Philippa Kaur

Analytical method validationThe results of the precision study with relative standard deviation (RSD), and accuracy are shown in Table 1. Through the precision study we found the value of RSD as less than 5%. Moreover, accuracy was done with percent recovery experiments. The results showed that the percentage recoveries for spiked samples were in the range of 95.7–103.7%.Table 1 Shows percent (%) recovery and relative standard deviation.Full size tablePhysicochemical properties and water quality indexThe investigations of the water quality properties of the Narora channel are shown in Table 2. The temperature, TDS, turbidity, and alkalinity were within the standards of the country18 and WHO19 (taken from UNEPGEMS). While pH and dissolved oxygen (D.O) were above the recommended standards indicating poor water quality. Moreover, the detected heavy metals were in the following order Ni  > Fe  > Cd  > Zn  > Cr  > Cu  > Mn. Among these heavy metals Mn, Cu, and Zn were within the recommended limits whereas Cr, Fe, Ni, and Cd were crossing the limits18 contributing to the poor quality. Furthermore, the WQI calculation will give more insights into the overall quality of water as it explains the combined effect of several physicochemical properties12. Its calculation is done simply by converting numerous variables of water quality into a single number12,20. In addition to this, WQI simplifies all the data and helps in clarifying water quality issues by combining the complex data and producing a score that shows the status of water quality2,12,21. The WQI classifies water quality status into five groups such as if WQI  Cu  > Zn  > Fe  > Zn  > Ni  > Cr from root to stalk; and Mn  > Cd  > Zn  > Cu  > Fe  > Ni  > Cr from stalk to leaves.Table 5 Heavy metal concentrations in Eichhornia crassipes (mg/kg.dw).Full size tableFigure 3MPI values in E. crassipes.Full size imageTable 6 Bioaccumulation factor (BAF), transfer factor (TF), and mobility factor (MF) in plant E. crassipes.Full size tableThese factors BAF, TF, and MF are utilized to monitor the level of anthropogenic pollution in plants and their surrounding medium2,15,32,34,35. BAF shows the concentrations of heavy metals bioaccumulated by plants from the water. If the BAF  > 1 it indicates hyperaccumulation36. So, in the present study, all the concerned heavy metals were hyperaccumulated in the plant. The TF elucidates the capability of the plant to translocate the accumulated metals to its other parts. The roots of E. crassipes showed the highest translocation capacity for Ni (1.57) as well as Zn (1.30) to other parts. If the value of TF exceeds 1, then it represents the high accumulation efficiency37,38, therefore, plants will be considered as the hyperaccumulators for the Ni and Zn. Although the Cd was the highest accumulated metal in the plant, it could have been because of its may be because of its low TF. Whereas, TF values lower than 1 for Cr, Mn, Fe, Cu, and Cd pointed out that this plant’s roots act as a non-hyperaccumulator for these heavy metals. Furthermore, the highest MF values were depicted for Mn in both cases which reflects that E. crassipes can suitably be used for phytoextraction of Mn as well as for Cd, Zn, Fe, Ni, and Cu. The BAF, TF, and MF of Cr are low in the present study, which implies that roots are limiting the Cr. Moreover, if the BAF ≤ 1.00 then it shows the capability of absorption only rather than accumulation36,37. In addition, if the values of BAF, TF, and MF exceed 1, plants can also work for phytoextraction. Furthermore, if the BAF  > 1 and TF  More

During the COVID-19 pandemic, behavioral restrictions were imposed, after which various health problems were reported in many countries45,46. The pandemic has also increased food insecurity worldwide; consequently, panic buying has been observed in many countries, including Japan47. However, even in such situations, we found that diversity in local food access, ranging from self-cultivation to direct-to-consumer sales, was significantly associated with health and food security variables. Specifically, our results revealed the following five key discussion points.Urban agriculture in walkable neighborhoods bore fruit for health and food system resilience. However, the magnitude of its contribution differed depending on the type of urban agricultureThe results of this study showed that those who grew food by themselves at allotment farms and home gardens had significantly better subjective well-being and physical activity levels than those who did not. This result is in line with previous studies conducted during times free from the impact of infectious disease pandemics38,39,40. The use of direct sales was not related to subjective well-being but was significantly associated with physical activity. The reason might be that farm stand users tend to live in areas with farmland and travel to purchase fruits and vegetables at farm stands on foot or by bicycle. This result is consistent with that of a previous study demonstrating that the food environment in neighborhoods is an important component in promoting physical activity17.Our results also showed that those who grew food by themselves at allotment farms and those who purchased local foods at farm stands were significantly less anxious about the availability of fresh food both during the state of emergency and in the future than their counterparts. In contrast, home garden users showed significant differences only for the state of emergency. This result might be due to the differences in the size and yield of cultivation at allotment farms and home gardens. One lot in allotment farms in Tokyo can produce as much as or more than the average annual vegetable consumption per household in Japan48. However, home gardens are generally smaller and produce limited fresh foods for consumption, which may have influenced food security concerns.As in other countries, Japan imports much food from overseas and is deeply integrated into the large-scale global food system. However, as shown in this study, urban agriculture in Japanese suburbs forms small-scale, decentralized, and community-based local food systems. This multilayered food system can complement the disruptions and shortages of the global system when various problems occur for climatic, sociopolitical, or other reasons, such as pandemics. In fact, our empirical evidence suggests that urban agriculture in walkable neighborhoods, particularly allotment farms and direct-to-consumer sales at farm stands, contributed to the mitigation of food security concerns in neighborhood communities. This means that urban agriculture could enhance the resilience of the urban food system at a time when the global food system has been disrupted due to a pandemic. This validates recent discussions about the potential of urban agriculture to facilitate food system resilience10. Furthermore, our findings imply that the types of urban agriculture employed matter in determining the degree of contribution to food system resilience.To summarize the overall results, urban agriculture in walkable neighborhoods bore fruit for health and food system resilience during the COVID-19 pandemic. However, different types of urban agriculture exhibited varying associations with health and resilience. Allotment farms were positively related to all of the following: subjective well-being, physical activity, and food security concerns, both during the state of emergency and in the future. Home gardens were positively related to subjective well-being, physical activity, and food security concerns only during the state of emergency. Farm stands were positively related to physical activity and food security concerns both during the state of emergency and in the future.These differences may be due to the characteristics of the respective spaces. It is suggested that this diversity of urban agriculture has led to different types of people benefiting from various kinds of urban agriculture. Allotment farms were found to be associated with high subjective well-being, physical activity, and food security, but they may not be feasible for those who do not have enough physical strength because users are responsible for cultivating their lots, which measure 10–30 square meters40. In contrast, home gardens can be created even by those who are not confident in their physical strength. In fact, our study showed that women and older people engaged in home gardening more than men and younger people. In addition, direct-to-consumer sales at farm stands are the easiest way to obtain local fresh foods for those who do not have the time and space for allotment farms and home gardens. The need for urban agriculture has been argued in many countries2,3. However, little attention has been paid to its scale, accessibility, and diversity. Our study suggests that it is worthwhile to create diverse food production spaces within walkable neighborhoods while considering the diversity of people who access these spaces.Compared to other urban greenery and food retailers, the benefits of urban agriculture on subjective well-being and food security could be greaterCompared to the use of other urban green spaces, including urban parks, our results indicated that self-cultivation at allotment farms and home gardens was more strongly associated with subjective well-being. Previous studies have offered limited perspectives on the differences among various types of urban green spaces33. Our study further suggests that urban parks, allotment farms, and home gardens are differently associated with human health. However, as the reason was not determined, further research is needed.Furthermore, compared to other food retailers, such as supermarkets, convenience stores, and co-op deliveries, allotment farms and farm stands were more strongly associated with less anxiety about fresh food availability in the future. The availability of local fresh foods within walkable neighborhoods might have mitigated food security concerns because residents could grow food by themselves or directly observe farmers’ production processes, which may have made the difference from purchasing at places where the food systems were not visible.Flexibility in work style might promote urban agriculture in walkable neighborhoodsThere was an association between work style—working from home—and access to local food. According to the Ministry of Health, Labor and Welfare (https://www.mhlw.go.jp/english), 52% of Tokyo office workers worked from home during the first emergency declaration. Long commute times and high train congestion rates have been a problem in Tokyo suburbs, but remote workers have gained more time at and around their homes by reducing their commute times, increasing their opportunities to access local food in their walkable neighborhoods. Those who worked from home sought outdoor activities for refreshment and exercise and used a variety of urban green spaces during the pandemic49. Allotment farms and home gardens might be used as such urban green spaces. This result is consistent with previous studies assessing the characteristics of Canadian gardeners during the COVID-19 pandemic28,30.Until now, urban planners and policymakers have rarely taken work style into account. However, the flexibility of work styles and work hours may bring new insights; for example, those who work from home may become important players in urban agriculture. It has been pointed out that cities have a large hidden potential for urban agriculture by cultivating underused lands50. Our study suggests that such underused lands could be converted into productive urban landscapes for remote workers to engage in farming or gardening in between jobs as a hobby or as a side business.Food equity might be improved by urban agriculture in walkable neighborhoodsLocal fresh food is generally considered more expensive than junk food in high-income countries, creating social issues of food inequity. Therefore, past discussions on urban agriculture and food security have focused primarily on low-income households in socioeconomically disadvantaged areas24,25,26.In contrast, our study covered people from all income groups and found no statistically significant relationship between access to local food and income. This finding might be due to two urban cultural backgrounds regarding local food in Tokyo, that is, accessibility and affordability. First, residential segregation by income levels is not noteworthy in Tokyo and people from various income brackets live mixed in the same neighborhoods51. Therefore, most urban residents living in the suburbs have geographically equitable opportunities to access local foods. Second, local foods sold at farm stands are affordable. Prices are almost the same or cheaper than buying food at food retailers. While prices increase because of middleman margins related to shipping in the wholesale market, such increases are unnecessary when selling directly to consumers at farm stands. In addition, the allotment farm lots are not expensive to rent, particularly those operated by local municipalities (Supplementary Note 1).These two backgrounds make local fresh food physically and economically accessible to consumers of all income levels, resulting in food equity. This is particularly important because the concept of food system resilience includes the equitability perspective27.The integration of urban agriculture into walkable neighborhoods is a fruitful wayWhile the current discussion on walkable neighborhoods does not emphasize urban agriculture, our evidence indicated its effectiveness. The concept of walkable neighborhoods (e.g., the 15-min city model) stresses the decarbonization benefit of limiting vehicle travel, as well as the health benefits of promoting walking and cycling13,14,15,16. In addition, our research indicated that urban agriculture in walkable neighborhoods benefited health and well-being by increasing recreational outdoor opportunities to neighborhood communities, including remote workers. It also contributed to food system resilience by providing local foods to all people, including low-income households, when the global food system was disrupted due to the pandemic. Furthermore, recent studies on urban agriculture reported the decarbonization benefit of reducing carbon footprints in food production and distribution7,8. Small-scale and community-based urban agriculture in walkable neighborhoods might especially bring this benefit because neighborhood communities travel to farms on foot or by bicycle, which means almost no emission by distribution. While urban green spaces have various health benefits32,33,34,35, urban agriculture also contributes to food system resilience as well as carbon emission reduction, which makes it unique.Urban agriculture was once considered a failure of urban planning in Japan because it symbolized uncontrolled sprawl. This is analogous to the Western view, as urban agriculture was once considered the ultimate oxymoron1. However, our empirical evidence suggests that the urban‒rural mixture at neighborhood scales is a reasonable urban form that contributes to the resilience of the urban food system and to the health and well-being of neighborhood communities. It is no longer a failure of urban planning but a legacy of urban sprawl in the current urban context.Our study showed that integrating urban agriculture into walkable neighborhoods is a fruitful way of creating healthier cities and developing more resilient urban food systems during times of uncertainty. In cities where there is no farmland in intraurban areas, it would be considered effective to utilize underused spaces such as vacant lots and rooftops as productive urban landscapes. In growing cities where urban areas are still expanding, it would be advantageous to conserve agricultural landscapes within their urban fabrics. Our study could provide referential insights and robust evidence for urban policy to integrate urban agriculture into walkable neighborhoods.This study has potential limitations, including the timing of the survey and the measurement method that was utilized. We conducted the survey between June 4 and 8, 2020, just after the end of the first declaration of a state of emergency by the Japanese government. During this period, the main cultivation activities were planting and growing, and the harvest was just beginning. This seasonal constraint may have influenced the results. Because the survey was conducted during the pandemic, we used subjective methods to measure health and well-being status. However, the results might be different using objective methods52, thus further research is necessary. In addition, a longitudinal study is needed to determine whether the trends observed in this study were specific to the emergency period or whether they will persist after the COVID-19 pandemic. More

All the materials followed relevant institutional and national guidelines and legislation.MitesWe used a T. kanzawai population collected from trifoliate orange trees (Poncirus trifoliata [L.] Raf.) in 2018 in Kyoto, Japan, and a T. urticae population collected from chrysanthemum plants (Chrysanthemum morifolium Ramat.) in 1998 in Nara, Japan. These populations were reared on adaxial surfaces of kidney bean (Phaseolus vulgaris L.) primary leaves, which were pressed onto water-saturated cotton in Petri dishes (90 mm diameter, 14 mm depth). The water-saturated cotton served as a barrier to prevent mites from escaping. The dishes were maintained at 25 °C, 50% relative humidity, and a 16L:8D photoperiod. All experiments were conducted under these conditions. We only used mated adult females (i.e., the dispersal stage) of T. kanzawai or T. urticae mites.CaterpillarsWe used caterpillars of four lepidopteran species: Bombyx mori L., P. Xuthus, Spodoptera litura Fabricius and T. oldenlandiae. We collected eggs and larvae of T. oldenlandiae from C. japonica in 2021 in Kyoto, Japan, and reared them on C. japonica leaves until pupation. Theretra oldenlandiae shares Vitaceae host plants with T. kanzawai and T. urticae8,15. We collected eggs and larvae of P. xuthus from Ptelea trifoliata in 2021 in Kyoto, Japan, and reared them on Citrus unshiu Markov. leaves until pupation. Papilio. xuthus and T. kanzawai share P. trifoliata as a host plant in Kyoto (Kinto, personal observation).We obtained commercial populations of the B. mori Kinshu × Showa strain (Ueda-sanshu Co., Ltd, Nagano, Japan) or the w1-pnd strain. We reared B. mori larvae on an artificial diet produced at the Kyoto Institute of Technology. Although T. kanzawai use Morus alba, a food plant for the B. mori strain, the mite and the strain never encounter one another in the wild, because the B. mori strain has been domesticated for hundreds of years.We obtained a sub-cultured population of S. litura from the Kyoto Institute of Technology. We reared first to fourth instars of S. litura on an artificial diet (Insecta LFM, Nosan Insect Materials, Kanagawa, Japan), while final instars were fed P. vulgaris leaves. Because S. litura feeds on various wild and cultivated plants22,23, it may share some host plants with T. kanzawai and T. urticae, both of which also feed on many host plant species8,9,10.We reared caterpillars of T. oldenlandiae, P. xuthus, and S. litura in 900 mL transparent plastic cups and caterpillars of B. mori in transparent plastic containers (140 × 220 × 35 mm). All caterpillars were maintained under the same laboratory conditions described above.PlantsWe used several parts of P. vulgaris plants in the following experiments. This species is a preferred food for both mite species16,17 and S. litura24, but the other three caterpillar species do not feed on it (Kinto, personal observation). We thus used P. vulgaris rather than shared host plants, because some caterpillars and mites (T. urticae and P. xuthus, for example) do not share any host plant.Avoidance of caterpillar traces on leaf surfaces by spider mitesTo examine whether spider mites avoid settling on host plant surfaces bearing caterpillar traces, we conducted dual-choice tests using paired adjacent leaf squares with and without caterpillar traces. We did not use whole plants because, in practice, it was difficult to induce caterpillar traces on whole plants. We used two spider mite species (T. kanzawai and T. urticae) and four caterpillar species (T. oldenlandiae, P. xuthus, B. mori, and S. litura). We cut a 10 × 20 mm leaf piece from a fully expanded primary kidney bean leaf and then cut the piece into two equal squares (10 × 10 mm). To introduce caterpillar traces to one square, we arranged them on a separate piece of paper towel on water-saturated cotton. This procedure was necessary because the caterpillars used were larger than individual leaf squares. Then we placed a fourth or final instar caterpillar on the squares and induced the caterpillar to walk across every leaf square three times (Fig. 1a). We carefully removed all caterpillar-produced silk threads from the squares. Within 30 min, we arranged the square (trace +) to touch against the other square (trace −) on water-saturated cotton in a Petri dish. Subsequently, a 2- to 4-day-old mated adult female of T. kanzawai or T. urticae was introduced onto a pointed piece of Parafilm in contact with both leaf edges using a fine brush (Fig. 1a). We recorded the leaf square onto which the mite had settled at 2 h after its introduction, as preliminary observations confirmed that all females would settle on a particular leaf within that period. Each female mite and pair of leaf squares were used only once. All tests described below were conducted between 13:00 and 17:00 h, when adult female spider mites actively disperse by walking. There were 14 replicates using traces of T. oldenlandiae, 48 of P. xuthus, 20 of B. mori, and 26 of S. litura for T. kanzawai, as well as 18, 32, 16, and 47, respectively, for T. urticae. Data were subjected to two-tailed binomial tests with the common null hypothesis that a spider mite would settle on the two squares with equal probability (i.e., 0.5).Figure 1(a) Procedure used to observe avoidance of caterpillar traces by spider mites. (b) Experimental setup used to observe avoidance of B. mori traces on plant stems by T. kanzawai. (c) Experimental setup used to observe avoidance of B. mori trace extracts by T. kanzawai.Full size imageDuration of B. mori trace avoidance by T. kanzawai
To examine whether the effects of caterpillar traces on spider mite avoidance decline over time, we used T. kanzawai mites and B. mori caterpillars. We used B. mori because populations can be easily maintained over many generations. We prepared bean leaf squares with B. mori traces in the same manner descried above and preserved the traced square on water-saturated cotton for 0 h (n = 30), 24 h (n = 29), 48 h (n = 28), or 72 h (n = 28). Then we arranged the square (trace +) to lie in close proximity to the control square (trace −) that had been preserved for the same periods of time. Then we compared the avoidance response of T. kanzawai females in the same manner described above.Avoidance of B. mori traces on plant stems by T. kanzawai
To examine whether T. kanzawai females avoid walking along plant stems bearing caterpillar traces, we used Y-shaped kidney bean stems (Fig. 1b). We cut symmetric bean plants ca. 15 days after sowing from their base and inserted them perpendicularly into a 5 mL glass bottle filled with water and wet cotton. To induce caterpillar traces on one branch of the stem, we allowed a silkworm to crawl from the branching point to the far end of one branch three times for each stem (n = 20). Then we introduced a T. kanzawai adult female at a release point 35 mm below the branch point (Fig. 1b). We recorded the branch along which the female walked to the far end. Each female mite and each Y-shaped stem were used only once. The numbers of females were compared using binomial tests in the same manner described above.Avoidance of B. mori trace extracts by T. kanzawai
To extract chemical traces of caterpillar, we introduced 10 third instar B. mori to a glass Petri dish (120 mm diameter, 60 mm depth). After 1 h, we removed all caterpillars and washed the inside bottom of the dish with 1.0 mL acetone. We replicated the procedure twice using different individuals to combine all extracts and to acquire enough extract for the following experiment.To examine avoidance of B. mori trace extracts by T. kanzawai females, we conducted dual-choice experiments using T-shaped pathways of filter paper (35 × 35 mm; width, 2 mm; Fig. 1c). Using disposable micropipettes (Drummond Scientific Co., PA, USA), 1.75 caterpillar equivalents (i.e., 60 µL) of acetone extract were applied to an alternately selected branch (17.5 mm long) of each pathway (i.e., 0.10 caterpillar equivalent/mm), with control acetone applied to the other branch. We applied each solution dropwise at the junction point to minimize mixing. After evaporating the solvent from those pathways, we perpendicularly suspended them (Fig. 1c) and introduced an adult female mite at 2 days post-maturation onto the bottom of each pathway using a fine brush and recorded the branch along which the female first walked to the far end. Each female mite and each T-shaped filter paper were used only once, with 19 replicates. Each female mite made a choice within 10 min. The avoidance response of T. kanzawai was analysed in the same manner described above.Indirect effects of B. mori traces on T. kanzawai via plantsTo determine whether B. mori traces on plants indirectly affect the performance of T. kanzawai on plants, we introduced 70–80 randomly selected quiescent female deutonymphs of T. kanzawai onto kidney bean leaf disks. Immediately after synchronized adult emergence, we introduced the same number of adult males to allow mating; the detailed procedure is described elsewhere25. After 24 h, we transferred the females singly onto 10 × 10 mm bean leaf squares with or without B. mori traces prepared as described above. Because the number of eggs laid within a certain period is considered the most sensitive performance index of spider mite females26,27, any plant-mediated indirect interaction, such as defence induction in response to caterpillar traces, should result in lower egg numbers laid by the test females. We counted the eggs laid on the leaf squares 24 h after their introduction. One female that laid no eggs during the 24 h period (n = 1, trace +) was excluded from the analysis. We obtained 33 and 36 replicates for the trail+ and trail– conditions, respectively. We compared the numbers of eggs laid on leaves with and without B. mori traces using a generalized linear model with a Poisson error distribution using the SAS 9.22 software (SAS Institute Inc., Cary, NC, USA).EthicsThis article does not contain any studies with human participants or animals. More

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Effects of cropland conversion on OC pool in bulk soilCropland restoration identified as an efficient ecological project to promote soil C sequestration in karst erosion areas28,30. The conversion from MS to FG resulted in the total soil OC content and stock across 0–30 cm layers increasing by 46.12% and 43.73% respectively. The result was highly coincident with previous studies observed at 0–10 cm layer, which reported that FG cultivation replaced from MS cultivation could remarkably increase soil OC pool in karst region, Southwest China28. In our study, the lower OC content and stock in MS may be partially attributed to the non-returned crop residues and increased exposure of deep soil OM to oxygen under tillage disturbance, resulting in decreased soil OC accumulation through reducing the input of OM and accelerating OM decomposition28,30,37,38. Nevertheless, the conversion from MS to FG can increase the soil OC pool by increasing inputs from crops. For detail, laregly aboverground crops are harvested and removed from the fields each every year for economic production, there is thus a lack of aboverground OC input. Therefore, the root biomass became the main source of OM inputs, and even slight changes in biomass can substantially alter soil C level39. In the present study, the root biomass in FG field was approximately 6 times that in MS field (110.06 ± 17.24 kg hm−2 averagely) (Table S2). Consequently, the higher root biomass in FG are responsible for the corresponding higher C storage of fine root in FG, which is supported by the fact that higher amount of C were stored in the fine roots of FG field compared with that of MS field (Table S2). In fact, several studies have demonstrated that cultivation of perennial grasses is efficient in stimulating soil OC accumulation owing to its great amount of fine roots and underground biomass33,40. Soil disturbance (such as tillage) is one of the main causes of soil C depletion in agricultural systems, and increased tillage practice can result in greater soil C loss41,42,43. Therefore, the frequent tillage conducted in MS field resulted in lower levels of OC than that in FG field under minimal tillage disturbance.Impacts of cropland conversion on soil aggregates structure and stabilitySoil structure plays an important role in soil environment and quality, which is strongly characterized by soil aggregates and their stability43,44. In our study, soil macro-aggregates dominated the largest portion of total soil while meso-aggregates and micro-aggregates were only accounted for a small portion, indicating that cropland conversion could facilitated the formation of macro-aggregates (Table 2). These findings are in line with other studies, wherein that macro-aggregates occupied the major portion of total soil following farmland or vegetation restoration19,30. Tillage disturbance often disrupts aggregates by bringing subsurface soil to the surface, which can readily promote soil C turnover and hinder macro-aggregate formation45. Conversely, minimal tillage experienced and greater accumulation of root residues resulted in higher C accumulation in the FG field. Furthermore, fine roots improved the soil aggregate stability via the interaction with mycorrhizal fungi, which produced exudates and binding agents and promoted the formation of soil aggregates46,47. Therefore, higher inputs of root residue in the soil could enhance the capacity of aggregate re-formation. In fact, these can be supported by the higher value of root biomass and its C stock in the FG field. In addition, forage grass cultivation can enhance the formation of large and stable soil aggregates by fine roots and fungal hyphae through the production of exudates and binding agents, such as humic compounds, polymers and roots48,49. Thus, few tillage disturbance and higher inputs of root biomass in FG field resulted in soil aggregation enhanced, especially macro-aggregates.Soil aggregate stability can also be characterized by the values of MWD and GMD. Higher MWD or GMD values indicate greater aggregate stability due to more agglomerate ability. The value of MWD in the current study varied from 1.36 to 1.96, which was classified as “stable” by LeBissonnais’ categorization of aggregate stability50.Regardless of soil depth, the FG field had the greatest MWD and GMD values, indicating that its soil aggregates were more stable than those of the other three cropland use types. We may thus draw the conclusion that FG cropland conversion can improve the stability of aggregates based on MWD and GMD.Changes in OC stocks associated –aggregates following cropland conversionCropland use change generally affects soil C sequestration through changing OM inputs and decomposition19. Our study revealed that aggregate-associated OC was significantly higher in FG field than in MS field. These increases were mainly attributed to the new C derived from root residues inputs and decreased losses of OC associated-aggregate by C mineralization in FG soil49. Generally, tillage can breakdown large aggregates into small aggregates, and thus decrease the formation of soil macro-aggregates41,42. Thus, the lower OC content and stock associated-aggregate in MS field can be attributed to the OC loss resulting from soil erosion, and OM input reduction with tillage disturbance8,30,45.In this study, the effects of cropland conversion on OC content associated-aggregate fractions occurred in the top 20 cm soil layers. In the karst region, approximate 57–89% of crop roots are concentrated in the surface soil layer, which directly affects OM inputs from underground root residues51,52. Meanwhile, tillage practices also happened on top 20 cm soil layer6,28,29. As a result, in soils below 20 cm, little or no tillage disturbance and limited OM inputs resulted in fewer or no distinctly changing levels of OC content associated with aggregate following cropland use change.Cropland use change not only affected the OC stocks in bulk soil, but also affected the OC stocks associated-aggregates (Table 1). The difference of sensitivity of OC associated-aggregate to cropland use change may affect its contribution to bulk soil OC accumulation30,38. In our study, the macro-aggregate fraction was the most important contributor to total OC stock increase, followed by meso-aggregate and micro-aggregate (Fig. 4). This is primarily due to the higher amount and OC content of macro-aggregates. Overall all cropland use types, the OC stock associated with macro-aggregate in FG field was higher than that in other three cropland types regardless of soil depth (Fig. 4). For instance, OC stocks within macro-aggregate accounted for about 85.40%, 77.72% and 97.55% of total soil OC stock at 0–10 cm, 10–20 cm and 20–30 cm, respectively, under the conversion from MS to FG. Thus, the accumulation pattern of bulk soil OC stocks could closely related with changes of OC stocks associated with macro-aggregate under cropland use change.The physical protection of OC in aggregates is regarded as one of the main mechanisms for soil OC accumulation through diminishing soil OC degradation and preventing its interaction with mineral particles53,54. In the present study, OC stock in bulk soil correlated substantially with the OC content-associated aggregate following cropland conversion (Fig. 5). Further analysised revealed that OC stocks in bulk soil was significantly correlated to OC stock associated with macro-aggregate (R2 = 0.83, p  More

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