system error. Figure 1 has been improved and has also been confirmed in the revised manuscript.

Comments 19: L229: The text incorrectly refers to "Veen diagrams" instead of "Venn diagrams. refers to "Veen diagrams" instead of "Venn diagrams.

Response 19: I'm sorry for this careless mistake. "Veen diagrams" has been revised as "Venn diagrams”.

Comments 20: L229-236: The current presentation lacks a clear comparative analysis with the control treatment (CK), which is vital for understanding the impact of different treatment durations. While specific overlaps between treatments and CK are mentioned, the implications of these overlaps are not fully explored.

Response 20: The paragraph has been revised as follows: Venn diagrams were constructed to visually represent the unique and shared ASVs among the various treatments. The overlaps between Y5 and CK, Y9 and CK, as well as Y13 and CK, revealed 611, 453, and 281 shared soil bacterial ASVs, respectively. These shared ASVs constituted 16.3%, 11.8%, and 7.5% of the ASVs observed in the CK treatment. Consequently, alterations in crop species and continuous planting led to variations in soil bacterial composition, with the differences gradually increasing. The plant transformation strongly altered the species composition while the endemic soil bacteria proportion remained stable during the continuous tomato cultivation. The unique bacterial ASVs for CK, Y5, Y9, and Y13 were 2960, 2108, 2098, and 1935, respectively, accounting for 79.2%, 55.1%, 55.8%, and 57.0% of the total ASVs in each treatment (Figure 3).

Comments 21: The percentages of shared and unique ASVs presented in the text appear to be inaccurately calculated or inconsistently reported. The calculation of 1.15% for 207 common ASVs, in particular, seems mathematically implausible given the reported total ASVs for each treatment.

Response 21: In the submitted papers, the total number of shared treatments is 207. While the total number of ASVs of all treatments is 17898. Therefore, the calculated ratio is 1.15%. To ensure clarity and avoid any potential confusion, this sentence has been omitted from the revised manuscript.

Comments 22: After the results are substantially revised based on the statistical analysis, the discussion section should be modified accordingly to reflect the updated findings.

Response 22: The discussion section has been revised.

Comments 23: Title: Suggest to replace the “Reduces” into “decreases” in title “Continue planting decreases soil bacterial diversity and alters community structure in a greenhouse tomato cultivation

Response 23: The title has been revised to “Conversion to Greenhouse Cultivation from Continuous Corn Production Decreases Soil Bacterial Diversity and Alters Community Structure”.

Comments 24: Abstract: Include specific percentages or fold-changes for changes in bacterial abundance.

Provide specific values or ranges for diversity indices (Chao1, Observed species, Pielou_e.g).

Add specific examples of functional predictions and metabolic pathways.

Response 24: The abstract section has been revised according to the comments.

Abstract: Changes in crop types and long-term monoculture substantially impact soil microbial communities. Exploring these changes and their influencing factors is of great significance for addressing the challenges of continuous cropping. Soil Surface layers samples from greenhouse tomatoes fields cultivated for 5 (Y5), 9 (Y9), 13 years (Y13), or a surrounding corn field (CK) as control were analyzed. Y13 significantly increased the relative abundance of Pseudomonadota (43.1%)and decreased Actinobacteria (50.3%) compared to CK. Soil bacterial alpha diversity generally declined from CK to Y13 (0.1%-22.2%), with a small peak in Y9 for Chao1 and Observed_ species. Significant differences in Chao1 and Observed_ species were observed between CK and Y13. The Beta diversity analysis revealed a pronounced variation in soil bacterial community structure across planting years, with the divergence from CK intensifying. In comparison to Y5 vs. CK, Y9 and Y13 exhibit marked differences from CK across a broader and same metabolic pathways, suggesting a potential convergence of microbial activities over time. Y9 and Y13 showed the significant higher biosynthesis abundance (7.50% and 6.36%, respectively) than CK. In terms of soil physicochemical indices, the carbon-nitrogen ratio was the primary factor influencing soil bacterial composition. In conclusion, crop alteration and continued planting change the soil bacterial composition, and increasing planting years suppressed soil bacterial diversity, leading to a stable bacterial ecology after nine years. Implementing appropriate measures during this critical period is vital for optimal soil utilization.

Comments 25: Introduction

General Comments:

Some sentences are overly complex and could be simplified for better readability.

Ensure consistent use of terminology. For instance, "facility vegetable planting" and "greenhouse vegetable production" should be standardized.

Response 25: The introduction has undergone a comprehensive revision to enhance its logical flow and clarity. The order of paragraphs has been rearranged, and the topics presented within them have been refined to ensure a seamless narrative. The writing style has also been fine-tuned to make the content more coherent and easily understandable.

Comments 26: Line 34-35: "The method of facility vegetable planting has been rapidly evolving, resulting in the cultivation of over 4 million hectares of land in 2021[1]."

Suggestion: "The method of greenhouse vegetable planting has rapidly evolved, resulting in over 4 million hectares being cultivated in 2021[1]."

Response 26: The sentence has been revised to “The method of greenhouse vegetable planting has rapidly evolved in China, resulting in over 4 million hectares being cultivated in 2021”.

Comments 27: Line 39-40: "Tomato, a globally popular vegetable is commonly grown under greenhouse conditions in northern China [2,3]."

Suggestion: "Tomatoes, a globally popular vegetable, are commonly grown in greenhouses in northern China [2,3]."

Response 27: The sentence has been revised according to the comment.

Comments 28: Line 40-41: "In practical production setting, continuous planting of greenhouse tomatoes is a conventional scenario due to cost consideration and the desire to maximize soil utilization."

Suggestion: "In practical production, continuous planting of greenhouse tomatoes is common due to cost considerations and the desire to maximize soil utilization."

Response 28: The sentence has been revised according to the comment.

Comments 29: Line 42-43: "However, this practice often leads to obstacles such as the accumulation of autotoxic substances, degradation of soil physiochemical properties, disruption of native soil microbial communities, and the buildup of soil-borne pathogens [4,5]."

Response 29: To connect with the previous sentence, “however” has been changed to “on the other hand”, and the subsequent sentence has been modified according to the comment.

Comments 30: Line 47-49: "Consequently, many studies indicated that mono-cropped or intensively managed greenhouse production systems resulted in the risk of soil acidification and salinization, and prolonged cultivation in such systems can lead to great changes in the structure and abundance of soil community[9]."

Response 30: The sentence has been revised according to the comment.

Comments 31: Line 50-51: "Soil microorganisms play a pivotal role in nutrient cycles and energy flow within the soil, serving as indicators of soil structure, fertility, stability, and sustainable utilization."

Suggestion: "Soil microorganisms play a pivotal role in nutrient cycles and energy flow, serving as indicators of soil structure, fertility, stability, and sustainable use."

Response 31: The sentence has been revised according to the comment.

Comments 32: Line 61-64: "The composition of these bacteria is influenced by nitrogen levels or types, copiotrophic groups (e.g., Alphaproteobacteria and Acidobacteria), which grow rapidly, are more prone to proliferate in nutrient-rich environments, whereas oligotrophic groups (e.g., Actinobacteria), which are considered K-strategists that have a slower growth rate, would likely decline[15]."

Clearly articulate the study's aim and hypotheses towards the end of the introduction.

Response 32: The sentence has been revised according to the comment. Furthermore, the paragraph detailing the study's aims and hypotheses has been refined in accordance with both general and specific comments provided, resulting in a more focused and comprehensive presentation of the research objectives.

Comments 33: Line 97-98: "The present study investigated the impact of the planting years on the structure and diversity of the soil microbial community in greenhouse tomatoes using real-time PCR."

Suggestion: "This study investigates the impact of planting duration on the structure and diversity of soil microbial communities in greenhouse tomatoes using real-time PCR."

Response 33: This sentence has been deleted, and according to the previous comments, this part has been written in the materials and methods section.

Thank you very much for your consideration and look forward to hearing from you soon.

Most Sincerely,

A/Prof. Xinmei Hao

College of Water Resources and Civil Engineering

China Agricultural University

No.17 Qinghua East Road, Haidian, Beijing, 100083, P. R. China

E-mail: [email protected]

Author Response File: Author Response.docx

Reviewer 2 Report

Comments and Suggestions for Authors

The problem of monocultures may be more serious, because people like to get higher yields and more “standard” yields so that many old varieties will no more be cultivated – despite of fact that these varieties might have properties which are useful if the climate is not standard or there will be more problems with pests and pesticides etc. In addition, the red tomatoes with mean size and round form are nice but sometimes the yellow small tomatoes with stronger taste may be more attractive. These factors increase the need of this paper. Especially climate is a factor that records (heat, more evaporation of water etc.), The manus should be partly corrected.

General: Open all symbols! Use italic for all scientific names! Be careful with all subscripts in chemical names, which I cannot use. See empty space before [ and after ].

Abstract: Line 17 chance “and” to “or”!

Nine years monoculture time may have been ok in this case, but it might be something else in other cases depending on climate, variety or plant species. You have done many analyses but not all and therefore in line 28 you could add “at least”

Introduction: Line 37: Is the consumption of vegetables been reduced in Chinese diet? Or is it becoming more one-sided so that tomatoes may have replaced some (rural and local) leaf or root vegetables?

Materials and methods

Number of parallels? What was the variety of tomatoes?

Results

The tables 1 and 2 must be corrected to that the rows will be columns and the columns will be rows (as they are in the figures).

You must totally rewrite the texts in Fig, 1, since now the letter texts in Figures are unclear.

Fig 5. Use yellow either for Y5 or Y9 (as used in the Fig. 6)!

Line 337: Salinity should be discussed more.

Fig. 7. Add all symbols! For instance ORNDEC? Is it ornithine degradation? What are the others? One possibility is to explain them in supplemented material.

However, amino acids belong to very essential compounds so that the availability of them might be very crucial. Thus, I would discuss this, too.

Author Response

Dear Reviewer

We appreciate your comments very much, which have greatly helped to improve quality of the manuscript. We hope that the current version of the manuscript can be considered for further review. The following details our responses to the comments.

Comments 1: The problem of monocultures may be more serious, because people like to get higher yields and more “standard” yields so that many old varieties will no more be cultivated – despite of fact that these varieties might have properties which are useful if the climate is not standard or there will be more problems with pests and pesticides etc. In addition, the red tomatoes with mean size and round form are nice but sometimes the yellow small tomatoes with stronger taste may be more attractive. These factors increase the need of this paper. Especially climate is a factor that records (heat, more evaporation of water etc.), The manus should be partly corrected.

Response 1: This paper has been enhanced by the inclusion of record tables detailing various treatment locations and water and fertilizer management strategies. Additionally, every section of the manuscript has been modified and improved to ensure clarity and comprehensiveness.

Comments 2: General: Open all symbols! Use italic for all scientific names! Be careful with all subscripts in chemical names, which I cannot use. See space before [ and after ].

Response 2: The proper nouns, chemical names, and other relevant formats throughout the manuscript have been checked and revised accordingly.

Comments 3: Abstract: Line 17 chance “and” to “or”!

Response 3: the sentence in line 17 has been revised to “Soil Surface layers samples from greenhouse tomatoes fields cultivated for 5 (Y5), 9 (Y9), 13 years (Y13), or a surrounding corn field (CK) as control were analyzed”. The sentence in line 28 has been revised to “In conclusion, crop alteration and continued planting change the soil bacterial composition, and increasing planting years suppressed soil bacterial diversity, leading to a stable bacterial ecology after nine years”.

Comments 4: Introduction: Line 37: Is the consumption of vegetables been reduced in Chinese diet? Or is it becoming more one-sided so that tomatoes may have replaced some (rural and local) leaf or root vegetables?

Response 4: The rational and balanced diet structure in China has led to an ever-increasing proportion of various types of vegetables in the diet. Concurrently, the escalating population in China has fueled a corresponding rise in the demand for vegetables. From 1995 to 2018, the per capita daily vegetable supply in China surged from 592 grams to 1262 grams, and this figure is projected to remain stable in 2030 and 2050. In comparison to horticulture-developed nations such as the US, the Netherlands, Greece, Japan, and South Korea, China's higher vegetable supply is primarily attributed to larger harvested areas rather than superior yields (Tang et al., 2003). Enhancing yield and quality has consistently been a pivotal research focus in horticulture. In recent years, while increasing fertilizer application has yielded certain positive results in boosting yields, it has also inadvertently led to issues like soil nutrient excess and soil health problems. These concerns will be paramount in future endeavors, as the focus shifts towards ensuring sustainable soil use while maintaining optimal yield quality.

Reference

Tang, Y., Dong, J., Gruda, N., and Jiang, H. China Requires a Sustainable Transition of Vegetable Supply from Area-Dependent to Yield-Dependent and Decreased Vegetable Loss and Waste. Int J Environ Res Public Health. 2023, 20, 1223

Comments 5: Materials and methods

Number of parallels? What was the variety of tomatoes?

Response 5: in this study, three greenhouse or corn fields were selected to be investigated. The variety of tomatoes is red tomato (Seminis 4224). This information has been added to the revised manuscript.

Comments 6: Results: The tables 1 and 2 must be corrected to that the rows will be columns and the columns will be rows (as they are in the figures).

Response 6: Table 1 and Table 2 have been revised as Table 2 and Table 3, respectively. Soil physiochemical properties have been listed in Table 1.

Table 1 Soil physicochemical properties for different treatments.

Treatment	CK ¹	Y5	Y9	Y13
ρ ²	1.58 ± 0.11 a ³	1.45 ± 0.20 a	1.41 ± 0.09 a	1.29 ± 0.16 a
EC (μS/cm)	185.83 ± 31.47 a	236.40 ± 31.36 a	449.93 ± 31.71 a	367.57 ± 7.16 a
pH	7.99 ± 0.19 a	8.01 ± 0.07 a	7.73 ± 0.14 a	7.84 ± 1.16 a
TN (mg/kg)	762.33 ± 145.31 b	1632.00 ± 145.82 a	1843.67 ± 145.94 a	1901.00 ± 9.16 a
AP (mg/kg)	26.33 ± 9.66 b	310.00 ± 9.69 a	318.33 ± 9.83 a	263.67 ± 8.16 a
AK (mg/kg)	100.67 ± 47.68 b	375.33 ± 47.96 a	266.67 ± 47.77 ab	353.67 ± 7.16 a
OC (g/kg)	9.05 ± 1.32 b	17.01 ± 1.73 a	18.68 ± 1.02 a	19.55 ± 0.16 a
C:N (mg/kg)	11.96 ± 0.49 a	10.46 ± 0.31 b	10.31 ± 0.62 b	10.34 ± 6.16 b
NH₄(mg/kg)	0.05 ± 0.01 b	0.68 ± 0.07 a	0.73 ± 0.01 a	0.65 ± 0.16 a
NO₃(mg/kg)	101.39 ± 27.58 a	5.77 ± 27.85 c	49.83 ± 27.02 b	54.92 ± 0.16 b

¹ CK: the field with corn planting; Y5: the greenhouse with a five-year planting duration; Y9: the greenhouse with a nine-year planting duration; Y13: the greenhouse with a thirteen-year planting duration.

²ρ: soil bulk density, EC: soil electric conductivity, TN: soil total nitrogen, AP: soil available phosphorus, AK: soil available potassium, OC: soil organic carbon, C:N: soil carbon-nitrogen ratio, NH₄: soil NH₄⁺-N content, NO₃: soil NO₃^--N content.

³ Values are means ± standard deviation (n = 3). Different lowercase letters indicate significant differences among treatments based on one-way ANOVA followed by an LSD test (p<0.05).

Table 2 The ASV number in taxa for different treatments.

Treatment	CK ¹	Y5	Y9	Y13
Phylum	27±1 a ²	28±1 a	28±1 a	29±1 a
Class	69±3 ab	67±5 b	76±4 a	75±3 a
Order	141±7 b	148±7 ab	159±3 a	163±9 a
Family	193±18 a	207±15 a	217±4 a	220±7 a
Genus	265±35 a	286±32 a	292±12 a	286±4 a
Species	43±13 b	69±3 a	72±3 a	65±5 a

² Values are means ± standard deviation (n = 3). Different lowercase letters indicate significant differences among treatments based on one-way ANOVA followed by an LSD test (p<0.05).

Table 3 Soil bacterial alpha-diversity indexes for different treatments.

Treatment	CK¹	Y5	Y9	Y13
Chao1	1757.7±225.6 a ²	1540.8±128.8 ab	1579.0±122.6 ab	1368.3±31.9 b
Observed species	1661.4±185.5 a	1489.7±103.5 ab	1515.3±116.6 ab	1334.7±9.3 b
Pielou_e	0.9063±0.0262 a	0.9125±0.0062 a	0.9021±0.0188 a	0.9054±0.0160 a
Shannon	9.69±0.42 a	9.62±0.10 a	9.53±0.28 a	9.40±0.16 a
Simpson	0.9963±0.0027 a	0.9976±0.0003 a	0.9968±0.0012 a	0.9969±0.0012 a

²Values are means ± standard deviation (n = 3). Different lowercase letters indicate significant differences among treatments based on one-way ANOVA followed by an LSD test (p<0.05).

Comments 7: You must totally rewrite the texts in Fig, 1, since now the letter texts in Figures are unclear.

Response 7: In the submitted paper, this issue may have arisen due to the system error. Figure 1 has been improved and has also been confirmed in the revised manuscript.

Comments 8: Fig 5. Use yellow either for Y5 or Y9 (as used in the Fig. 6)!

Response 8: The color scheme of all figures in the revised manuscript has undergone alterations to enhance clarity and improve visual comprehension.

Comments 9: Line 337: Salinity should be discussed more.

Response 9: the discussion section has been highly improved according to the comment. The discussion paragraph of salinity has been revised as follows:

During greenhouse vegetable cultivation, the soil bacterial abundance and composition exhibited a distinct pattern as the number of vegetable planting years increased. For example, bacterial phyla Actinobacteria and Proteobacteria were dominant phyla that play an important role in the soil carbon cycle and can degrade recalcitrant carbon[29]. Interestingly, the combined relative abundance of Actinobacteriota and Pseudomonadota was observed to be lower in Y9 and Y13 compared to Y5, displaying an initial decline followed by a subsequent increase. This variation was related to the changes in soil salinity, despite the observed increase in soil organic carbon with the extension of cultivation years. The increase in salinity in Y9 and Y13 limits the efficient use of soil carbon, leading to a lower relative abundance of these two dominant phyla. The same result was found in a study of surface soils based on a natural sodicity/salinity gradient, which can strongly structure soil microbial communities [30]. An et al. [26] also found lowest relative abundance of α-and γ-Proeteobacteria was related to an increase in soil salinity.

Comments 10: Fig. 7. Add all symbols! For instance ORNDEC? Is it ornithine degradation?

Response 10: The Description of each metabolic pathways was provided in supplementary materials (Table S4).

Comments 11: What are the others? One possibility is to explain them in supplemented material.

However, amino acids belong to very essential compounds so that the availability of them might be very crucial. Thus, I would discuss this, too.

Response 11: The relevant results and discussion paragraphs have been revised as follows:

The PICRUSt analysis revealed distinct metabolic pathway patterns among the four treatments, as evidenced by the second-level metabolism of the MetaCyc database. The core components of soil bacterial metabolism encompassed biosynthesis, degradation/utilization/assimilation, and the generation of precursor metabolites and energy (Figure 6). The results showed that the metabolic pathway of biosynthesis, and macromolecule modification were generally increased with the increasing of planting years, Y13 had a significantly higher relative abundance than CK treatment. Amino Acid Biosynthesis constitutes the paramount aspect of the biosynthesis process, contributing approximately a quarter of its overall functionality. Notably, Y9 and Y13 exhibited significantly elevated levels compared to Y5. The specific second-level metabolism difference was shown in the supplementary material (Table S3).

Furthermore, the peak in the metabolic pathway for Amino Acid Biosynthesis was observed in Y9, which was intimately tied to the level of soil organic matter. A previous study proved a significant linear correlation between organic matter and protease activities [36]. Thus, a higher organic matter level and micronutrient fertilizer utilization in greenhouse cultivation created a favorable environment for protease enhancement, thereby potentially providing more available substrates for Amino Acid Biosynthesis.

Thank you very much for your consideration and look forward to hearing from you soon.

Most Sincerely,

A/Prof. Xinmei Hao

College of Water Resources and Civil Engineering

China Agricultural University

No.17 Qinghua East Road, Haidian, Beijing, 100083, P. R. China

E-mail: [email protected]

Author Response File: Author Response.docx

Reviewer 3 Report

Comments and Suggestions for Authors

The article is dedicated to studying the changes in the microbial community during the cultivation of tomatoes in greenhouses over 13 years. The article contains several issues that need to be addressed:

Proteobacteria should be changed to Pseudomonadota.
Lines 62 and 64: Acidobacteria and Actinobacteria are confused. Acidobacteria are slow-growing organisms and not copiotrophs.
Materials and Methods 2.2: No details about bioinformatic processing are provided. Please include the platform, the database used for taxonomy assignment, and the similarity threshold for this taxonomy assignment.
Clarify the number of replicates used in DNA extraction and sequencing. This should be clearly explained in the text.
Results: It is standard practice in microbial community sequencing studies to provide statistics on the number of sequences obtained. Please include the total number of sequences obtained, the number remaining after quality filtration, and the number of sequences per sample.
Table 1: What do the letters 'a' and 'b' after the numbers signify? What follows the symbol ± SD or SE? Please clarify this in the table caption.
Figure 1: The format is unreadable and in an unclear language; please correct this.
Table 2: The same comments apply as for Table 1.
Page 7 and Figure 4: The alphabetical designations of genera carry no biological meaning. Include a higher taxon name to clarify the context.

Author Response

Dear Reviewer

We greatly appreciate your comments and suggestions about the manuscript, which indeed helps to improve the quality of our paper. We have studied the comments carefully and made revisions accordingly. The following are our detailed responses:

Comments 1: Proteobacteria should be changed to Pseudomonadota.

Response 1: “Proteobacteria” has been revised to “Pseudomonadota” in the manuscript.

Comments 2: Lines 62 and 64: Acidobacteria and Actinobacteria are confused. Acidobacteria are slow-growing organisms and not copiotrophs.

Response 2: After searching and comparing the references again, I am sorry that the quotation here is not rigorous enough, there are certain errors present in the relevant article. In a previous study, bacteria belonging to the Acidobacteria phylum were abundant in soils with very low resource availability, could be classified into oligotrophic, and a-Proteobacteria, Firmicutes, and Actino-bacteria, could not be assigned into copiotrophic or oligotrophic categories (Fierer, et al., 2007). The sentence in the submitted manuscript has been deleted. The total paragraph has been revised as:

The structure and activity of microbial communities vary significantly across cultivation years due to soil property changes[14]. Dominant bacterial abundance and diversity correlate with soil carbon, nitrogen, and phosphorus levels[15-17]. Zhang et al. [18] discovered that the relative abundance of the Acidobacteria phylum significantly increased with an increase in soil nitrogen input, whereas the Actinobacteria phylum showed contrasting results, indicating a decrease in its relative abundance under similar conditions. Furthermore, factors such as higher soil organic content and varying carbon-to-nitrogen ratios can significantly affect soil microbial diversity [19] and the relative abundance of Proteobacteria, Bacteroidetes, and Acidobacteria [20,21]. Specifically, the relative abundance of the Acidobacteria phylum was observed to be diminished in soils enriched with high concentrations of organic carbon. In contrast, b-Proteobacteria and Bacteroidetes, both exhibiting copiotrophic characteristics, demonstrated the highest relative abundances in soils with high availability of carbon[20].

Comments 3：Materials and Methods 2.2: No details about bioinformatic processing are provided. Please include the platform, the database used for taxonomy assignment, and the similarity threshold for this taxonomy assignment.

Clarify the number of replicates used in DNA extraction and sequencing. This should be clearly explained in the text.

Response 3: The relevant information has been added to the revised manuscript. At the same time, soil samples were analyzed using the latest database as suggested by the academic editor. The determination methods of soil physicochemical indicators also have been supplied. The section has been revised as follows:

2.3. Soil sample collection and determination of soil physicochemical and microbial properties

Comments 4：Results: It is standard practice in microbial community sequencing studies to provide statistics on the number of sequences obtained. Please include the total number of sequences obtained, the number remaining after quality filtration, and the number of sequences per sample.

Response 4: The specific information has been provided in supplementary materials (Table S2)

Comments 5: Table 1: What do the letters 'a' and 'b' after the numbers signify? What follows the symbol ± SD or SE? Please clarify this in the table caption.

Response 5: All tables presented have been revised according to the comments, and comprehensive table notes have been appended to provide additional clarity and context.

Comments 6：Figure 1: The format is unreadable and in an unclear language; please correct this.

Response 6: In the submitted paper, this issue may have arisen due to the system error. Figure 1 has been improved and has also been confirmed in the revised manuscript.

Comments 7：Table 2: The same comments apply as for Table 1.

Response 7: All tables presented have been revised according to the comments, and comprehensive table notes have been appended to provide additional clarity and context.

Comments 8：Page 7 and Figure 4: The alphabetical designations of genera carry no biological meaning. Include a higher taxon name to clarify the context.

Response 8: Upon comparative analysis, the clustering outcomes of four treatments within this study exhibit a similarity across different classification levels. Consequently, the revised manuscript adopts a phylum-level cluster analysis approach, and the corresponding results have been revised.

In the cluster heat map of the top 20 phyla based on their relative abundance in the soil bacterial community, each treatment was clustered into individual categories, except for Y9 and Y13, which formed a single clade (Figure 4). The dominant soil bacterial phyla in CK and different planting years treatments were distinctly different and the dominant soil genera could be clustered into two groups. Specifically, the relative abundance of Actinobacteriota, Entotheonellaeota, Acidobacteriota, and Gemmatimonadota was notably higher in the CK treatment than in others, gradually decreasing with an increase in planting years. Conversely, the bacterial phyla Planctomycetota, Desulfobacterota, Latescibacterota, NB1-j, Pseudomonadota, Bacteroidota, and Patescibacteria dominated the Y13 treatment.

Figure 4. Cluster heat map analysis of the top 20 phyla by relative abundance in the soil bacterial community among four treatments. Where CK is the field with corn planting; Y5 is the greenhouse with a five-year planting duration; Y9 is the greenhouse with a nine-year planting duration; Y13 is the greenhouse with a thirteen-year planting duration.

Thank you very much for your consideration and look forward to hearing from you soon.

Most Sincerely,

A/Prof. Xinmei Hao

College of Water Resources and Civil Engineering

China Agricultural University

No.17 Qinghua East Road, Haidian, Beijing, 100083, P. R. China

E-mail: [email protected]

Author Response File: Author Response.docx

Round 2

Reviewer 1 Report

Comments and Suggestions for Authors

Please see the attachment

Comments for author File: Comments.pdf

Comments on the Quality of English Language

It is fine.

Author Response

Comments 1: Title

The authors significantly altered the title, shifting the focus from continuous maize planting to the conversion of maize fields into tomato cultivation. What are the implications of this change, and how does it impact the study? Does this alteration indicate the same research focus or an entirely different study?

Response 1: The revised manuscript has the same focus as the original version. The title was modified to reflex the study more precisely. In the study, the soil properties including bacterial diversity and composition was compared between greenhouse soils with different lengths of cultivation years after conversion from growing corn for a long time, with soils of continuous corn cultivation. As the reviewer pointed out early, the title of the original version implied that the comparison was conducted between soils after conversion to greenhouse cultivation and those prior to the conversion. But this is not the case. Therefore, we think the revised title represents our study more appropriately.

Comments 2: Abstract

L23: The authors changed the control (CK) from maize to a different corn species. I am still unsure whether the research materials are the same as in the previous manuscript or if they have been changed.

Response 2: The materials are the same as the previous manuscript. We used ‘corn’ and ‘maize’ interchangeably. To keep it consistent, we changed to use “corn” throughout the manuscript. Therefore, this does not involve changes in corn species or other research materials.

Comments 3: Introduction:

The second paragraph in the Introduction section does not connect to the first. Additionally, the entire section lacks sufficient information on tomato and corn cultivation in greenhouse settings.

Line 199: It is not necessary to include a citation here.

Response 3: We have reorganized this section of the introduction. The first paragraph wrote about the importance of soil microorganisms, and the second and third paragraphs wrote about the research progress of the impact of continuous cropping on microorganisms. The fourth to seventh paragraphs discuss the shortcomings of the current research and the actual planting conditions. Against this backdrop, the greenhouse in the tomato planting area was selected as the research object. Since most greenhouses were converted from corn fields, the corn fields were also included as a control group in the study. The last paragraph writes the research goal of this article.

In addition, it should be noted that this study is not a controlled experiment, and the corn is not cultivated in a greenhouse. The reason why this article has always mentioned corn cultivation as a control is that local tomato cultivation involves destroying the corn field, building greenhouses, and then starting to plant tomatoes. It is not a new land that is cleared out to build greenhouses. During the research process, we did not find a greenhouse that had just destroyed the corn field and was about to start planting tomatoes. Therefore, we assumed that a piece of land nearby would be converted and directly sampled from the corn field as a control. This is the treatment of planting “zero year” tomatoes that has been mentioned throughout the article. In the following text, we have removed the term "zero-year" to avoid ambiguity. This may be your confusion point, which is also the reason why the introduction does not mention the setting conditions, planting methods, and so on of tomatoes and corn. It is not the main research background of this paper.

I apologize for the confusion. Upon checking, I did not find a citation in line 199 of the introduction section that you mentioned should be deleted. Additionally, line 199 of the full document discusses the method of measuring microbial indicators and does not involve any citations.

Comments 4: Experimental design:

“The study selected centralized greenhouse tomato planting and corn planting as sampling sites. Notably, corn cultivation has a rich history here, spanning over two decades. Conversely, the greenhouse tomato sites were recently established, having been restructured from portions of these established corn fields in recent years. Consequently, the adjacent corn fields naturally served as the control (CK), with a planting duration of zero years, providing a valuable benchmark for comparison. Additionally, tomato greenhouses that had undergone continuous monocropping for 5, 9, and 13 years were chosen in the vicinity (designated as Y5, Y9, and Y13, respectively). To ensure reproducibility, three greenhouses/corn fields were included for each study year.”

The experimental design is still nuclear.

I am still confused about the experimental design. Originally, the study focused on maize cultivation, but it has now shifted to corn. What does the 'zero year' control for the corn field mean? Does it refer to soil that was previously used for corn cultivation, or does it involve planting corn seedlings? Additionally, how was replication set up in the design? Was a Completely Randomized Design (CRD) or a Randomized Complete Block Design (RCBD) used? Please verify these details and consider including Figure 1 in the manuscript for clarity.

Response 4: Again, we used corn and maize interchangeably. Basically, the two terms had the exactly same meaning in the study. We have modified the manuscript to use the same term throughout the entire manuscript to avoid confusion. Strictly, our study was not a controlled experiment study, but a survey study. We collected soil samples from inside greenhouses with different years of vegetable cultivation (5-yr, 9-yr, and 13-yr), as well as nearby corn field with continuous monoculture history. We chose three sites in the region that meet the requirement. “Zero years” here means continuous maize cultivation, and no conversion to greenhouse. Therefore, there was no CRD or RCBD in the study.

The section titled of “Experimental design” has been revised as “Survey respondents”, and the first paragraph has been revised as “The study selected centralized greenhouse tomato planting and corn planting as sampling sites. Notably, corn cultivation has a rich history here, spanning over two decades. Conversely, the greenhouse tomato sites were recently established, having been restructured from portions of these established corn fields in recent years. Consequently, tomato greenhouses that had undergone continuous monocropping for 5, 9, and 13 years were chosen in the vicinity (designated as Y5, Y9, and Y13, respectively), and the adjacent corn fields naturally served as the control (CK), providing a valuable benchmark for comparison. To ensure reproducibility, three greenhouses/corn fields were included for each study year”

In addition, the word "treatment" in this article has been changed to "situation", which can better express the comparison object of this manuscript.

Figure 1 can be clearly displayed in a .doc or .docx file. Figure 1 has been uploaded separately as an attachment.

Comments 5: Results: The information presented in Table 1 for Section 3.1, 'Changes in Soil Physicochemical Properties,' is lacking. Specifically, there is insufficient statistical analysis and comparisons between the treatments and the control (CK).

Response 5: the paragraph has been revised as “The results showed that soil ρ decreased with increasing planting years while soil TN exhibited an opposite trend. Notably, soil EC, pH, AP, AK, and NH₄⁺-N values generally reached a small peak or dipped to a trough in the Y9 treatment with the continued planting. Differently, Y5 and Y13 had the significant higher soil TN, AP, AK, OC and NH₄⁺-N than CK while the opposite results were found in soil C: N and NO₃^--N levels (Table 1). No significant differences in soil ρ, EC, and pH between CK and other planting years”.

Comments 6: Discussion:

I did not find any discussion related to Section 3.1, 'Changes in Soil Physicochemical Properties, such as soil EC, pH, AP, AK, and NH4+-N ' Instead, the discussion begins with soil bacterial communities.The discussion section lacks citations and explanations regarding the conversion from corn to tomato cultivation. Additionally, it does not provide evidence or examples to support the observed changes in soil physicochemical properties or bacterial communities resulting from the change in cultivation. The discussion should align more closely with the results presented in the results section.

Response 6: In the first paragraph of Section 4.1, the change in soil bacterial composition caused by crop transition is cited. Unfortunately, we have not found any relevant research directly indicating the conversion of corn cultivation to tomato cultivation. Moreover, through the above modifications, we believe that readers can also use the results of corn cultivation as background values, rather than as a "treatment" equivalent to a controlled experiment.

The title and research objectives of this study emphasize that it is a study on the response of soil bacterial composition and diversity. In the process of explaining soil bacterial communities changes, it was found that soil physicochemical properties are the main factors affecting these changes, which is mainly reflected in the perspective of limit year effects. Therefore, we have added a citation about the changing patterns of soil physicochemical properties in section 4.2. In addition, there is also an explanation of the impact of changes in soil conductivity on microorganisms in Section 4.1.

Comments 7: Conclusions:

The research purpose and methods are not clearly outlined at the beginning. Similar issues are evident regarding soil physicochemical properties and bacterial communities. Additionally, there is a lack of comparative analysis concerning the impact of the cultivation conversion.

Response 7: The conversion of crop types is a local reality and a background situation for planting, which explains the significant differences between the research results of Y5, Y9, and Y13 and the control group. So we briefly added a sentence that "When the open field soil that has been planted with corn for many years is converted to greenhouse tomato, the soil bacterial composition has undergone significant changes".

Comments 8: Overall, the manuscript has improved, but it still contains weaknesses and insufficiencies. For example, there are inconsistencies with spacing before or after citations, such as on lines 64, 68, 76……., and throughout the manuscript.

Response 8: The format and order of references have been corrected in the revised manuscript.

Thank you very much for your consideration.

Author Response File: Author Response.docx

Reviewer 3 Report

Comments and Suggestions for Authors

All comments have been addressed, and the article can be accepted for publication. Please note that Figure 1 is still displayed incorrectly in the PDF version

Author Response

Comment 1: All comments have been addressed, and the article can be accepted for publication. Please note that Figure 1 is still displayed incorrectly in the PDF version

Response 1: Thank you very much for your recognition. Figure 1 can be clearly displayed in a .doc or .docx file. Figure 1 has been uploaded separately as an attachment.

Author Response File: Author Response.docx

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Conversion to Greenhouse Cultivation from Continuous Corn Production Decreases Soil Bacterial Diversity and Alters Community Structure

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