Nutrient Limitation and Ecological Chemicalometry Reveal the Impacts of Long-Term Continuous Cropping on Lavender Rhizosphere Soil

Comments 1: [The 20-year field plot exhibited a 177%–1033% increase in enzyme activity compared to the 1-year plot". This statement is too vague and misleading. The term 'enzyme activity' encompasses a variety of enzymes, with significant variations in their activity levels. According to Figure 1 in the article, the carbon harvesting enzyme (EEC) increased from approximately 130 to 390 μ mol/g h from Y1 to Y20, with an increase of approximately 200%; And the nitrogen harvesting enzyme (EEN) increased from about 700 to 1400 μ mol/g · h, an increase of about 100%. The increase of "1033%" mentioned in the original text did not find clear corresponding data in the main text, and it was not specified which enzyme it was. It is suggested that the author clearly list the specific range of increase for each enzyme, or change it to precise statements such as "carbon capture enzyme activity increases by 200%, nitrogen capture enzyme activity increases by 100%".]

Response 1: Thank you for pointing this out. We agree with this comment. Therefore, we have made the necessary changes.[We replaced the general statements in the original text with more specific expressions. The modified content is as follows:”The activity of carbon-related acquisition enzymes increases from 134.81 μmol/g·h in the first year to 393.86 μmol/g·h in the 20th year, an increase of 192%; the activity of nitrogen acquisition enzymes increases from 686.11 μmol/g·h in the first year to 1430.58 μmol/g·h in the 20th year, an increase of 108%. This indicates that the decomposition of organic matter and the nutrient cycling capacity continue to enhance.”The revision is located on Page 1, Paragraph 1, Line 21. ]

Comments 2: [The author must verify and unify the interpretation standards of the enzyme vector model. According to Moorhead et al. (2016, Soil Biology and Biochemistry), a vector angle>45 ° typically indicates phosphorus limitation, while<45 ° indicates nitrogen limitation. However, the 1:1 line division method in Figure 3D is different from conventional explanations, and it is necessary to clarify the definition of the coordinate axis of the figure and the criteria for determining the type of restriction, in order to ensure consistency between the abstract, main text, and caption.]

Response 2: Thank you for pointing this out. We agree with this comment. Therefore, we have made the necessary changes.[Based on the suggestions you provided, we have explained Figure 3(d) in the text. The content is as follows: "Figure 3(d) has the abscissa as (carbon-related acquisition enzyme) / (carbon-related acquisition enzyme + phosphorus-related acquisition enzyme), and the ordinate as (carbon-related acquisition enzyme) / (carbon-related acquisition enzyme + nitrogen-related acquisition enzyme + phosphorus-related acquisition enzyme). The upper left quadrant indicates that the contribution of carbon-related enzymes is relatively high, while the contribution of phosphorus-related enzymes is relatively low. The upper right quadrant, in this quadrant, on the upper side of the 1:1 dividing line, the activity of carbon-related enzymes dominates, while the contribution of phosphorus-related enzymes is less; on the lower side of the dividing line, the activity of carbon-related enzymes dominates, while the contribution of nitrogen-related enzymes is less. The lower left quadrant, on the upper side of the 1:1 dividing line, corresponds to the situation where the activities of carbon and phosphorus-related enzymes are low; on the lower side of the 1:1 dividing line, it corresponds to the situation where the activities of carbon and nitrogen-related enzymes are low. The lower right quadrant is characterized by the dominance of nitrogen-related enzymes, while the activity of carbon-related enzymes is relatively low."The modification is located on page 13, line 368. ]

Comments 3: [The title and abstract of the entire article use 'continuous cropping', but the first paragraph of the introduction uses' monoculture '. In agriculture and soil ecology, there is a clear distinction between these two terms. Monoculture: refers to the continuous planting of the same crop in the same field, with a time scale that can be long or short. Continuous cropping/Consecutive cropping: specifically refers to the long-term continuous planting of the same crop, usually associated with cohesive cropping obstacles. This article studies long-term continuous cropping for 1-20 years, and using 'continuous cropping' is more accurate. However, the introduction extensively uses the term 'monoculture' without distinction. It is recommended to unify the terminology or clearly define the concept in the introduction.]

Response 3: Thank you for pointing this out. We agree with this comment. Therefore, we have made the necessary changes.Thank you very much for pointing out the issues. [We have made the necessary revisions based on the suggestions you provided. We have replaced 'monoculture' with 'continuous cropping' in the text. The revisions are located on page 1, in the 1st paragraph, lines 12, 13, and 39; on page 2, in the 1st paragraph, lines 44, 45, 54, 56, and 64; on page 3, in the 3rd paragraph, lines 129, 132, and 140; on page 4, in the 1st paragraph, line 145; on page 17, in the 3rd paragraph, line 491; on page 18, in the 3rd paragraph, lines 538 and 551; on page 19, in the 1st paragraph, line 565; and on page 20, in the 2nd paragraph, lines 617 and 635.]

Comments 4:[Add 2-3 articles on soil stoichiometry in arid/oasis agriculture; Provide a clear reference threshold for the stoichiometric ratio of microbial biomass; Increase the discussion on the specificity of irrigated agriculture.]

Response 4:Thank you for pointing this out. We agree with this comment. Therefore, we have made the necessary changes.Thank you very much for pointing out the issues.[The discussion part has already been covered and relevant literature has been consulted for reference.The revision is made at line 459 of the first paragraph on page 17 and at line 501 of the third paragraph.]

Comments 5:[Research hypothesis (1) proposes that "MBC and MBN continue to accumulate, while phosphorus is relatively scarce", but does not explain why carbon and nitrogen are expected to "continue" to accumulate rather than reach equilibrium. Hypothesis (2) proposes a change in restriction type from nitrogen limitation to phosphorus limitation, but the introduction does not provide sufficient theoretical basis to predict this transition. According to ecological stoichiometry theory, long-term continuous cropping leads to an increase in soil C: N ratio and a decrease in N: P ratio (at the end of the second paragraph of the introduction), which should lead to an increase in carbon limitation and a relief of phosphorus limitation, rather than the opposite.Adding specific discussions on root exudates input, soil organic matter accumulation, and phosphorus fixation mechanisms in the hypothesis derivation makes the theoretical foundation of the hypothesis more solid.]

Response 5:Thank you for pointing this out. We agree with this comment. Thank you very much for your suggestions.[ Regarding the issues related to the assumptions mentioned in the text that you raised, we have provided a more persuasive theoretical basis for the assumptions proposed in the text: "Long-term continuous cultivation often leads to a nutrient imbalance, with an increase in the carbon-to-nitrogen ratio (C:N) in the soil and a decrease in the nitrogen-to-phosphorus ratio (N:P). This imbalance causes carbon and nitrogen cycles to dominate, while phosphorus remains a limiting factor. The input of root exudates and organic matter plays a crucial role in the nutrient cycle, influencing the composition of microbial biomass and nutrient limitations. Additionally, the fixation mechanism of phosphorus, such as adsorption onto soil particles, further limits the availability of phosphorus, and the limitation of phosphorus will continue or intensify."The modification is located on page 3, paragraph 3, line 132.]

Comments 6:[The article does not specify in which season the sampling was conducted. Lavender in the Ili River Valley usually blooms from June to August, and there is significant seasonal variation in the microbial activity of the rhizosphere soil. The sampling time is crucial for the reproducibility of the results.]

Response 6:Thank you for pointing this out. We agree with this comment. Thank you very much for your suggestions.[Due to our negligence, the sampling time was not mentioned in the text. I have now added the sampling time to the text: "Sampling was conducted in early August."The modification is located on page 4, in the third paragraph, at line 174.]

Comments 7:[The definition and collection method of "rhizosphere soil": The article only states that "Soil samples are collected within the lavender planting rows", using a 5 cm diameter soil drill to collect the 0-20 cm soil layer. But the strict definition of rhizosphere soil should be the soil in direct contact with the root system (usually 1-4 mm from the root surface). The methods described in the article are more like root zone soil or inter row soil. If the collected soil is indeed rhizosphere soil, it should be indicated whether the shaking off method or root washing method was used.]

Response 7:Thank you for pointing this out. We agree with this comment. Thank you very much for your suggestions.[Based on the suggestions you provided, we have re-described the methods and details used for sampling. "Within a 10-cm radius around the root system of each target plant, the soil was carefully excavated to retrieve intact roots. The loosely attached soil was gently shaken off and discarded, while the soil that was tightly adhering to the root surface (strictly defined as rhizosphere soil, < 2 mm from the root surface) was collected using a sterile brush. The sampling depth was 0–20 cm. For each treatment, three representative plants with consistent growth status were selected per row, and the rhizosphere soils were pooled to form one composite sample. A total of 10 composite samples were collected per treatment, yielding 60 samples across the six treatments. All samples were immediately placed in a cooler with ice packs, transported to the laboratory, and stored at 4°C prior to analyses of soil enzyme activities, microbial biomass, and soil physicochemical properties. "The modification is located on page 4, in the third paragraph, at line 180.]

Comments 8:[The spatial independence of repeated sampling points: The spatial distribution of 10 mixed sampling points within a 1-hectare field is not specified, which may lead to spatial autocorrelation issues.]

Response 8:Thank you for pointing this out. We agree with this comment. Thank you very much for your suggestions.[Now we will elaborate on the spatial distribution of the sampling points in this text: "Ten sampling points are arranged in a systematic grid pattern within fields larger than 1 hectare. The spacing between the points is fixed at 20 meters, and they are distributed in a "zigzag" pattern to avoid directional deviations. A 5-meter field edge buffer zone is also set up. Each sample is composed of 5 sub-samples within a 2-meter by 2-meter area around the sampling point (collected diagonally, with sub-sample spacing ≥ 50 cm), in order to balance the micro-variation of the root zone and the representativeness of the sampling."The modification is located on page 4, in the third paragraph, at line 188.]

Comments 9:[The units of EEC, EEN, and EEP in Figures 1d-f are "μ mol/g · h", but the enzyme activity units commonly measured by fluorescence method are "nmol/g · h" or "μ mol/g · h". According to the numerical values (EEC about 100-400, EIN about 700-1400), this unit seems to be too large and needs to be confirmed whether it is an error of "nmol".]

Response 9:Thank you for pointing this out. We agree with this comment. Thank you very much for your suggestions.[After reviewing the data, we did mistakenly mix up the units. Now, we will change "μmol/g·h" to "nmol/g·h".The modification is located on page 9, line 317.]

Comments 10:[Revise formula layout; Clearly state the parameter order of the ATAN2 function; Provide complete calculation codes or Excel templates in the supplementary materials.]

Response 10:Thank you for pointing this out. We agree with this comment. Thank you very much for your suggestions.[We have revised the formula in the text and submitted the supplementary materials.The modification is located on page 6, line 5, at position 245.]

Comments 11:[Clarify the calculation method and parameter sources of CUE; If indirect estimation is used, specific literature should be cited and the formula should be explained.]

Response 11:Thank you for pointing this out. We agree with this comment. Thank you very much for your suggestions.[Based on the valuable suggestions you provided, we will improve the CUE formula and related contents.The revision is made in the 6th page, on lines 1 and 3 of paragraphs 230 and 239.]

Comments 12:[Review all statistical results; Explain the possible mechanisms behind abnormal years (such as the MBP peak of Y4 and the ratio trough of Y10) during the discussion; Consider adding years as continuous variables (rather than categorical variables) for trend analysis.]

Response 12:Thank you for pointing this out. We agree with this comment. Thank you very much for your suggestions.In response to your suggestions, we have incorporated the explanations needed into the article.[The MBP fluctuation is significant over the years. The peak in the 4th year might be due to an increase in organic matter input or improved temperature and humidity conditions that promote microbial activity; the trough in the 10th year might be related to the slowdown of organic matter turnover, reduced phosphorus input, or drought/nutrient imbalance leading to a decrease in microbial biomass, and it might also be influenced by the change in crop rotation in the previous period that alters the community structure. After adjusting the years from a categorical variable to a continuous variable, the long-term trend of the MBP fluctuation can be identified rather than isolated anomalies, revealing the periodic change patterns driven by environmental factors such as soil moisture, nutrient availability, and temperature, thereby more accurately assessing the impact of long-term environmental changes on the microbial phosphorus dynamics.The modification is located on page 18, paragraph 2, line 509.]

Comments 13:[According to Figure 3b, the vector angles of Y1 and Y4 are approximately 30 ° -35 ° (<45 °), indicating nitrogen limitation according to the standard; The vector angles of Y15 and Y20 are greater than 45 °, indicating phosphorus limitation. This contradicts the description in the main text once again.]

Response 13:Thank you for pointing this out. We agree with this comment. Thank you very much for your suggestions.[Due to some issues in data processing, after reprocessing the data, we have now modified the figures in the paper to be in line with the description in the text.The modification is located on page 13, line 367.]

Comments 14:[The mechanism by which MBP sharply decreases after reaching its peak at Y4 remains unexplained.]

Response 14:Thank you for pointing this out. We agree with this comment. Thank you very much for your suggestions.[We now incorporate the reason for the sharp decline in MBP4 after its peak into the paper: "After lavender was grown in succession for four years, the phenolic acid allelochemicals secreted by the root system accumulated in the rhizosphere soil and exceeded the threshold for microbial inhibition, directly inhibiting the activity of phosphatase and disrupting the structure of the phosphorus-decomposing microbial community, resulting in a shift from the brief release of available phosphorus at the beginning of cultivation to its fixation and depletion. At the same time, the vigorous growth of the plants and microorganisms intensified the competition for the limited available phosphorus, forcing the microbial phosphorus assimilation capacity to collapse, ultimately leading to a cliff-like decline in microbial biomass phosphorus after reaching its peak."The modification is located on page 8, paragraph 2, line 303.]

Comments 15:[The trend of changes in EEP (phosphorus harvesting enzyme) contradicts the conclusion of phosphorus limitation.]

Response 15:Thank you for pointing this out. We agree with this comment. Thank you very much for your suggestions.[By reprocessing the data, the contradictory statements have been eliminated.The modification is located on page 12, paragraph 2, line 357.]

Comments 16:[CUE first rise and then fall mode lacks mechanism explanation.]

Response 16:Thank you for pointing this out. We agree with this comment. Thank you very much for your suggestions.[We have now incorporated the CUE change mechanism into the text: "The core reason for the initial increase and subsequent decline of microbial carbon utilization efficiency (CUE) lies in the following: In the early stage of cultivation, there is an abundant supply of easily decomposable carbon sources and relatively weak nutrient limitation, allowing microorganisms to maintain a high CUE through efficient assimilation strategies. However, as the consecutive cropping years increase, phenolic acid allelochemicals accumulate, leading to the depletion of available phosphorus. Microorganisms are then forced to shift a large amount of carbon from biomass construction to extracellular enzyme synthesis and stress maintenance of respiration. The imbalance between investment and return in carbon and nutrient acquisition eventually causes a cliff-like decline in CUE."The revision is located on page 14, in the second paragraph, at line 391.]

Comments 17:[Increase the discussion on the mechanism of phosphorus fixation in irrigated agriculture in arid areas; Comparison of chemical stoichiometry studies between supplementary and shrub/semi shrub ecosystems; Be cautious of direct analogies with research on different ecosystems.]

Response 17:Thank you for pointing this out. We agree with this comment. Thank you very much for your suggestions.[We have added the following to the discussion section: "Unlike natural shrub ecosystems that gradually adapt to phosphorus limitation through community succession and mycorrhizal symbiosis, the monoculture system of lavender forms a unique "chemical-biological" synergistic fixation mechanism under irrigation conditions: Ca²⁺ and HCO₃⁻ in the irrigation water drive the continuous precipitation of phosphorus, the pulse concentration changes caused by dry-wet alternation intensify the fixation strength, and the phenolic acid substances released by the roots further block the desorption sites of phosphorus. This process is driven by secondary metabolism of the roots, the chemistry of the irrigation water and the coupling of soil minerals, and an independent theoretical framework is needed to explain it."The revision is located on page 17, line 459 of the first paragraph.]

Comments 18:[Thoroughly verify the consistency between the conclusion and the main text data; Revise the names of key factors; Limit the expression of 'steady state' or use more accurate terms such as' regime shift '.]

Response 18:Thank you for pointing this out. We agree with this comment. Thank you very much for your suggestions.[Due to previous issues with data processing, the data has now been revised and improved. The conclusion is consistent with the data.The revision is located on page 7, line 296.]

Comments 19:[The existing PCA and RDA analyses reveal correlations, but cannot resolve causal paths. Suggest constructing SEM to clarify the cascading effects of continuous cropping years, soil physicochemical properties, microbial biomass, enzyme activity, and nutrient limitations.]

Response 19:Thank you for pointing this out. We agree with this comment. Thank you very much for your suggestions.[Thank you for your valuable feedback. However, due to the insufficient sample size for the experiment, it is not possible to meet the requirements for constructing the SEM. We have explained the causal path in the text as follows: "The gradient structure revealed by PCA shows that PC1 (38.37%) constitutes the main gradient axis of 'continuous cropping years - nutrient availability': the left side gathers 4-year samples, accompanied by high MBP, SOC and AP loads, representing the initial root-zone nutrient enrichment state during continuous cropping; the right side distributes 15-year and 20-year samples, carrying high EC, TN and enzyme activity vectors, reflecting the coupling of soil salinization and microbial metabolic stress under long-term continuous cropping. PC2 (19.15%) constitutes the secondary axis of 'acidification - salinization', with the upper part of high NO₃⁻-N and EC loads corresponding to the nitrogen leaching and salt accumulation of 20-year samples, and the lower part of the low pH vector pointing to the extreme soil acidification of 10-year samples. This orthogonal gradient structure confirms that the continuous cropping years are not driven by a single dimension, but are jointly driven by two independent paths of nutrient depletion and acidification/salinization to promote soil functional degradation. The constrained sorting of RDA further quantifies the driving mechanism of MBP as a key response variable: The MBP vector is significantly negatively correlated with pH, EC, TER_E-EEA and TER_E-L (P < 0.001), while it is positively correlated with SOC, AP, and NH₄⁺-N. Thus, the causal chain thus constructed is: prolonged continuous cropping → accumulation of phenolic acid substances in roots (supported by literature) → soil acidification (P↓) and salinization (EC↑) → inhibition of phosphatase activity → immobilization of available phosphorus (AP↓) and depletion of microbial phosphorus pool (MBP↓) → microbial forced increase in extracellular enzyme investment (TER↑/EEA↑) to exploit scarce phosphorus → redistribution of carbon resources from biomass construction to enzyme synthesis and maintenance of respiration → decline in carbon utilization efficiency (CUE↓). Among them, soil acidification and effective phosphorus depletion constitute a dual bottleneck, the former exacerbating phosphorus fixation through Al³⁺/Fe³⁺ activation, and the latter directly triggering microbial phosphorus starvation responses; and the increase in enzyme investment is not an adaptive gain, but a costly, low-return stress compensation, ultimately leading to the loss of soil ecosystem stability."The modification is located on page 15, paragraph 2, line 420.]

· Comments 1: [Long phrases can be made simpler and more clear with thorough, expert English editing.]

Response 1: Thank you for your valuable suggestions. [We have revised the article again.The entire text was carefully edited and polished in English.]

Comments 2: [Include a conceptual schematic mechanism.]

Response 2: Thank you for pointing this out. We agree with this comment. Therefore, we have made the necessary changes.[We added mechanism diagrams to the text.The modification is located on page 16, line 454.]

Comments 3: [Several phrases are misused or inconsistent. For example, "intercropping" is sometimes used when monoculture or continuous cropping is intended. "Ecological chemicalometry" should be used instead of "ecological stoichiometry," and "chemical metric characteristics" should be defined more clearly. This needs to be fixed throughout because it causes conceptual confusion.]

Response 3: Thank you for pointing this out. We agree with this comment. Therefore, we have made the necessary changes.[We have changed 'intercropping' to 'continuous cropping'.We changed 'ecological stoichiometry' to 'Ecological stoichiometry'In the article, we clearly defined the concept of stoichiometric characteristics: "Stoichiometric characteristics refer to the quantitative proportion relationships of key elements such as carbon (C), nitrogen (N), and phosphorus (P) in organisms or ecosystems. The core lies in revealing the constraint laws of element balance - the growth, metabolism, and community structure of organisms are not determined by the absolute content of a single element, but are regulated by the relative proportions of elements."The revision is located on page 1, line 2, page 5, paragraph 2, line 199, page 17, paragraph 3, line 483, and page 19, paragraph 2, line 592.]

· Comments 4:[The Materials and Methods section have several ambiguous or inadequate methodological details, such as the sample design (are the fields independently or pseudo-replicated? Soil depth is restricted to 0–20 cm, and environmental variability between locations is uncontrolled. There was no explanation given. Additionally, the measurement parameters (temperature, incubation time) and enzyme activity (units) are not consistently provided. Additionally, statistical analysis includes some tests that are superfluous or inadequately explained, as well as an excessively complicated description. Therefore, make the experimental design (replication, site selection) clearer, include measurement information, and streamline the statistical part.]

Response 4:Thank you for pointing this out. We agree with this comment. Thank you very much for your suggestions.[We have already provided an explanation in the text.The modification is located on page 4, in the third paragraph, at line 184.]

Comments 5:[Enhance figure captions, cut down on repetition, and make sure wording and images are consistent in the results section.]

Response 5:Thank you for pointing this out. We agree with this comment. Thank you very much for your suggestions.[After the revision, the wording in the result section is consistent with the pictures.The graphics in the entire text have been modified to ensure consistency between the text and the graphics.]

· Comments 6:[Discussion: Results (Fig. 3d) indicate that P limitation is already present in the early phases, while the abstract shifts from N limitation to P limitation + C limitation. Therefore, interpretation needs to be reconciled because it is inconsistent. Lack of mechanistic depth also includes the absence of functional genes, root exudates, direct measurement of P availability methods, and microbial community study (such as sequencing).]

Response 6:Thank you for pointing this out. We agree with this comment. Thank you very much for your suggestions.[Previously, due to a problem with data processing, the graphics were incorrect. However, the issue has now been resolved through modifications.The modification is located on page 13, line 367.]

· Comments 7:[Incorporate microbial community data in the conclusion section (future work).]

Response 7:Thank you for pointing this out. We agree with this comment. Thank you very much for your suggestions.[The causal chain in the current conclusion, which is "phenolic acid accumulation → phosphatase inhibition → effective phosphorus depletion → CUE decline", has formed a closed loop. In future follow-up studies, we will incorporate microbial community data to quantify the contribution rate of community structure changes to the soil phosphorus cycling function.The revision is located on page 21, line 659 of the first paragraph.]