Study on the influence of different fertilization regimes on the yield and bulb quality of garlic (Allium Sativum L.)

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Yongtao Zhang, Jiabin Song, Xiaoping Zeng, Haimei Zhu, Lingdi Zhou, and 2 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-2063527/v1 This work is licensed under a CC BY 4.0 License Status: Posted Version 1 posted You are reading this latest preprint version Abstract Purpose: The purpose of this study was to assess the effect of microbial biofertilizer and nanofertilizer on the growth, allicin content and elemental content of garlic in China. Method: We conducted field trial of microbial biofertilizer and germanium-containing controlled release nanofertilizer along two consecutive winter periods of 2020/2021 and 2021/2022 in a major garlic production area at Pizhou city to determine the effect of treatment in comparison to the local fertilization practice. Result: The results revealed that the application of microbial biofertilizer and germanium-containing controlled release nanofertilizer increased the bulb yield by approximately 6-28% and 14%, respectively, compared with local fertilization practice. Meanwhile, the application of microbial biofertilizer consistently increased the allicin content of garlic in year 2021 and 2022. Furthermore, we also observed a strong positive correlation between shoot nitrate concentration at spring garlic stage and the final garlic bulb yield, implying that the accumulation of nitrate content in garlic at spring garlic stage maybe beneficial to biomass accumulation through either nutritional effect and increased chilling tolerance. Conclusion: Taken together, our results revealed that the application of microbial biofertilizer and germanium-containing controlled release nanofertilizer can increase growth and nitrogen use efficiency of garlic in our experiment. Allium sativum Nitrogen Nitrate Microbial biofertilizer Nanofertilizer Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 1 Introduction Garlic ( Allium sativum L.) is one of the oldest known and popular bulb vegetable crops that is growing globally in temperate and tropical regions. Garlic belongs to the family Alliaceae, which also includes onion, chive and shallot (Takhtajan 1997 ). Garlic has often been used worldwide as flavoring for dishes attributed to its unique aroma. In addition, garlic has been proven to be the excellent nutritional source of manganese, vitamin C and vitamin B6 (Petropoulus et al. 2018). In traditional medicine, garlic is used to treat various illnesses such as hypertension, cancer, cold and coughing (Gebreyohannes et al. 2013). Besides that, garlic has been found to have been reported to have high antibacterial, antifungal, anti-protozoal and antiviral activities, indicating its potential in treating different diseases (Batiha et al. 2020 ). According to a latest population-based cohort study, anti-aging protective effect of garlic might be associated with a lower risk of all-cause mortality (Shi et al. 2019 ). According to the FAO database, the total global production of garlic reached 30.7 million tons with a land area of 1.6 million hectares in 2019 (FAO 2019 ). Among garlic-producing countries, China is the world largest garlic producer with an annual output of 23.3 million tons and the production is in increasing trend to meet the demand of growing population. The total cultivated area for garlic in China has reached 0.83 million hectares (FAO 2019 ). The growth, yield and quality attributes of garlic are strongly affected by environmental factors such as genotypes and environmental factors such as climatic conditions, soil properties and agronomic practices (Atif et al. 2019 ). Among the agronomic practices, the type and the rate of fertilizer application have a profound effect on the yield of garlic and on the sustainability of soil productivity (González et al. 2009 ). As a shallow rooted vegetable, garlic have relatively higher demand for fertilizer for optimal growth and development due to its lower nutrient extraction capacity. Lower nutrient extraction capacity and higher nutrient requirement of garlic will often lead to overapplication of fertilizer by farmers that subsequently caused environmental problems (Jiku et al. 2020 ). Hence, a better fertilizer practices that can reduce the application rate of fertilizer without compromising the yield of garlic is very much needed. Conventional agricultural practices are highly dependent on chemical fertilizers and pesticides. While the chemical fertilizer can quickly increase crop yields, pro-longed use of chemical fertilizer can lead to harmful effect on the soil quality and environment (Savci 2012 ). Furthermore, the recent global energy crisis has also led to a tight supply of chemical fertilizer, and the price of chemical fertilizers has risen sharply, triggering food security concerns. Therefore, there is an urgent need for environmentally friendly agricultural practices that can improve fertilizer use efficiency. Microbial biofertilizer is one of the specific groups of fertilizer that has been shown to increase crop productivity through enhancing the availability and uptake of mineral nutrients for plants (Okur 2018 ). The underlying mechanisms by which microbial biofertilizer enhance plant growth are diverse, including biological nitrogen fixation, production of plant growth hormones, promoting solubilization of potassium and phosphorus (Kour et al. 2020 ). In garlic cultivation, studies reported that the combination of NPK fertilizer and microbial biofertilizer application can reduce fertilizer use without compromising the crop yield (Chanchan et al. 2018 ). Chanchan et al. ( 2018 ) showed that the production of garlic increased by about 15% after adding microbial biofertilizer, although the NPK doses was reduced by 25%. Hence, microbial biofertilizers have superior importance in nutrient management system to ensure agricultural sustainability, particularly for garlic cultivation. Nanofertilizer is a new type of smart fertilizer developed by using nano-technology. Due to its controlled release properties and increased nutrient bioavailability, it has been well regarded as a novel technique towards attaining sustainable agriculture (Raliya et al. 2018 ). Nanofertilizer can be categorized into three different classes i.e., nanoscale supplements, nanoscale additives and fertilizers with nanoparticles coating (Dhir 2021 ). Among which, nutrient encapsulated/coated with nanomaterial coatings has been shown to have the effect of slowing down the release of nutrient and increase nutrient use efficiency of crops by minimizing nutrient leaching (Kottegoda et al. 2017 ). Besides that, the applica-tion of nanofertilizer can increase the bioavailability of nutrient due to its high specific surface area and high reactivity (Zulfiqar et al. 2019 ). Previous study showed that the application of “nano-urea” was beneficial to the growth of many crops including rice, radish, celery, cabbage, eggplant, pepper, tomato and so on. It was observed that the grain yield and N uptake of rice was significantly increased under “nano-urea” treatment (Sahu et al. 2022 ). However, the application of nanofertilizer in garlic cultivation is rarely reported. Therefore, in this study, we investigated the effect of microbial biofertilizer and nanofertilizer towards the yield, nutrient status and nutrient use efficiency of garlic 2. Materials And Method 2.1 Description of study site and experimental design The garlic cultivar used in this study was Daqingke, a Jiangsu local guideline cultivar, which originated from Shandong province, China. The experiment was conducted in the experimental site located at Pizhou city (34º17’N, 117º41’E), Jiangsu province, China (Figure S1). The physicochemical properties of the soil in the first-year study were as follows: pH 7.63, 36.95 ± 11.06 g·kg − 1 organic matter, 1.60 ± 0.22 g·kg-1 N, 96.57 ± 15.60 g·kg-1 P 2 O 5 and 231.11 ± 48.50 mg·kg − 1 K 2 O. The size of each plot was 1000 m 2 . The garlic was planted at 22 cm apart rows with 15 cm of individual spacing. The garlic was sown on 10 Oct 2020 and harvested on 14 May 2021. The variation in the ambient temperature throughout the growth period of garlic in this study was shown in Figure S2. In the second-year study, the physicochemical properties of the soil were as follows: pH 7.89, 16.82 ± 2.16 g·kg − 1 organic matter, 0.85 ± 0.12 g·kg − 1 N, 32.30 ± 6.98 g·kg − 1 P 2 O 5 and 274.03 ± 18.21 mg·kg − 1 K 2 O. The garlic was sown on 22 Oct 2021 and harvested on 20 May 2022. Table 1 showed the fertilization treatments used in the study. The first fertilizer used in this study was microbial biofertilizer (the product number in the market, Sunongfei2021lianzhuzhi003). The second fertilizer used in this study was germanium-containing controlled release nanofertilizer (the product number in the market, Sunongfei2021lianzhuzhi006). The preparation of germanium-containing controlled release nanofertilizer has been described in China (CN) Chinese patent documents with the publication number of CN108794114A. Table 1 Treatment carried out on garlic ( Allium sativum L.) Treatment Rate (kg·ha − 1 ) N (kg·ha − 1 ) P 2 O 5 (kg·ha − 1 ) K 2 O (kg·ha − 1 ) Other 40% 10-14-16 (Control) 1200 120 168 192 - 30% 8-10-12 (T1) 1500 120 150 180 Microbial Biofertilizer 30% 8-10-12 (T2) 1500 120 150 180 Germanium-containing Control Release Nanofertilizer 2.2 Biomass, total nitrogen (N) measurement and calculation of NUE The garlic plant samples were divided into shoots and roots, and weighed to determine the fresh weight. The plant sample was then heated at 105°C for 30 min to inactivate cellular enzymatic activities. The total N concentration in garlic plant samples were determined using Kjeldahl method (Kjeldahl 1883 ). Briefly, the finely ground garlic plant sample (3 g) were moistened with small amount of deionized water, then mixed with 5 ml of concentrated sulfuric acid and stirred overnight. The tubes containing sample solution were heated at 230°C for 30 minutes. The temperature was adjusted to 280°C and maintained for 30 minutes once smoke appeared in the tube. After that, H 2 O 2 solution was added into the sample solution and shake gently until the solution become clear. The sample solution was then heated for another 10 minutes and the nitrogen concentration in the sample were analyzed using continuous Flow AutoAnalyzer III (AA3). The total N accumulated in different parts of garlic plant samples were calculated by multiplying the N concentration with the corresponding biomass weight. The nitrogen use efficiency (kg·kg − 1 N) was calculated as: NUE = Y/N, where Y is the yield of garlic (kg.ha − 1 ) and N is the total amount of N fertilizer applied (kg·ha − 1 ); nitrogen absorption efficiency (NAE, kg.kg − 1 ) was calculated by total N content in whole plant (kg.ha − 1 )/total amount of N fertilizer applied (kg.ha − 1 ); physiological N use efficiency (PNUE, kg.kg − 1 ) was calculated as bulb yield/total N accumulation at maturity, and nitrogen harvest index (NHI, %) was calculated as total N accumulation in garlic bulb at maturity/total N accumulation at maturity (Chen et al. 2016 ). 2.3 Plant nitrate content analysis For the determination of nitrate content, plant samples (0.5 g) were homogenized in 5 mL distilled water and transferred to 10 mL centrifuge tubes. The samples were boiled for 30 minutes in 100°C water and allowed to cool down to room temperature. The sample solution was then centrifuged at 5000 ×g for 10 min, and the supernatant was transferred to a clean tube. The supernatant (0.1 mL) was then added with 0.4 mL of 5% sulfuric acid-salicylic acid solution, mixed thoroughly on a vortex mixer and kept at room temperature for 20 minutes. After that, 9.5 mL of sodium hydroxide solution (8%) was slowly added into the solution. Nitrate concentration was then measured using absorbance at 410 nm via spectrophotometer. 2.4 Elemental analysis of garlic For the determination of other elements, plant samples were first ground into powder in liquid nitrogen. The finely ground plant sample (0.5 g) was then placed in the digestion tube and mixed overnight with 5 mL concentrated nitric acid. The tubes were placed on a digestion block and heated at 120°C until frothing stopped. Then, the temperature was raised to 180°C and an appropriate amount of HClO 4 was added until the solution become clear (Chen et al. 2016 ). The concentration of phosphorus, potassium, magnesium, calcium, zinc, boron, iron and sulfur in the garlic sample was then analyzed via ICP-OES (ScientificTM iCAP6300). 2.5 Allicin content analysis For the determination of allicin, peeled fresh garlic (10 g) was mixed with 30 mL of methanol solution (methanol: water = 85:15) and mashed with tissue masher. Then, the sample solution was topped up with methanol solution to 50 ml. Then, the sample solution was centrifuged at 12,000 rpm at 4°C for 20 min. A total of 1 mL of supernatant was added with 1.5 ml of methanol solution. Later, the solution was filtered using 0.45 µm pore size filter. The filtered solution (20 µL) was then subjected to HPLC analysis using YMC-Pack ODS-AC18 (25 cm x 4.6 mm) with a gradient program at a flow rate of 0.8 mL/min. 2.6 Statistical analysis The research data were analyzed by one-way ANOVA using Duncan’s multiple range tests (P < 0.05) to compare the means. Different letters indicated statistically significant differences at p < 0.05 among treatments. All statistical evaluations were performed by applying the IBM SPSS Statistics version 20 software (SPSS Inc., Chicago, IL). 3. Results 3.1 Effect of different fertilizer treatments on growth and yield of garlic In this study, the fresh weight of garlic plants, garlic bulb and bulb yield per m 2 was determined to evaluate the effects of different fertilizer treatments on the growth of garlic under field condition (Fig. 1 and Table 2 ). In 2021, the average fresh weight of garlic plants under T1 treatment was the highest (200.7 ± 0.67 g), followed by T2 treatment (164.3 ± 2.73 g) and control treatment (136 ± 4.73 g). This corresponded well with the average fresh bulbs weight per plant. Garlic plants under T1 treatment recorded the highest average fresh bulbs weight per plant (74.3 ± 0.33 g), whereas the garlic plants under control treatment have the lowest average fresh bulbs weight per plant (58 ± 1.16 g). At harvest, the bulb yield per m 2 was the highest under T1 treatment (2.23 ± 0.01 kg), followed by T2 and control treatment (Table 2 ). A similar finding was observed in year 2022. For the average fresh weight of garlic plant, the highest was recorded under T2 treatment (182.0 ± 7.02 g), followed by T1 treatment (168.5 ± 4.19 g) and control treatment (146.3 ± 11.06 g). Meanwhile, the average fresh weight of garlic bulb was the highest under T1 treatment (97.3 ± 9.88 g), followed by T2 treatment (91.2 ± 5.66 g) and control treatment (69.2 ± 8.41 g). Similarly, the bulb yield per m 2 of the garlic grown under T2 and T1 treatment were approximately 14% and 6% higher than that of control, respectively (Table 2 ). These findings indicated that the addition of microbial biofertilizer and germanium-containing controlled release nanofertilizer had positive effects on the growth and yield of garlic even under the conditions of reduced P and K supply. Furthermore, we showed that the allicin concentration in garlic increased significantly with the application of microbial biofertilizer. In 2021, the allicin concentration in the garlic under T1 treatment was the highest (0.355 g/kg), however there was no significant difference between control (0.243 g/kg) and T2 treatment (0.283 g/kg) (Fig. 2a). On the other hand, in 2022, both treatments significantly increased the allicin content with T1 treatment recorded the highest allicin concentration (0.65 g/kg) followed by T2 treatment (0.53 g/kg) (Fig. 2b). Table 2 Fresh weight (FW) of garlic plant, garlic bulb and bulb yield per m 2 of garlic harvested in 2021 and 2022. Values are means ± SE (n≥6). Significant difference (P<0.05) is indicated by different lower case letters above bars as determined by Duncan’s multiple range test. 3.2 The nitrate concentration and content in garlic under different fertilization treatments In this study, we observed that the fluctuation patterns of nitrate concentration in the shoot and root of garlic plants at different growth stages are consistent, regardless of the types of fertilizer treatment (Fig. 3). The concentration of nitrate in garlic leaf peaked at the spring garlic stage and began to decline sharply during the bulb development stage. However, we found that the shoot nitrate concentration at spring garlic stage increased significantly under T1 and T2 treatment. In 2021, T1 and T2 treatment increased the shoot nitrate concentration by 44% and 37%, respectively (Fig. 3a). Meanwhile, in 2022, the shoot nitrate concentration of garlic at spring garlic stage increased by 51–52% under the treatment of both biofertilizers (Fig. 3b). In 2021, the garlic under T2 treatment had the highest root nitrate concentration, which was approximately 39% and 53% higher than that of control and T1 treatment, respectively (Fig. 3c). In 2022, T1 treatment recorded the highest root nitrate concentration at spring garlic stage, which was about 36.6% and 33.6% higher than that of T2 treatment and control treatment, respectively (Fig. 3d). However, there was no significant difference in nitrate concentration in the shoot and root of garlic plants under different treatments at harvest stage. 3.3 The nitrogen concentration and content of garlic under different fertilization treatments In 2021, the shoot concentration of nitrogen in garlic peaked at the spring garlic stage and declined gradually from the bulb developmental stage to the harvesting stage (Fig. 4). The highest shoot concentration of nitrogen was recorded by control treatment (25.24 ± 0.75 g/kg) at spring garlic stage, followed by T2 (15.54 ± 1.9 g/kg) and T1 treatment (11.41 ± 0.33 g/kg) (Fig. 4a). In 2022, the shoot concentration of nitrogen peaked at the seedling stage and reached its lowest value at harvest stage, irrespective of the treatments. The highest shoot concentration of nitrogen was recorded by the control treatment which was 23.55 ± 1.41 g/kg (Fig. 4a). Meanwhile, the maximum nitrogen content in the shoot was recorded at bulb developmental stage, irrespective of treatments and location (Fig. 4b). In 2021, we observed that the shoot nitrogen content in garlic plant under control treatment was the highest throughout different growth stages, except for harvest stage. At bulb developmental stage, the shoot nitrogen content of control garlic plant was 59% and 38% higher than that of T1 and T2 treatments, respectively. However, in 2022, the garlic under T1 treatment had the highest shoot nitrogen content (192.93 ± 0.99 mg) during bulb development stage (Fig. 4b). For the root concentration of nitrogen, the garlic grown in 2021 had the highest root concentration of nitrogen in spring garlic stage. Among which, T2 treatment recorded the highest root concentration of nitrogen (27.29 ± 2.55 g/kg), followed by control (24.35 ± 2.48 g/kg) and T1 treatment (16.12 ± 2.24 g/kg) (Fig. 4c). Similarly, the garlic grown in 2022 had the highest concentration of nitrogen during the spring garlic stage. Among the treatment, the garlic grown under control treatment recorded the highest root concentration, which was 23.05 ± 0.33 g/kg (Fig. 4c). In 2021, the root nitrogen content of garlic under most treatments peaked at the bulb development stage. Among which, the highest root nitrogen content was recorded by the garlic under T2 treatment. In contrast, the root nitrogen contents of garlic planted in 2022 reached its peak at spring garlic stage and the highest root nitrogen content was recorded by control treatment (Fig. 4d). In this study, the potassium content in the shoot and root of garlic plants was also quantitatively determined to explore the effect of different treatments on the potassium accumulation in garlic plants (Fig. 5). From the results, we observed that there is no significant difference between treatments except for the root potassium concentration. For the two-years study, the root potassium concentration of garlic under treatment T1 and T2 consistently higher than that of control treatment. In 2021, the root potassium concentration of garlic under treatment T1 and T2 was approximately 41% and 32% higher than those of control treatment, respectively (Fig. 5c). Similarly, in 2022, the root potassium concentration of garlic under treatment T1 and T2 was approximately 30% and 19% higher than those of control treatment, respectively (Fig. 5c). 3.5 Elemental analysis of garlic bulb Plant Mg, Ca, Fe, B and Zn content were measured to investigate the effect of different fertilization treatments on these nutrients in garlic bulb at harvest stage (Table 3 ). In 2021, it was observed that the Fe content in garlic bulb under T1 and T2 treatment was significantly higher than that in the control. The accumulation of Fe in garlic bulb under T1 and T2 treatment was approximately 63% and 64% higher than that of control treatment, respectively. Meanwhile, there was no significant difference in Mg, Ca, B and Zn content among different treatments. In 2022, the Ca content in garlic bulbs under T1 treatment was significantly higher than that in the control and T2 treatment. Contrary to the data from year 2021, the Fe content in the garlic bulb under T1 and T2 treatment was significantly lower than that in the control. The accumulation of Fe in garlic under T1 and T2 treatment was approximately 17% and 23% lower than that in the control, respectively. Table 3 Elemental analysis of garlic bulb cultivated under different fertilization treatments. Values are means ± SE (n ≥ 3). Significant difference (P < 0.05) is indicated by different lower-case letters as determined by Duncan’s multiple range test. Year Element (mg.kg − 1 ) Control T1 T2 2021 Mg 3698.90 ± 227.83a 4111.22 ± 152.45a 4248.40 ± 322.40a Ca 2422.80 ± 114.36b 2573.72 ± 57.07ab 2630.87 ± 131.75ab Fe 190.12 ± 3.41b 310.94 ± 16.83a 312.00 ± 7.41a B 11.61 ± 0.49a 12.62 ± 0.69a 13.57 ± 1.36a Zn 11.94 ± 0.58a 11.14 ± 1.36a 10.92 ± 1.65a 2022 Mg 4130.73 ± 120.81a 3865.33 ± 139.59a 3887.10 ± 125.94a Ca 2382.15 ± 110.72b 2737.28 ± 203.61a 2448.31 ± 86.43ab Fe 196.14 ± 4.50a 161.93 ± 3.92b 150.22 ± 2.95c B 12.82 ± 0.63a 13.64 ± 1.13a 12.89 ± 1.27a Zn 9.90 ± 1.32a 8.89 ± 0.71a 9.26 ± 0.45a Note: Mg: Magnesium; Ca: Calcium; Fe: Ferum; B: Boron; Zn: Zinc. 3.6 Nitrogen use efficiency (NUE) in garlic under different fertilization treatments In view of the increase in biomass and bulb fresh weight of garlic grown under T1 and T2 treatment, we calculated the nitrogen use efficiency (NUE), nitrogen absorption efficiency (NAE), physiological N use efficiency (PNUE) and nitrogen harvest index (NHI) of garlic at the harvesting stage (Table 4 ). In 2021, the NUE of garlic under T1 and T2 treatments increased by about 28% and 14%, respectively. Likewise, the PNUE of garlic grown under T1 and T2 treatment increased by 46% and 112%, respectively. For NHI, the garlic under T2 treatment increased by about 60%. In contrast, the NAE of the garlic under T1 and T2 treatment decreased by approximately 12% and 46%. However, the NAE and NHI of T2 treatment decreased by about 46% and 15%, respectively compared to those of control. Similarly, in 2022, the NUE of the garlic under T2 treatment increased by about 5% relative to that of control treatment. Meanwhile, the NAE of the garlic under T1 and T2 treatment increased by 46% and 92%, respectively. In contrast, the PNUE of garlic under T1 and T2 treatment decreased by about 21% and 22%, respectively. There was no significant difference in NHI among different treatment. Table 4 Comparison of nitrogen use efficiency, nitrogen absorption efficiency, physiological N-use efficiency and nitrogen harvest index between garlic grown under different fertilization treatments. Values are means ± SE (n ≥ 3). Significant difference (P < 0.05) is indicated by different lower-case letters as determined by Duncan’s multiple range test. Parameter Control T1 T2 2021 NUE (kg.kg − 1 ) 145.0 ± 2.89c 185.8 ± 0.83b 165.8 ± 2.20a NAE (kg.kg − 1 ) 1.17 ± 0.05a 1.03 ± 0.02b 0.63 ± 0.01b PNUE (kg.kg − 1 ) 124.39 ± 5.10c 181.24 ± 3.97b 263.75 ± 1.70a NHI (%) 48.0 ± 0.10a 47.0 ± 0.10b 76.8 ± 0.4c 2022 NUE (kg.kg − 1 ) 201.8 ± 19.56a 203.5 ± 21.17a 210.94 ± 12.81a NAE (kg.kg − 1 ) 1.06 ± 0.53a 1.55 ± 0.21a 2.04 ± 0.15b PNUE (kg.kg − 1 ) 134.39 ± 4.65a 132.40 ± 5.34a 105.23 ± 13.87a NHI (%) 46.0 ± 0.40b 46.0 ± 0.10b 47.0 ± 0.20a Note: NUE: Nitrogen Use Efficiency; NAE: Nitrogen Absorption Efficiency; PNUE: Physiological N-use efficiency; NHI: Nitrogen Harvest Index. 3.7 Correlation between nitrate concentration at spring garlic stage and garlic biomass In this study, Pearson correlation analysis was conducted to investigate the relationship between nitrate concentration at spring garlic stage and the biomass of garlic at harvesting stage (Fig. 6). We observed that nitrate concentration in the shoot of garlic at spring garlic stage was positively correlated with to the biomass of garlic at harvest stage (R2 = 0.797; p ≤ 0.01). Meanwhile, there was no correlation observed between nitrate concentration in the root of garlic and biomass of garlic at harvesting stage. The finding indicated that the shoot nitrate concentration at spring garlic stage was somewhat related to the final biomass of garlic. 4. Discussion 4.1 Nanofertilizer improve the biomass accumulation and quality parameter of garlic In this two-years study, we showed that the application of microbial biofertilizer and germanium containing controlled release nanofertilizer significantly increased garlic plant biomass accumulation and bulb yield in a major garlic production area (Pizhou city) in China (Fig. 1; Table 2 ). In addition, the NUE and NAE of the garlic under the treatment of both fertilizers was also higher than those of control treatment. The yield increasing effect of beneficial microorganisms on garlic has also been reported in several previous study. For example, the introduction of genetically engineered strain of the nitrogen-fixing bacterium Pseudomonas protegens CHA0 has been found to increase the bulb yield of garlic by 12.03% (Wang et al. 2020 ). Besides that, the study also revealed that the introduction of Pseudomonas protegens can lower disease index and increased the allicin content in garlic (Wang et al. 2020 ). This was consistent with our result that the allicin content in garlic was significantly increased under treatment of microbial biofertilizer. For nanofertilizer, a similar observation was also made by many previous studies that the application of CuO nanoparticle and Ag nanoparticle can significantly improve the agronomic traits of onion such as biomass accumulation, nutrient elements and yield (Fouda et al. 2020 ; Wang et al. 2020 ). 4.2 Pattern of nitrate accumulation throughout growth stage In aerobic agricultural soil, nitrate is arguably the main source of mineral nitrogen for plants (Dechorgnat et al. 2011 ). Since garlic performed well when planted in well-drained soil, hence it is reasonable to assume that nitrate is essential for its growth and development. In plant, nitrate is actively taken up by root cells and mobilized to other plant organs through the help of nitrate transporters (Fan et al. 2017 ). Within plant cells, some of the absorbed nitrate is metabolized directly into nitrogen-containing compounds and the excessive nitrate is translocated into vacuole for storage (Wang et al. 2014 ). It was shown that cold temperature can have multiple negative effects on plant morphology and physiology, especially on root metabolic activity, leaf water potential and nitrate uptake (Laine et al. 1994 ; Burchett et al. 2006 ; Sun et al. 2017 ). In this study, we observed that the nitrate accumulation in the whole plant of garlic increased drastically during spring garlic stage. This nitrate accumulation in plants may be associated to the low temperature and then low photosynthesis and C accumulation during winter season. However, high nitrate from N accumulation at the later stage would result into a faster conversion rate of nitrate into N-containing compounds (such as protein) and therefore a faster biomass accumulation with high photosynthesis together at the late growth stage. However, at the later growth stage (i.e., bulb development stage and harvest stage), the conversion of nitrate into N-containing compounds become faster than the nitrate absorbed from the root, leading to lower nitrate accumulation. 4.3 Nitrate concentration at spring garlic stage was positively correlated with garlic biomass at harvest In this study, we observed a strong positive correlation between the shoot nitrate concentration at spring garlic stage and garlic biomass. In contrast, there was no significant correlation between the root nitrate concentration and garlic biomass. This observation suggested that the shoot nitrate concentration in garlic at spring garlic stage exert significant impact on biomass accumulation of garlic at harvest. The observed phenomenon may be either due to the role of nitrate as nutrient or as the major anionic osmotica that increase plant tolerance to chilling stress (Heerden et al. 2004 ). Chilling stress is one of the environmental factors that restrict the growth and productivity of plant. Generally, chilling stress causes a decrease in the hydraulic conductivity of the roots and subsequently a significant decrease in leaf water potential. The reduced leaf water potential leads to the loss of leaf turgor pressure, resulting in severe plant wilting (Hussain et al. 2018 ). Hence, osmotic adjustment is crucial to assists the plant to maintain higher turgor potential. It has been well established that nitrate is the major osmolyte that help maintaining the cell turgor by reducing the osmotic potential of vacuolar sap in many crops (Mott and Steward 1972 ). Besides that, previous study reported a linear correlation between plant nitrate and water content (Cárdenas-Navarro et al. 1999 ). For example, nitrate has been shown to involve in stomatal functioning by controlling the turgor pressure of guard cell (Guo et al. 2003 ). In addition, study showed that intracellular nitrate can also control the root hydraulic properties in plants, resulting into higher water uptake (Gorska et al. 2008 ). A previous study also showed that nitrate supplementation can significantly reduce the detrimental effect of chilling stress on crops (Heerden et al. 2004 ). This observation suggested that the higher shoot nitrate concentration at spring garlic stage can increase the chilling tolerance of garlic, which may be achieved by maintaining leaf water potential. In addition to nitrate, studies showed that potassium (K + ) is also important for cold tolerance in plant by regulating the osmotic and water potential, increasing antioxidant levels to reduce ROS production and reducing electrolyte leakage caused by chilling stress (Oosterhuis et al. 2013 ; Hasanuzzaman et al. 2018 ). Hence, the relatively higher root concentration of K + maybe a protective strategy for garlic to survive under chilling stress (Fig. 5c). As the major cation in plant cell, K + has been shown to be closely linked with NO 3 − (counterion of K + ) in translocation, transportation and distribution due to the effect of anion-cation balance in plant cells (Raddatz et al. 2020 ). Hence, it is possible that nitrate and potassium act synergistically in protecting the garlic from chilling stress. It has been reported that exogenous application of potassium nitrate can promote the cold tolerance in Panax ginseng by enhancing the antioxidant level (Devi et al. 2012 ). However, a more detailed study at the molecular level should be conducted in order to further understand the underlying mechanism. 5. Conclusions Overall, the microbial biofertilizer and germanium-containing controlled release fertilizer used in this study could significantly increase the growth, nitrogen use efficiency and allicin content. This study also observed a strong positive correlation between the shoot nitrate concentration at spring garlic stage and garlic biomass. It indicated that higher shoot nitrate at spring garlic stage may promote the growth of garlic at later stage, which may be due to the synergistic effect of nitrate and potassium in enhancing the cold tolerance of garlic. Our study provides a scientific theoretical basis for high yield garlic using the microbial biofertilizer. Declarations Ethics approval and consent to participate ： Here is no ethics conflicts in these data Consent to publish: we are consent to publish these data Availability of data and materials: The data and materials in the manuscript are available. Competing interests: The authors declare no competing interests. Funding: This work was supported by the sub-project 4 of Jiangsu middle and late mature garlic industrial cluster construction: Garlic mechanical intelligent operation technology and demonstration and promotion of green production technology. Authors' Contributions: ZY produced the fertilizer; ZY, FX, ZX designed the experiment; SJ, LZ, FX conducted and wrote the manuscript; ZH and ZL supplied the experiment land. Acknowledgements: We would like to thank the assistance of Agricultural and rural Office of Nianzhuang Town, Pizhou city, Jiangsu, China. References Atif MJ, Amin B, Ghani MI, Hayat S, Ali M, Zhang Y, Cheng Z (2019) Influence of different photoperiod and temperature regimes on growth and bulb quality of garlic ( Allium sativum L.) cultivars. 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Res Crop 19(1):127. https://doi.org/10.5958/2348-7542.2018.00021.9 Chen J, Zhang Y, Tan Y, Zhang M, Zhu L, Xu G, Fan X (2016) Agronomic nitrogen‐use efficiency of rice can be increased by driving OsNRT2.1 expression with the OsNAR2.1 promoter. Plant Biotechnol J 14(8):1705-1715. https://doi.org/10.1111/pbi.12531 Dechorgnat J, Nguyen CT, Armengaud P, Jossier M, Diatloff E, Filleur S, Daniel-Vedele F (2011) From the soil to the seeds: the long journey of nitrate in plants. J Exp Bot 62(4):1349–1359. https://doi.org/10.1093/jxb/erq409 Devi BSR, Kim YJ, Selvi SK, Gayathri S, Altanzul K, Parvin S, Yang DU, Lee OR, Lee S, Yang DC (2012) Influence of potassium nitrate on antioxidant level and secondary metabolite genes under cold stress in Panax ginseng . Russ J Plant Physiol 59, 318–325. https://doi.org/10.1134/S1021443712030041 Dhir B (2021) Nanofertilizers and their applications. In: Kumar, R.; Kumar, R.; Kaur, G. (eds) New Frontiers of Nanomaterials in Environmental Science. Springer, Singapore, pp 229-241 Fan X, Naz M, Fan X, Xuan W, Miller AJ, Xu G (2017) Plant nitrate transporters: from gene function to application. J Exp Bot 68(10):2463–2475. https://doi.org/10.1093/jxb/erx011 FAO 2019. Statistical Yearbook. Food and Agriculture Organization (FAO). http://www.fao.org/faostat/en/#data/QC. Fouda MMG, Abdelsalam NR, El-Naggar ME, Zaitoun AF, Salim BMA, Bin-Jumah M, Allam AA, Abo-Marzoka SA, Kandil EE (2020) Impact of high throughput green synthesized silver nanoparticles on agronomic traits of onion. Int J Biol Macromol 149(2020):1304-1317. https://doi.org/10.1016/j.ijbiomac.2020.02.004 Gebreyohannes G, Gebreyohannes M (2013) Medicinal values of garlic: A Review. 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(2018) Potassium: A Vital Regulator of Plant Responses and Tolerance to Abiotic Stresses. Agron 2018 , 8(3), 31. https://doi.org/10.3390/agronomy8030031 Heerden P, Strasser R, Krüger G (2004) Reduction of dark chilling stress in N 2 -fixing soybean by nitrate as indicated by chlorophyll a fluorescence kinetics. Physiol Plant 121(2):239–249. https://doi.org/10.1111/j.0031-9317.2004.0312.x Hussain HA, Hussain S, Khaliq A, Ashraf U, Anjum SA, Men S, Wang L (2018) Chilling and drought stresses in crop plants: implications, cross talk, and potential management opportunities. Front Plant Sci 9:393. https://doi.org/10.3389/fpls.2018.00393 Jiku MA, Alimuzzaman M, Singha A, Rahaman M, Ganapati RK, Alam M, Sinha S (2020) Response and productivity of Garlic ( Allium Sativum L.) by different levels of potassium fertilizer in farm soils. Bull Natl Res Cent 44:9(2020). https://doi.org/10.1186/s42269-020-0267-7 Kjeldahl J (1883) Neue Methode zur Bestimmung des Stickstoffs in organischen Körpern. Zeitschrift für Anal Chemie 22: 366–382. Kottegoda N, Sandaruwan C, Priyadarshana G, Siriwardhana A, Rathnayake UA, Arachchige DMB, Kumarasinghe AR, Dahanayake D, Karunaratne V, Amaratunga GAJ (2017) Urea-hydroxyapatite nanohybrids for slow release of nitrogen. ACS Nano 11(2):1214-1221. https://doi.org/10.1021/acsnano.6b07781 Kour D, Rana KL, Yadav AN, Yadav N, Kumar M, Kumar V, Vyas P, Dhaliwal HS, Saxena AK (2020) Microbial biofertilizers: Bioresources and eco-friendly technologies for agricultural and environmental sustainability. Biocatal Agric Biotechnol 23:101487. https://doi.org/10.1016/j.bcab.2019.101487 Laine P, Bigot J, Ourry A, Boucaud J (1994) Effects of low temperature on nitrate uptake, and xylem and phloem flows of nitrogen, in Secale cereale L. and Brassica napus L. New Phytol 127(4):675–683. https://doi.org/10.1111/j.1469-8137.1994.tb02970.x Mott RL, Steward FC (1972) Solute accumulation in plant cells V. An aspect of nutrition and development. Ann Bot 36:915–937. Okur NA (2018) A review: Bio-fertilizers- power of beneficial microorganisms in soils. Biomed J Sci Tech Res 4(4): 4028-4029. https://doi.org/ 10.26717/BJSTR.2018.04.001076 Oosterhuis DM, Loka DA, Raper TB (2013) Potassium and stress alleviation: Physiological functions and management of cotton. J Plant Nutr Soil Sci 176:331–343. Petropoulos SA, Fernandes Â, Ntatsi G, Petrotos K, Barros L, Ferreira ICFR (2018) Nutritional value, chemical characterization and bulb morphology of greek garlic landraces. Mol 23(2):319. https://doi.org/10.3390/molecules23020319 Raddatz N, Morales de los Ríos L, Lindahl M, Quintero FJ, Pardo JM (2020) Coordinated Transport of Nitrate, Potassium, and Sodium. Front Plant Sci 11:247. https://doi.org/10.3389/fpls.2020.00247 Raliya R, Saharan V, Dimkpa C, Biswas P (2018) Nanofertilizer for precision and sustainable agriculture: Current state and future perspectives. J Agric Food Chem 66(26):6487-6503. https://doi.org/10.1021/acs.jafc.7b02178. Sahu TK, Kumar M, Kumar N, Chandrakar T, Singh DP (2022) Effect of nano urea application on growth and productivity of rice ( Oryza sativa L.) under midland situation of Bastar region. Pharma innov 11(6):185-187. Savci S (2012) Investigation of effect of chemical fertilizers on environment. APCBEE Procedia 1:287–292. https://doi.org/10.1016/j.apcbee.2012.03.047 Shi X, Lv Y, Mao C, Yuan J, Yin Z, Gao X, Zhang Z (2019) Garlic consumption and all-cause mortality among chinese oldest-old individuals: A population-based cohort study. Nutrients 11(7): 1504. https://doi.org/10.3390/nu11071504 Sun B, Liu GL, Phan TT, Yang LT, Li YR, Xing YX (2017) Effects of Cold Stress on Root Growth and Physiological Metabolisms in Seedlings of Different Sugarcane Varieties. Sugar Tech 19:165–175. https://doi.org/10.1007/s12355-016-0452-z Takhtajan A (1997) Diversity and classification of flowering plants; Columbia University, New York Press, pp 643 Wang M, Bian Z, Shi J, Wu Y, Yu X, Yang Y, Ni H, Chen H, Bian X, Li T, Zhang Y, Jiang L, Tu Qiang (2020) Effect of the nitrogen-fixing bacterium Pseudomonas protegens CHA0-ΔretS-nif on garlic growth under different field conditions. Ind. Crops Prod. 145:111982. https://doi.org/10.1016/j.indcrop.2019.111982 Wang M, Shen Q, Xu G, Guo S (2014) Chapter one-new insight into the strategy for nitrogen metabolism in plant cells. In; Jeon, K.W.B.T.-I.R. of C. and M.B., Ed.; Academic Press, 2014; Vol. 310, pp. 1–37 ISBN 1937-6448. Wang Y, Deng C, Cota-Ruiz K, Peralta-Videa JR, Sun Y, Rawat S, Tan W, Reyes A, Hernandez-Viezcas JA, Niu G, Li C, Gardea-Torresdey JL (2020) Improvement of nutrient elements and allicin content in green onion ( Allium fistulosum ) plant exposed to CuO nanoparticles. Sci Total Environ 725:138387. https://doi.org/10.1016/j.scitotenv.2020.138387 Zulfiqar F, Navarro M, Ashraf M, Akram NA, Munné-Bosch S (2019) Nanofertilizer use for sustainable agriculture: advantages and limitations. Plant Sci 289:110270. https://doi.org/10.1016/j.plantsci.2019.110270 Supplementary Files SupplementaryInformation.docx Cite Share Download PDF Status: Posted Version 1 posted You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. As a division of Research Square Company, we’re committed to making research communication faster, fairer, and more useful. We do this by developing innovative software and high quality services for the global research community. Our growing team is made up of researchers and industry professionals working together to solve the most critical problems facing scientific publishing. Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-2063527","acceptedTermsAndConditions":true,"allowDirectSubmit":true,"archivedVersions":[],"articleType":"Research Article","associatedPublications":[],"authors":[{"id":183490396,"identity":"f2a5c8ec-0859-4469-99b5-90d741d53096","order_by":0,"name":"Yongtao Zhang","email":"","orcid":"","institution":"Jiangsu Noni Huinong Agricultural Technology Co., Ltd.","correspondingAuthor":false,"submittingAuthor":false,"prefix":"","firstName":"Yongtao","middleName":"","lastName":"Zhang","suffix":""},{"id":183490397,"identity":"3a70cb0c-b578-4a7a-b480-2e66ed4e21fc","order_by":1,"name":"Jiabin Song","email":"","orcid":"","institution":"Nanjing 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06:54:34","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-2063527/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-2063527/v1","draftVersion":[],"editorialEvents":[],"editorialNote":"","failedWorkflow":false,"files":[{"id":34373690,"identity":"813b50f5-fddd-42f6-9cd5-150eb5a14105","added_by":"auto","created_at":"2023-03-16 18:51:47","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":136324,"visible":true,"origin":"","legend":"\u003cp\u003eLegend not included with this version\u003c/p\u003e","description":"","filename":"OnlineFig.1.png","url":"https://assets-eu.researchsquare.com/files/rs-2063527/v1/b139fe904dccfb533aae0c24.png"},{"id":34374317,"identity":"2bc36377-0159-4e85-8ef1-46124cb7f35e","added_by":"auto","created_at":"2023-03-16 18:59:47","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":23868,"visible":true,"origin":"","legend":"\u003cp\u003eLegend not included with this version\u003c/p\u003e","description":"","filename":"OnlineFig.2.png","url":"https://assets-eu.researchsquare.com/files/rs-2063527/v1/277fa4da7030b4fd9bd66384.png"},{"id":34374320,"identity":"8f282acf-9eb3-47d1-b1af-608941e619da","added_by":"auto","created_at":"2023-03-16 18:59:47","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":111973,"visible":true,"origin":"","legend":"\u003cp\u003eFluctuation of nitrate concentration and content in the shoot and root of garlic plant at different growth stages under varying fertilizer treatments. \u003cstrong\u003ea\u003c/strong\u003e Shoot nitrate concentration (g·kg\u003csup\u003e-1\u003c/sup\u003e) of garlic harvested in 2021, \u003cstrong\u003eb\u003c/strong\u003e Shoot nitrate concentration (g·kg\u003csup\u003e-1\u003c/sup\u003e) of garlic harvested in 2022, \u003cstrong\u003ec\u003c/strong\u003e Root nitrate concentration (g·kg\u003csup\u003e-1\u003c/sup\u003e) of garlic harvested in 2021, \u003cstrong\u003ed\u003c/strong\u003e Root nitrate concentration (g·kg\u003csup\u003e-1\u003c/sup\u003e) of garlic harvested in 2022. Values are means ± SE (n ≥ 3). Significant difference (P\u0026lt;0.05) is indicated by asterisk as determined by Duncan’s multiple range test\u003c/p\u003e","description":"","filename":"OnlineFig.3.png","url":"https://assets-eu.researchsquare.com/files/rs-2063527/v1/8a71391167433fbad36d21e3.png"},{"id":34373688,"identity":"64535789-0216-4dfa-a303-64e4dbbad984","added_by":"auto","created_at":"2023-03-16 18:51:47","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":119405,"visible":true,"origin":"","legend":"\u003cp\u003eNitrogen concentration and content in the shoot and root of garlic plant under different fertilizer treatments. \u003cstrong\u003ea\u003c/strong\u003e Nitrogen concentration in the shoot of garlic plant. \u003cstrong\u003eb\u003c/strong\u003eNitrogen content in the shoot of garlic plant. \u003cstrong\u003ec\u003c/strong\u003e Nitrogen concentration in the root of garlic plant. \u003cstrong\u003ed\u003c/strong\u003e Nitrogen content in the root of garlic plant. Values are means±SE (n ≥ 3). Significant difference (P\u0026lt;0.05) is indicated by asterisk as determined by Duncan’s multiple range test\u003c/p\u003e","description":"","filename":"OnlineFig.4.png","url":"https://assets-eu.researchsquare.com/files/rs-2063527/v1/a57d9b14cb3ae14c870f720c.png"},{"id":34374498,"identity":"34bd3922-fd8a-4ec8-916b-886d822b2eea","added_by":"auto","created_at":"2023-03-16 19:07:47","extension":"png","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":56681,"visible":true,"origin":"","legend":"\u003cp\u003eAvailable potassium concentration and content in the shoot and root of garlic plant under different fertilizer treatments. \u003cstrong\u003ea\u003c/strong\u003e Available potassium concentration in the shoot of garlic plant. \u003cstrong\u003eb\u003c/strong\u003e Available potassium content in the shoot of garlic plant. \u003cstrong\u003ec\u003c/strong\u003e Available potassium concentration in the root of garlic plant. \u003cstrong\u003ed\u003c/strong\u003e Available potassium content in the root of garlic plant. Values are means ± SE (n ≥ 3). Significant difference (P\u0026lt;0.05) is indicated by asterisk as determined by Duncan’s multiple range test\u003c/p\u003e","description":"","filename":"OnlineFig.5.png","url":"https://assets-eu.researchsquare.com/files/rs-2063527/v1/ff531383a231d8c4b70e11fb.png"},{"id":34374318,"identity":"3b67fc71-0a62-4d27-ace0-61f6636cc76c","added_by":"auto","created_at":"2023-03-16 18:59:47","extension":"png","order_by":6,"title":"Figure 6","display":"","copyAsset":false,"role":"figure","size":30565,"visible":true,"origin":"","legend":"\u003cp\u003ePearson correlation analysis between nitrate concentration at the spring garlic stage and garlic biomass at harvest. Blue colored line represents pearson correlation analysis between shoot nitrate concentration at spring garlic stage and garlic biomass at harvest. Orange colored line represents pearson correlation analysis between root nitrate concentration at spring garlic stage and garlic biomass at harvest.\u003c/p\u003e","description":"","filename":"OnlineFig.6.png","url":"https://assets-eu.researchsquare.com/files/rs-2063527/v1/1fccdcdd5b1b538952d5766e.png"},{"id":43648536,"identity":"219b8373-6d6a-4d82-a760-b3768e5213b3","added_by":"auto","created_at":"2023-09-25 17:18:12","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":1323377,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-2063527/v1/0af38427-6e5d-4c42-860d-29198c8fcdc8.pdf"},{"id":34373694,"identity":"1d0bb957-1dc2-450c-aa9d-1fb8ad7711fb","added_by":"auto","created_at":"2023-03-16 18:51:47","extension":"docx","order_by":10,"title":"","display":"","copyAsset":false,"role":"supplement","size":439577,"visible":true,"origin":"","legend":"","description":"","filename":"SupplementaryInformation.docx","url":"https://assets-eu.researchsquare.com/files/rs-2063527/v1/51c97759be3f96b0e8ec9558.docx"}],"financialInterests":"","formattedTitle":"Study on the influence of different fertilization regimes on the yield and bulb quality of garlic (Allium Sativum L.)","fulltext":[{"header":"1 Introduction","content":"\u003cp\u003eGarlic (\u003cem\u003eAllium sativum\u003c/em\u003e L.) is one of the oldest known and popular bulb vegetable crops that is growing globally in temperate and tropical regions. Garlic belongs to the family Alliaceae, which also includes onion, chive and shallot (Takhtajan \u003cspan citationid=\"CR35\" class=\"CitationRef\"\u003e1997\u003c/span\u003e). Garlic has often been used worldwide as flavoring for dishes attributed to its unique aroma. In addition, garlic has been proven to be the excellent nutritional source of manganese, vitamin C and vitamin B6 (Petropoulus et al. 2018). In traditional medicine, garlic is used to treat various illnesses such as hypertension, cancer, cold and coughing (Gebreyohannes et al. 2013). Besides that, garlic has been found to have been reported to have high antibacterial, antifungal, anti-protozoal and antiviral activities, indicating its potential in treating different diseases (Batiha et al. \u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2020\u003c/span\u003e). According to a latest population-based cohort study, anti-aging protective effect of garlic might be associated with a lower risk of all-cause mortality (Shi et al. \u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e2019\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eAccording to the FAO database, the total global production of garlic reached 30.7\u0026nbsp;million tons with a land area of 1.6\u0026nbsp;million hectares in 2019 (FAO \u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e2019\u003c/span\u003e). Among garlic-producing countries, China is the world largest garlic producer with an annual output of 23.3\u0026nbsp;million tons and the production is in increasing trend to meet the demand of growing population. The total cultivated area for garlic in China has reached 0.83\u0026nbsp;million hectares (FAO \u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e2019\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eThe growth, yield and quality attributes of garlic are strongly affected by environmental factors such as genotypes and environmental factors such as climatic conditions, soil properties and agronomic practices (Atif et al. \u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e2019\u003c/span\u003e). Among the agronomic practices, the type and the rate of fertilizer application have a profound effect on the yield of garlic and on the sustainability of soil productivity (Gonz\u0026aacute;lez et al. \u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e2009\u003c/span\u003e). As a shallow rooted vegetable, garlic have relatively higher demand for fertilizer for optimal growth and development due to its lower nutrient extraction capacity. Lower nutrient extraction capacity and higher nutrient requirement of garlic will often lead to overapplication of fertilizer by farmers that subsequently caused environmental problems (Jiku et al. \u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e2020\u003c/span\u003e). Hence, a better fertilizer practices that can reduce the application rate of fertilizer without compromising the yield of garlic is very much needed.\u003c/p\u003e \u003cp\u003eConventional agricultural practices are highly dependent on chemical fertilizers and pesticides. While the chemical fertilizer can quickly increase crop yields, pro-longed use of chemical fertilizer can lead to harmful effect on the soil quality and environment (Savci \u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e2012\u003c/span\u003e). Furthermore, the recent global energy crisis has also led to a tight supply of chemical fertilizer, and the price of chemical fertilizers has risen sharply, triggering food security concerns. Therefore, there is an urgent need for environmentally friendly agricultural practices that can improve fertilizer use efficiency. Microbial biofertilizer is one of the specific groups of fertilizer that has been shown to increase crop productivity through enhancing the availability and uptake of mineral nutrients for plants (Okur \u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e2018\u003c/span\u003e). The underlying mechanisms by which microbial biofertilizer enhance plant growth are diverse, including biological nitrogen fixation, production of plant growth hormones, promoting solubilization of potassium and phosphorus (Kour et al. \u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e2020\u003c/span\u003e). In garlic cultivation, studies reported that the combination of NPK fertilizer and microbial biofertilizer application can reduce fertilizer use without compromising the crop yield (Chanchan et al. \u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e2018\u003c/span\u003e). Chanchan et al. (\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e2018\u003c/span\u003e) showed that the production of garlic increased by about 15% after adding microbial biofertilizer, although the NPK doses was reduced by 25%. Hence, microbial biofertilizers have superior importance in nutrient management system to ensure agricultural sustainability, particularly for garlic cultivation.\u003c/p\u003e \u003cp\u003eNanofertilizer is a new type of smart fertilizer developed by using nano-technology. Due to its controlled release properties and increased nutrient bioavailability, it has been well regarded as a novel technique towards attaining sustainable agriculture (Raliya et al. \u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e2018\u003c/span\u003e). Nanofertilizer can be categorized into three different classes i.e., nanoscale supplements, nanoscale additives and fertilizers with nanoparticles coating (Dhir \u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e2021\u003c/span\u003e). Among which, nutrient encapsulated/coated with nanomaterial coatings has been shown to have the effect of slowing down the release of nutrient and increase nutrient use efficiency of crops by minimizing nutrient leaching (Kottegoda et al. \u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e2017\u003c/span\u003e). Besides that, the applica-tion of nanofertilizer can increase the bioavailability of nutrient due to its high specific surface area and high reactivity (Zulfiqar et al. \u003cspan citationid=\"CR39\" class=\"CitationRef\"\u003e2019\u003c/span\u003e). Previous study showed that the application of \u0026ldquo;nano-urea\u0026rdquo; was beneficial to the growth of many crops including rice, radish, celery, cabbage, eggplant, pepper, tomato and so on. It was observed that the grain yield and N uptake of rice was significantly increased under \u0026ldquo;nano-urea\u0026rdquo; treatment (Sahu et al. \u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e2022\u003c/span\u003e). However, the application of nanofertilizer in garlic cultivation is rarely reported. Therefore, in this study, we investigated the effect of microbial biofertilizer and nanofertilizer towards the yield, nutrient status and nutrient use efficiency of garlic\u003c/p\u003e"},{"header":"2. Materials And Method","content":"\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e \u003ch2\u003e2.1 Description of study site and experimental design\u003c/h2\u003e \u003cp\u003eThe garlic cultivar used in this study was Daqingke, a Jiangsu local guideline cultivar, which originated from Shandong province, China. The experiment was conducted in the experimental site located at Pizhou city (34\u0026ordm;17\u0026rsquo;N, 117\u0026ordm;41\u0026rsquo;E), Jiangsu province, China (Figure S1). The physicochemical properties of the soil in the first-year study were as follows: pH 7.63, 36.95\u0026thinsp;\u0026plusmn;\u0026thinsp;11.06 g\u0026middot;kg\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e organic matter, 1.60\u0026thinsp;\u0026plusmn;\u0026thinsp;0.22 g\u0026middot;kg-1 N, 96.57\u0026thinsp;\u0026plusmn;\u0026thinsp;15.60 g\u0026middot;kg-1 P\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e5\u003c/sub\u003e and 231.11\u0026thinsp;\u0026plusmn;\u0026thinsp;48.50 mg\u0026middot;kg\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e K\u003csub\u003e2\u003c/sub\u003eO. The size of each plot was 1000 m\u003csup\u003e2\u003c/sup\u003e. The garlic was planted at 22 cm apart rows with 15 cm of individual spacing. The garlic was sown on 10 Oct 2020 and harvested on 14 May 2021. The variation in the ambient temperature throughout the growth period of garlic in this study was shown in Figure S2. In the second-year study, the physicochemical properties of the soil were as follows: pH 7.89, 16.82\u0026thinsp;\u0026plusmn;\u0026thinsp;2.16 g\u0026middot;kg\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e organic matter, 0.85\u0026thinsp;\u0026plusmn;\u0026thinsp;0.12 g\u0026middot;kg\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e N, 32.30\u0026thinsp;\u0026plusmn;\u0026thinsp;6.98 g\u0026middot;kg\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e P\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e5\u003c/sub\u003e and 274.03\u0026thinsp;\u0026plusmn;\u0026thinsp;18.21 mg\u0026middot;kg\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e K\u003csub\u003e2\u003c/sub\u003eO. The garlic was sown on 22 Oct 2021 and harvested on 20 May 2022. Table\u0026nbsp;\u003cspan refid=\"Tab1\" class=\"InternalRef\"\u003e1\u003c/span\u003e showed the fertilization treatments used in the study. The first fertilizer used in this study was microbial biofertilizer (the product number in the market, Sunongfei2021lianzhuzhi003). The second fertilizer used in this study was germanium-containing controlled release nanofertilizer (the product number in the market, Sunongfei2021lianzhuzhi006). The preparation of germanium-containing controlled release nanofertilizer has been described in China (CN) Chinese patent documents with the publication number of CN108794114A.\u003c/p\u003e \u003cp\u003e \u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab1\" border=\"1\"\u003e \u003ccaption language=\"En\"\u003e \u003cdiv class=\"CaptionNumber\"\u003eTable 1\u003c/div\u003e \u003cdiv class=\"CaptionContent\"\u003e \u003cp\u003eTreatment carried out on garlic (\u003cem\u003eAllium sativum\u003c/em\u003e L.)\u003c/p\u003e \u003c/div\u003e \u003c/caption\u003e \u003ccolgroup cols=\"6\"\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c4\" colnum=\"4\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c5\" colnum=\"5\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c6\" colnum=\"6\"\u003e\u003c/div\u003e \u003cthead\u003e \u003ctr\u003e \u003cth align=\"left\" colname=\"c1\"\u003e \u003cp\u003eTreatment\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c2\"\u003e \u003cp\u003eRate\u003c/p\u003e \u003cp\u003e(kg\u0026middot;ha\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e)\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c3\"\u003e \u003cp\u003eN\u003c/p\u003e \u003cp\u003e(kg\u0026middot;ha\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e)\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c4\"\u003e \u003cp\u003eP\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e5\u003c/sub\u003e\u003c/p\u003e \u003cp\u003e(kg\u0026middot;ha\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e)\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c5\"\u003e \u003cp\u003eK\u003csub\u003e2\u003c/sub\u003eO\u003c/p\u003e \u003cp\u003e(kg\u0026middot;ha\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e)\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c6\"\u003e \u003cp\u003eOther\u003c/p\u003e \u003c/th\u003e \u003c/tr\u003e \u003c/thead\u003e \u003ctbody\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cb\u003e40% 10-14-16 (Control)\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e1200\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e120\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e168\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e192\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e-\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cb\u003e30% 8-10-12\u003c/b\u003e\u003c/p\u003e \u003cp\u003e\u003cb\u003e(T1)\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e1500\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e120\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e150\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e180\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003eMicrobial Biofertilizer\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cb\u003e30% 8-10-12\u003c/b\u003e\u003c/p\u003e \u003cp\u003e\u003cb\u003e(T2)\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e1500\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e120\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e150\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e180\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003eGermanium-containing Control Release Nanofertilizer\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003c/tbody\u003e \u003c/colgroup\u003e \u003c/table\u003e\u003c/div\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec4\" class=\"Section2\"\u003e \u003ch2\u003e2.2 Biomass, total nitrogen (N) measurement and calculation of NUE\u003c/h2\u003e \u003cp\u003eThe garlic plant samples were divided into shoots and roots, and weighed to determine the fresh weight. The plant sample was then heated at 105\u0026deg;C for 30 min to inactivate cellular enzymatic activities. The total N concentration in garlic plant samples were determined using Kjeldahl method (Kjeldahl \u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e1883\u003c/span\u003e). Briefly, the finely ground garlic plant sample (3 g) were moistened with small amount of deionized water, then mixed with 5 ml of concentrated sulfuric acid and stirred overnight. The tubes containing sample solution were heated at 230\u0026deg;C for 30 minutes. The temperature was adjusted to 280\u0026deg;C and maintained for 30 minutes once smoke appeared in the tube. After that, H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e solution was added into the sample solution and shake gently until the solution become clear. The sample solution was then heated for another 10 minutes and the nitrogen concentration in the sample were analyzed using continuous Flow AutoAnalyzer III (AA3).\u003c/p\u003e \u003cp\u003eThe total N accumulated in different parts of garlic plant samples were calculated by multiplying the N concentration with the corresponding biomass weight. The nitrogen use efficiency (kg\u0026middot;kg\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e N) was calculated as: NUE\u0026thinsp;=\u0026thinsp;Y/N, where Y is the yield of garlic (kg.ha\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e) and N is the total amount of N fertilizer applied (kg\u0026middot;ha\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e); nitrogen absorption efficiency (NAE, kg.kg\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e) was calculated by total N content in whole plant (kg.ha\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e)/total amount of N fertilizer applied (kg.ha\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e); physiological N use efficiency (PNUE, kg.kg\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e) was calculated as bulb yield/total N accumulation at maturity, and nitrogen harvest index (NHI, %) was calculated as total N accumulation in garlic bulb at maturity/total N accumulation at maturity (Chen et al. \u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e2016\u003c/span\u003e).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec5\" class=\"Section2\"\u003e \u003ch2\u003e2.3 Plant nitrate content analysis\u003c/h2\u003e \u003cp\u003eFor the determination of nitrate content, plant samples (0.5 g) were homogenized in 5 mL distilled water and transferred to 10 mL centrifuge tubes. The samples were boiled for 30 minutes in 100\u0026deg;C water and allowed to cool down to room temperature. The sample solution was then centrifuged at 5000 \u0026times;g for 10 min, and the supernatant was transferred to a clean tube. The supernatant (0.1 mL) was then added with 0.4 mL of 5% sulfuric acid-salicylic acid solution, mixed thoroughly on a vortex mixer and kept at room temperature for 20 minutes. After that, 9.5 mL of sodium hydroxide solution (8%) was slowly added into the solution. Nitrate concentration was then measured using absorbance at 410 nm via spectrophotometer.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec6\" class=\"Section2\"\u003e \u003ch2\u003e2.4 Elemental analysis of garlic\u003c/h2\u003e \u003cp\u003eFor the determination of other elements, plant samples were first ground into powder in liquid nitrogen. The finely ground plant sample (0.5 g) was then placed in the digestion tube and mixed overnight with 5 mL concentrated nitric acid. The tubes were placed on a digestion block and heated at 120\u0026deg;C until frothing stopped. Then, the temperature was raised to 180\u0026deg;C and an appropriate amount of HClO\u003csub\u003e4\u003c/sub\u003e was added until the solution become clear (Chen et al. \u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e2016\u003c/span\u003e). The concentration of phosphorus, potassium, magnesium, calcium, zinc, boron, iron and sulfur in the garlic sample was then analyzed via ICP-OES (ScientificTM iCAP6300).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec7\" class=\"Section2\"\u003e \u003ch2\u003e2.5 Allicin content analysis\u003c/h2\u003e \u003cp\u003eFor the determination of allicin, peeled fresh garlic (10 g) was mixed with 30 mL of methanol solution (methanol: water\u0026thinsp;=\u0026thinsp;85:15) and mashed with tissue masher. Then, the sample solution was topped up with methanol solution to 50 ml. Then, the sample solution was centrifuged at 12,000 rpm at 4\u0026deg;C for 20 min. A total of 1 mL of supernatant was added with 1.5 ml of methanol solution. Later, the solution was filtered using 0.45 \u0026micro;m pore size filter. The filtered solution (20 \u0026micro;L) was then subjected to HPLC analysis using YMC-Pack ODS-AC18 (25 cm x 4.6 mm) with a gradient program at a flow rate of 0.8 mL/min.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec8\" class=\"Section2\"\u003e \u003ch2\u003e2.6 Statistical analysis\u003c/h2\u003e \u003cp\u003eThe research data were analyzed by one-way ANOVA using Duncan\u0026rsquo;s multiple range tests (P\u0026thinsp;\u0026lt;\u0026thinsp;0.05) to compare the means. Different letters indicated statistically significant differences at p\u0026thinsp;\u0026lt;\u0026thinsp;0.05 among treatments. All statistical evaluations were performed by applying the IBM SPSS Statistics version 20 software (SPSS Inc., Chicago, IL).\u003c/p\u003e \u003c/div\u003e"},{"header":"3. Results","content":"\u003cdiv id=\"Sec10\"\u003e\n \u003ch2\u003e3.1 Effect of different fertilizer treatments on growth and yield of garlic\u003c/h2\u003e\n \u003cp\u003eIn this study, the fresh weight of garlic plants, garlic bulb and bulb yield per m\u003csup\u003e2\u003c/sup\u003e was determined to evaluate the effects of different fertilizer treatments on the growth of garlic under field condition (Fig. 1 and Table \u003cspan\u003e2\u003c/span\u003e). In 2021, the average fresh weight of garlic plants under T1 treatment was the highest (200.7\u0026thinsp;\u0026plusmn;\u0026thinsp;0.67 g), followed by T2 treatment (164.3\u0026thinsp;\u0026plusmn;\u0026thinsp;2.73 g) and control treatment (136\u0026thinsp;\u0026plusmn;\u0026thinsp;4.73 g). This corresponded well with the average fresh bulbs weight per plant. Garlic plants under T1 treatment recorded the highest average fresh bulbs weight per plant (74.3\u0026thinsp;\u0026plusmn;\u0026thinsp;0.33 g), whereas the garlic plants under control treatment have the lowest average fresh bulbs weight per plant (58\u0026thinsp;\u0026plusmn;\u0026thinsp;1.16 g). At harvest, the bulb yield per m\u003csup\u003e2\u003c/sup\u003e was the highest under T1 treatment (2.23\u0026thinsp;\u0026plusmn;\u0026thinsp;0.01 kg), followed by T2 and control treatment (Table \u003cspan\u003e2\u003c/span\u003e). A similar finding was observed in year 2022. For the average fresh weight of garlic plant, the highest was recorded under T2 treatment (182.0\u0026thinsp;\u0026plusmn;\u0026thinsp;7.02 g), followed by T1 treatment (168.5\u0026thinsp;\u0026plusmn;\u0026thinsp;4.19 g) and control treatment (146.3\u0026thinsp;\u0026plusmn;\u0026thinsp;11.06 g). Meanwhile, the average fresh weight of garlic bulb was the highest under T1 treatment (97.3\u0026thinsp;\u0026plusmn;\u0026thinsp;9.88 g), followed by T2 treatment (91.2\u0026thinsp;\u0026plusmn;\u0026thinsp;5.66 g) and control treatment (69.2\u0026thinsp;\u0026plusmn;\u0026thinsp;8.41 g). Similarly, the bulb yield per m\u003csup\u003e2\u003c/sup\u003e of the garlic grown under T2 and T1 treatment were approximately 14% and 6% higher than that of control, respectively (Table \u003cspan\u003e2\u003c/span\u003e). These findings indicated that the addition of microbial biofertilizer and germanium-containing controlled release nanofertilizer had positive effects on the growth and yield of garlic even under the conditions of reduced P and K supply.\u003c/p\u003e\n \u003cp\u003eFurthermore, we showed that the allicin concentration in garlic increased significantly with the application of microbial biofertilizer. In 2021, the allicin concentration in the garlic under T1 treatment was the highest (0.355 g/kg), however there was no significant difference between control (0.243 g/kg) and T2 treatment (0.283 g/kg) (Fig.\u0026nbsp;2a). On the other hand, in 2022, both treatments significantly increased the allicin content with T1 treatment recorded the highest allicin concentration (0.65 g/kg) followed by T2 treatment (0.53 g/kg) (Fig.\u0026nbsp;2b).\u003c/p\u003e\n \u003cp\u003e\u003cstrong\u003eTable 2\u003c/strong\u003e Fresh weight (FW) of garlic plant, garlic bulb and bulb yield per m\u003csup\u003e2\u003c/sup\u003e of garlic harvested in 2021 and 2022. Values are means \u0026plusmn; SE (n\u0026ge;6). Significant difference (P\u0026lt;0.05) is indicated by different lower case letters above bars as determined by Duncan\u0026rsquo;s multiple range test.\u0026nbsp;\u003c/p\u003e\n \u003cp\u003e\u003cimg 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\" style=\"width: 798px; height: 270.13px;\" width=\"798\" height=\"270.13\"\u003e\u003cbr\u003e\u003c/p\u003e\n \u003cdiv id=\"Sec11\"\u003e\n \u003ch2\u003e3.2 The nitrate concentration and content in garlic under different fertilization treatments\u003c/h2\u003e\n \u003cp\u003eIn this study, we observed that the fluctuation patterns of nitrate concentration in the shoot and root of garlic plants at different growth stages are consistent, regardless of the types of fertilizer treatment (Fig.\u0026nbsp;3). The concentration of nitrate in garlic leaf peaked at the spring garlic stage and began to decline sharply during the bulb development stage. However, we found that the shoot nitrate concentration at spring garlic stage increased significantly under T1 and T2 treatment. In 2021, T1 and T2 treatment increased the shoot nitrate concentration by 44% and 37%, respectively (Fig.\u0026nbsp;3a). Meanwhile, in 2022, the shoot nitrate concentration of garlic at spring garlic stage increased by 51\u0026ndash;52% under the treatment of both biofertilizers (Fig.\u0026nbsp;3b).\u003c/p\u003e\n \u003cp\u003eIn 2021, the garlic under T2 treatment had the highest root nitrate concentration, which was approximately 39% and 53% higher than that of control and T1 treatment, respectively (Fig. 3c). In 2022, T1 treatment recorded the highest root nitrate concentration at spring garlic stage, which was about 36.6% and 33.6% higher than that of T2 treatment and control treatment, respectively (Fig. 3d). However, there was no significant difference in nitrate concentration in the shoot and root of garlic plants under different treatments at harvest stage.\u003c/p\u003e\n \u003c/div\u003e\n \u003cdiv id=\"Sec12\"\u003e\n \u003ch2\u003e3.3 The nitrogen concentration and content of garlic under different fertilization treatments\u003c/h2\u003e\n \u003cp\u003eIn 2021, the shoot concentration of nitrogen in garlic peaked at the spring garlic stage and declined gradually from the bulb developmental stage to the harvesting stage (Fig.\u0026nbsp;4). The highest shoot concentration of nitrogen was recorded by control treatment (25.24\u0026thinsp;\u0026plusmn;\u0026thinsp;0.75 g/kg) at spring garlic stage, followed by T2 (15.54\u0026thinsp;\u0026plusmn;\u0026thinsp;1.9 g/kg) and T1 treatment (11.41\u0026thinsp;\u0026plusmn;\u0026thinsp;0.33 g/kg) (Fig.\u0026nbsp;4a). In 2022, the shoot concentration of nitrogen peaked at the seedling stage and reached its lowest value at harvest stage, irrespective of the treatments. The highest shoot concentration of nitrogen was recorded by the control treatment which was 23.55\u0026thinsp;\u0026plusmn;\u0026thinsp;1.41 g/kg (Fig.\u0026nbsp;4a). Meanwhile, the maximum nitrogen content in the shoot was recorded at bulb developmental stage, irrespective of treatments and location (Fig.\u0026nbsp;4b). In 2021, we observed that the shoot nitrogen content in garlic plant under control treatment was the highest throughout different growth stages, except for harvest stage. At bulb developmental stage, the shoot nitrogen content of control garlic plant was 59% and 38% higher than that of T1 and T2 treatments, respectively. However, in 2022, the garlic under T1 treatment had the highest shoot nitrogen content (192.93\u0026thinsp;\u0026plusmn;\u0026thinsp;0.99 mg) during bulb development stage (Fig.\u0026nbsp;4b).\u003c/p\u003e\n \u003cp\u003eFor the root concentration of nitrogen, the garlic grown in 2021 had the highest root concentration of nitrogen in spring garlic stage. Among which, T2 treatment recorded the highest root concentration of nitrogen (27.29\u0026thinsp;\u0026plusmn;\u0026thinsp;2.55 g/kg), followed by control (24.35\u0026thinsp;\u0026plusmn;\u0026thinsp;2.48 g/kg) and T1 treatment (16.12\u0026thinsp;\u0026plusmn;\u0026thinsp;2.24 g/kg) (Fig. 4c). Similarly, the garlic grown in 2022 had the highest concentration of nitrogen during the spring garlic stage. Among the treatment, the garlic grown under control treatment recorded the highest root concentration, which was 23.05\u0026thinsp;\u0026plusmn;\u0026thinsp;0.33 g/kg (Fig. 4c). In 2021, the root nitrogen content of garlic under most treatments peaked at the bulb development stage. Among which, the highest root nitrogen content was recorded by the garlic under T2 treatment. In contrast, the root nitrogen contents of garlic planted in 2022 reached its peak at spring garlic stage and the highest root nitrogen content was recorded by control treatment (Fig. 4d).\u003c/p\u003e\n \u003c/div\u003e\n \u003cdiv id=\"Sec13\"\u003e\n \u003cp\u003eIn this study, the potassium content in the shoot and root of garlic plants was also quantitatively determined to explore the effect of different treatments on the potassium accumulation in garlic plants (Fig. 5). From the results, we observed that there is no significant difference between treatments except for the root potassium concentration. For the two-years study, the root potassium concentration of garlic under treatment T1 and T2 consistently higher than that of control treatment. In 2021, the root potassium concentration of garlic under treatment T1 and T2 was approximately 41% and 32% higher than those of control treatment, respectively (Fig. 5c). Similarly, in 2022, the root potassium concentration of garlic under treatment T1 and T2 was approximately 30% and 19% higher than those of control treatment, respectively (Fig. 5c).\u003c/p\u003e\n \u003c/div\u003e\n \u003cdiv id=\"Sec14\"\u003e\n \u003ch2\u003e3.5 Elemental analysis of garlic bulb\u003c/h2\u003e\n \u003cp\u003ePlant Mg, Ca, Fe, B and Zn content were measured to investigate the effect of different fertilization treatments on these nutrients in garlic bulb at harvest stage (Table \u003cspan\u003e3\u003c/span\u003e). In 2021, it was observed that the Fe content in garlic bulb under T1 and T2 treatment was significantly higher than that in the control. The accumulation of Fe in garlic bulb under T1 and T2 treatment was approximately 63% and 64% higher than that of control treatment, respectively. Meanwhile, there was no significant difference in Mg, Ca, B and Zn content among different treatments.\u003c/p\u003e\n \u003cp\u003eIn 2022, the Ca content in garlic bulbs under T1 treatment was significantly higher than that in the control and T2 treatment. Contrary to the data from year 2021, the Fe content in the garlic bulb under T1 and T2 treatment was significantly lower than that in the control. The accumulation of Fe in garlic under T1 and T2 treatment was approximately 17% and 23% lower than that in the control, respectively.\u003c/p\u003e\n \u003ctable border=\"1\" id=\"Tab3\"\u003e\n \u003ccaption language=\"En\"\u003e\n \u003cdiv\u003eTable 3\u003c/div\u003e\n \u003cdiv\u003e\n \u003cp\u003eElemental analysis of garlic bulb cultivated under different fertilization treatments. Values are means\u0026thinsp;\u0026plusmn;\u0026thinsp;SE (n\u0026thinsp;\u0026ge;\u0026thinsp;3). Significant difference (P\u0026thinsp;\u0026lt;\u0026thinsp;0.05) is indicated by different lower-case letters as determined by Duncan\u0026rsquo;s multiple range test.\u003c/p\u003e\n \u003c/div\u003e\n \u003c/caption\u003e\n \u003cthead\u003e\n \u003ctr\u003e\n \u003cth align=\"left\"\u003e\n \u003cp\u003eYear\u003c/p\u003e\n \u003c/th\u003e\n \u003cth align=\"left\"\u003e\n \u003cp\u003eElement (mg.kg\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e)\u003c/p\u003e\n \u003c/th\u003e\n \u003cth align=\"left\"\u003e\n \u003cp\u003eControl\u003c/p\u003e\n \u003c/th\u003e\n \u003cth align=\"left\"\u003e\n \u003cp\u003eT1\u003c/p\u003e\n \u003c/th\u003e\n \u003cth align=\"left\"\u003e\n \u003cp\u003eT2\u003c/p\u003e\n \u003c/th\u003e\n \u003c/tr\u003e\n \u003c/thead\u003e\n \u003ctbody\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\" rowspan=\"5\"\u003e\n \u003cp\u003e\u003cstrong\u003e2021\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e\u003cstrong\u003eMg\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e3698.90\u0026thinsp;\u0026plusmn;\u0026thinsp;227.83a\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e4111.22\u0026thinsp;\u0026plusmn;\u0026thinsp;152.45a\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e4248.40\u0026thinsp;\u0026plusmn;\u0026thinsp;322.40a\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e\u003cstrong\u003eCa\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e2422.80\u0026thinsp;\u0026plusmn;\u0026thinsp;114.36b\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e2573.72\u0026thinsp;\u0026plusmn;\u0026thinsp;57.07ab\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e2630.87\u0026thinsp;\u0026plusmn;\u0026thinsp;131.75ab\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e\u003cstrong\u003eFe\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e190.12\u0026thinsp;\u0026plusmn;\u0026thinsp;3.41b\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e310.94\u0026thinsp;\u0026plusmn;\u0026thinsp;16.83a\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e312.00\u0026thinsp;\u0026plusmn;\u0026thinsp;7.41a\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e\u003cstrong\u003eB\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e11.61\u0026thinsp;\u0026plusmn;\u0026thinsp;0.49a\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e12.62\u0026thinsp;\u0026plusmn;\u0026thinsp;0.69a\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e13.57\u0026thinsp;\u0026plusmn;\u0026thinsp;1.36a\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e\u003cstrong\u003eZn\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e11.94\u0026thinsp;\u0026plusmn;\u0026thinsp;0.58a\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e11.14\u0026thinsp;\u0026plusmn;\u0026thinsp;1.36a\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e10.92\u0026thinsp;\u0026plusmn;\u0026thinsp;1.65a\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\" rowspan=\"5\"\u003e\n \u003cp\u003e\u003cstrong\u003e2022\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e\u003cstrong\u003eMg\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e4130.73\u0026thinsp;\u0026plusmn;\u0026thinsp;120.81a\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e3865.33\u0026thinsp;\u0026plusmn;\u0026thinsp;139.59a\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e3887.10\u0026thinsp;\u0026plusmn;\u0026thinsp;125.94a\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e\u003cstrong\u003eCa\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e2382.15\u0026thinsp;\u0026plusmn;\u0026thinsp;110.72b\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e2737.28\u0026thinsp;\u0026plusmn;\u0026thinsp;203.61a\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e2448.31\u0026thinsp;\u0026plusmn;\u0026thinsp;86.43ab\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e\u003cstrong\u003eFe\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e196.14\u0026thinsp;\u0026plusmn;\u0026thinsp;4.50a\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e161.93\u0026thinsp;\u0026plusmn;\u0026thinsp;3.92b\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e150.22\u0026thinsp;\u0026plusmn;\u0026thinsp;2.95c\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e\u003cstrong\u003eB\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e12.82\u0026thinsp;\u0026plusmn;\u0026thinsp;0.63a\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e13.64\u0026thinsp;\u0026plusmn;\u0026thinsp;1.13a\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e12.89\u0026thinsp;\u0026plusmn;\u0026thinsp;1.27a\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e\u003cstrong\u003eZn\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e9.90\u0026thinsp;\u0026plusmn;\u0026thinsp;1.32a\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e8.89\u0026thinsp;\u0026plusmn;\u0026thinsp;0.71a\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e9.26\u0026thinsp;\u0026plusmn;\u0026thinsp;0.45a\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003c/tbody\u003e\n \u003ctfoot\u003e\n \u003ctr\u003e\n \u003ctd colspan=\"5\"\u003eNote: Mg: Magnesium; Ca: Calcium; Fe: Ferum; B: Boron; Zn: Zinc.\u003c/td\u003e\n \u003c/tr\u003e\n \u003c/tfoot\u003e\n \u003c/table\u003e\n \u003c/div\u003e\n \u003cp\u003e\u003cbr\u003e\u003c/p\u003e\n\u003c/div\u003e\n\u003cdiv id=\"Sec15\"\u003e\n \u003ch2\u003e3.6 Nitrogen use efficiency (NUE) in garlic under different fertilization treatments\u003c/h2\u003e\n \u003cp\u003eIn view of the increase in biomass and bulb fresh weight of garlic grown under T1 and T2 treatment, we calculated the nitrogen use efficiency (NUE), nitrogen absorption efficiency (NAE), physiological N use efficiency (PNUE) and nitrogen harvest index (NHI) of garlic at the harvesting stage (Table \u003cspan\u003e4\u003c/span\u003e). In 2021, the NUE of garlic under T1 and T2 treatments increased by about 28% and 14%, respectively. Likewise, the PNUE of garlic grown under T1 and T2 treatment increased by 46% and 112%, respectively. For NHI, the garlic under T2 treatment increased by about 60%. In contrast, the NAE of the garlic under T1 and T2 treatment decreased by approximately 12% and 46%. However, the NAE and NHI of T2 treatment decreased by about 46% and 15%, respectively compared to those of control. Similarly, in 2022, the NUE of the garlic under T2 treatment increased by about 5% relative to that of control treatment. Meanwhile, the NAE of the garlic under T1 and T2 treatment increased by 46% and 92%, respectively. In contrast, the PNUE of garlic under T1 and T2 treatment decreased by about 21% and 22%, respectively. There was no significant difference in NHI among different treatment.\u003c/p\u003e\n \u003ctable border=\"1\" id=\"Tab4\"\u003e\n \u003ccaption language=\"En\"\u003e\n \u003cdiv\u003eTable 4\u003c/div\u003e\n \u003cdiv\u003e\n \u003cp\u003eComparison of nitrogen use efficiency, nitrogen absorption efficiency, physiological N-use efficiency and nitrogen harvest index between garlic grown under different fertilization treatments. Values are means\u0026thinsp;\u0026plusmn;\u0026thinsp;SE (n\u0026thinsp;\u0026ge;\u0026thinsp;3). Significant difference (P\u0026thinsp;\u0026lt;\u0026thinsp;0.05) is indicated by different lower-case letters as determined by Duncan\u0026rsquo;s multiple range test.\u003c/p\u003e\n \u003c/div\u003e\n \u003c/caption\u003e\n \u003cthead\u003e\n \u003ctr\u003e\n \u003cth align=\"left\"\u003e\u0026nbsp;\u003c/th\u003e\n \u003cth align=\"left\"\u003e\n \u003cp\u003eParameter\u003c/p\u003e\n \u003c/th\u003e\n \u003cth align=\"left\"\u003e\n \u003cp\u003eControl\u003c/p\u003e\n \u003c/th\u003e\n \u003cth align=\"left\"\u003e\n \u003cp\u003eT1\u003c/p\u003e\n \u003c/th\u003e\n \u003cth align=\"left\"\u003e\n \u003cp\u003eT2\u003c/p\u003e\n \u003c/th\u003e\n \u003c/tr\u003e\n \u003c/thead\u003e\n \u003ctbody\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\" rowspan=\"4\"\u003e\n \u003cp\u003e\u003cstrong\u003e2021\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e\u003cstrong\u003eNUE (kg.kg\u003c/strong\u003e\u003csup\u003e\u003cstrong\u003e\u0026minus;\u0026thinsp;1\u003c/strong\u003e\u003c/sup\u003e\u003cstrong\u003e)\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e145.0\u0026thinsp;\u0026plusmn;\u0026thinsp;2.89c\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e185.8\u0026thinsp;\u0026plusmn;\u0026thinsp;0.83b\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e165.8\u0026thinsp;\u0026plusmn;\u0026thinsp;2.20a\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e\u003cstrong\u003eNAE (kg.kg\u003c/strong\u003e\u003csup\u003e\u003cstrong\u003e\u0026minus;\u0026thinsp;1\u003c/strong\u003e\u003c/sup\u003e\u003cstrong\u003e)\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e1.17\u0026thinsp;\u0026plusmn;\u0026thinsp;0.05a\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e1.03\u0026thinsp;\u0026plusmn;\u0026thinsp;0.02b\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e0.63\u0026thinsp;\u0026plusmn;\u0026thinsp;0.01b\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e\u003cstrong\u003ePNUE (kg.kg\u003c/strong\u003e\u003csup\u003e\u003cstrong\u003e\u0026minus;\u0026thinsp;1\u003c/strong\u003e\u003c/sup\u003e\u003cstrong\u003e)\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e124.39\u0026thinsp;\u0026plusmn;\u0026thinsp;5.10c\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e181.24\u0026thinsp;\u0026plusmn;\u0026thinsp;3.97b\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e263.75\u0026thinsp;\u0026plusmn;\u0026thinsp;1.70a\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e\u003cstrong\u003eNHI (%)\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e48.0\u0026thinsp;\u0026plusmn;\u0026thinsp;0.10a\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e47.0\u0026thinsp;\u0026plusmn;\u0026thinsp;0.10b\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e76.8\u0026thinsp;\u0026plusmn;\u0026thinsp;0.4c\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\" rowspan=\"4\"\u003e\n \u003cp\u003e\u003cstrong\u003e2022\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e\u003cstrong\u003eNUE (kg.kg\u003c/strong\u003e\u003csup\u003e\u003cstrong\u003e\u0026minus;\u0026thinsp;1\u003c/strong\u003e\u003c/sup\u003e\u003cstrong\u003e)\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e201.8\u0026thinsp;\u0026plusmn;\u0026thinsp;19.56a\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e203.5\u0026thinsp;\u0026plusmn;\u0026thinsp;21.17a\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e210.94\u0026thinsp;\u0026plusmn;\u0026thinsp;12.81a\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e\u003cstrong\u003eNAE (kg.kg\u003c/strong\u003e\u003csup\u003e\u003cstrong\u003e\u0026minus;\u0026thinsp;1\u003c/strong\u003e\u003c/sup\u003e\u003cstrong\u003e)\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e1.06\u0026thinsp;\u0026plusmn;\u0026thinsp;0.53a\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e1.55\u0026thinsp;\u0026plusmn;\u0026thinsp;0.21a\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e2.04\u0026thinsp;\u0026plusmn;\u0026thinsp;0.15b\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e\u003cstrong\u003ePNUE (kg.kg\u003c/strong\u003e\u003csup\u003e\u003cstrong\u003e\u0026minus;\u0026thinsp;1\u003c/strong\u003e\u003c/sup\u003e\u003cstrong\u003e)\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e134.39\u0026thinsp;\u0026plusmn;\u0026thinsp;4.65a\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e132.40\u0026thinsp;\u0026plusmn;\u0026thinsp;5.34a\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e105.23\u0026thinsp;\u0026plusmn;\u0026thinsp;13.87a\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e\u003cstrong\u003eNHI (%)\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e46.0\u0026thinsp;\u0026plusmn;\u0026thinsp;0.40b\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e46.0\u0026thinsp;\u0026plusmn;\u0026thinsp;0.10b\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e47.0\u0026thinsp;\u0026plusmn;\u0026thinsp;0.20a\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003c/tbody\u003e\n \u003ctfoot\u003e\n \u003ctr\u003e\n \u003ctd colspan=\"5\"\u003eNote: NUE: Nitrogen Use Efficiency; NAE: Nitrogen Absorption Efficiency; PNUE: Physiological N-use efficiency; NHI: Nitrogen Harvest Index.\u003c/td\u003e\n \u003c/tr\u003e\n \u003c/tfoot\u003e\n \u003c/table\u003e\n\u003c/div\u003e\n\u003cdiv id=\"Sec16\"\u003e\n \u003ch2\u003e3.7 Correlation between nitrate concentration at spring garlic stage and garlic biomass\u003c/h2\u003e\n \u003cp\u003eIn this study, Pearson correlation analysis was conducted to investigate the relationship between nitrate concentration at spring garlic stage and the biomass of garlic at harvesting stage (Fig. 6). We observed that nitrate concentration in the shoot of garlic at spring garlic stage was positively correlated with to the biomass of garlic at harvest stage (R2\u0026thinsp;=\u0026thinsp;0.797; p\u0026thinsp;\u0026le;\u0026thinsp;0.01). Meanwhile, there was no correlation observed between nitrate concentration in the root of garlic and biomass of garlic at harvesting stage. The finding indicated that the shoot nitrate concentration at spring garlic stage was somewhat related to the final biomass of garlic.\u003c/p\u003e\n\u003c/div\u003e"},{"header":"4. Discussion","content":"\u003cdiv id=\"Sec18\" class=\"Section2\"\u003e \u003ch2\u003e4.1 Nanofertilizer improve the biomass accumulation and quality parameter of garlic\u003c/h2\u003e \u003cp\u003eIn this two-years study, we showed that the application of microbial biofertilizer and germanium containing controlled release nanofertilizer significantly increased garlic plant biomass accumulation and bulb yield in a major garlic production area (Pizhou city) in China (Fig.\u0026nbsp;1; Table\u0026nbsp;\u003cspan refid=\"Tab2\" class=\"InternalRef\"\u003e2\u003c/span\u003e). In addition, the NUE and NAE of the garlic under the treatment of both fertilizers was also higher than those of control treatment. The yield increasing effect of beneficial microorganisms on garlic has also been reported in several previous study. For example, the introduction of genetically engineered strain of the nitrogen-fixing bacterium \u003cem\u003ePseudomonas protegens\u003c/em\u003e CHA0 has been found to increase the bulb yield of garlic by 12.03% (Wang et al. \u003cspan citationid=\"CR36\" class=\"CitationRef\"\u003e2020\u003c/span\u003e). Besides that, the study also revealed that the introduction of \u003cem\u003ePseudomonas protegens\u003c/em\u003e can lower disease index and increased the allicin content in garlic (Wang et al. \u003cspan citationid=\"CR36\" class=\"CitationRef\"\u003e2020\u003c/span\u003e). This was consistent with our result that the allicin content in garlic was significantly increased under treatment of microbial biofertilizer. For nanofertilizer, a similar observation was also made by many previous studies that the application of CuO nanoparticle and Ag nanoparticle can significantly improve the agronomic traits of onion such as biomass accumulation, nutrient elements and yield (Fouda et al. \u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e2020\u003c/span\u003e; Wang et al. \u003cspan citationid=\"CR36\" class=\"CitationRef\"\u003e2020\u003c/span\u003e).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec19\" class=\"Section2\"\u003e \u003ch2\u003e4.2 Pattern of nitrate accumulation throughout growth stage\u003c/h2\u003e \u003cp\u003eIn aerobic agricultural soil, nitrate is arguably the main source of mineral nitrogen for plants (Dechorgnat et al. \u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e2011\u003c/span\u003e). Since garlic performed well when planted in well-drained soil, hence it is reasonable to assume that nitrate is essential for its growth and development. In plant, nitrate is actively taken up by root cells and mobilized to other plant organs through the help of nitrate transporters (Fan et al. \u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e2017\u003c/span\u003e). Within plant cells, some of the absorbed nitrate is metabolized directly into nitrogen-containing compounds and the excessive nitrate is translocated into vacuole for storage (Wang et al. \u003cspan citationid=\"CR37\" class=\"CitationRef\"\u003e2014\u003c/span\u003e). It was shown that cold temperature can have multiple negative effects on plant morphology and physiology, especially on root metabolic activity, leaf water potential and nitrate uptake (Laine et al. \u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e1994\u003c/span\u003e; Burchett et al. \u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e2006\u003c/span\u003e; Sun et al. \u003cspan citationid=\"CR34\" class=\"CitationRef\"\u003e2017\u003c/span\u003e). In this study, we observed that the nitrate accumulation in the whole plant of garlic increased drastically during spring garlic stage. This nitrate accumulation in plants may be associated to the low temperature and then low photosynthesis and C accumulation during winter season. However, high nitrate from N accumulation at the later stage would result into a faster conversion rate of nitrate into N-containing compounds (such as protein) and therefore a faster biomass accumulation with high photosynthesis together at the late growth stage. However, at the later growth stage (i.e., bulb development stage and harvest stage), the conversion of nitrate into N-containing compounds become faster than the nitrate absorbed from the root, leading to lower nitrate accumulation.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec20\" class=\"Section2\"\u003e \u003ch2\u003e4.3 Nitrate concentration at spring garlic stage was positively correlated with garlic biomass at harvest\u003c/h2\u003e \u003cp\u003eIn this study, we observed a strong positive correlation between the shoot nitrate concentration at spring garlic stage and garlic biomass. In contrast, there was no significant correlation between the root nitrate concentration and garlic biomass. This observation suggested that the shoot nitrate concentration in garlic at spring garlic stage exert significant impact on biomass accumulation of garlic at harvest. The observed phenomenon may be either due to the role of nitrate as nutrient or as the major anionic osmotica that increase plant tolerance to chilling stress (Heerden et al. \u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e2004\u003c/span\u003e). Chilling stress is one of the environmental factors that restrict the growth and productivity of plant. Generally, chilling stress causes a decrease in the hydraulic conductivity of the roots and subsequently a significant decrease in leaf water potential. The reduced leaf water potential leads to the loss of leaf turgor pressure, resulting in severe plant wilting (Hussain et al. \u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e2018\u003c/span\u003e). Hence, osmotic adjustment is crucial to assists the plant to maintain higher turgor potential. It has been well established that nitrate is the major osmolyte that help maintaining the cell turgor by reducing the osmotic potential of vacuolar sap in many crops (Mott and Steward \u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e1972\u003c/span\u003e). Besides that, previous study reported a linear correlation between plant nitrate and water content (C\u0026aacute;rdenas-Navarro et al. \u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e1999\u003c/span\u003e). For example, nitrate has been shown to involve in stomatal functioning by controlling the turgor pressure of guard cell (Guo et al. \u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e2003\u003c/span\u003e). In addition, study showed that intracellular nitrate can also control the root hydraulic properties in plants, resulting into higher water uptake (Gorska et al. \u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e2008\u003c/span\u003e). A previous study also showed that nitrate supplementation can significantly reduce the detrimental effect of chilling stress on crops (Heerden et al. \u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e2004\u003c/span\u003e). This observation suggested that the higher shoot nitrate concentration at spring garlic stage can increase the chilling tolerance of garlic, which may be achieved by maintaining leaf water potential. In addition to nitrate, studies showed that potassium (K\u003csup\u003e+\u003c/sup\u003e) is also important for cold tolerance in plant by regulating the osmotic and water potential, increasing antioxidant levels to reduce ROS production and reducing electrolyte leakage caused by chilling stress (Oosterhuis et al. \u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e2013\u003c/span\u003e; Hasanuzzaman et al. \u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e2018\u003c/span\u003e). Hence, the relatively higher root concentration of K\u003csup\u003e+\u003c/sup\u003e maybe a protective strategy for garlic to survive under chilling stress (Fig.\u0026nbsp;5c). As the major cation in plant cell, K\u003csup\u003e+\u003c/sup\u003e has been shown to be closely linked with NO\u003csub\u003e3\u003c/sub\u003e\u003csup\u003e\u0026minus;\u003c/sup\u003e (counterion of K\u003csup\u003e+\u003c/sup\u003e) in translocation, transportation and distribution due to the effect of anion-cation balance in plant cells (Raddatz et al. \u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e2020\u003c/span\u003e). Hence, it is possible that nitrate and potassium act synergistically in protecting the garlic from chilling stress. It has been reported that exogenous application of potassium nitrate can promote the cold tolerance in Panax ginseng by enhancing the antioxidant level (Devi et al. \u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e2012\u003c/span\u003e). However, a more detailed study at the molecular level should be conducted in order to further understand the underlying mechanism.\u003c/p\u003e \u003c/div\u003e"},{"header":"5. Conclusions","content":"\u003cp\u003eOverall, the microbial biofertilizer and germanium-containing controlled release fertilizer used in this study could significantly increase the growth, nitrogen use efficiency and allicin content. This study also observed a strong positive correlation between the shoot nitrate concentration at spring garlic stage and garlic biomass. It indicated that higher shoot nitrate at spring garlic stage may promote the growth of garlic at later stage, which may be due to the synergistic effect of nitrate and potassium in enhancing the cold tolerance of garlic. Our study provides a scientific theoretical basis for high yield garlic using the microbial biofertilizer.\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eEthics approval and consent to participate\u003c/strong\u003e\u003cstrong\u003e：\u003c/strong\u003eHere is no ethics conflicts in these data\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eConsent to publish:\u0026nbsp;\u003c/strong\u003ewe are consent to publish these data\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAvailability of data and materials:\u0026nbsp;\u003c/strong\u003eThe data and materials in the manuscript are available.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eCompeting interests:\u0026nbsp;\u003c/strong\u003eThe authors declare no competing interests.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eFunding:\u003c/strong\u003e This work was supported by the sub-project 4 of Jiangsu middle and late mature garlic industrial cluster construction: Garlic mechanical intelligent operation technology and demonstration and promotion of green production technology.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAuthors\u0026apos; Contributions:\u0026nbsp;\u003c/strong\u003eZY produced the fertilizer; ZY, FX, ZX designed the experiment; SJ, LZ, FX conducted and wrote the manuscript; ZH and ZL supplied the experiment land.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAcknowledgements:\u003c/strong\u003e We would like to thank the assistance of Agricultural and rural Office of Nianzhuang Town, Pizhou city, Jiangsu, China.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\n\u003cli\u003eAtif MJ, Amin B, Ghani MI, Hayat S, Ali M, Zhang Y, Cheng Z (2019) Influence of different photoperiod and temperature regimes on growth and bulb quality of garlic (\u003cem\u003eAllium sativum\u003c/em\u003e L.) cultivars. \u003cem\u003eAgron \u003c/em\u003e9(12): 879. https://doi.org/ 10.3390/agronomy9120879\u003c/li\u003e\n\u003cli\u003eBatiha GE, Beshbishy AM, Wasef LG, Elewa YHA, Al-Sagan AA, El-Hack MEA, Taha AE, Abd-Elhakim YM, Devkota HP (2020) Chemical constituents and pharmacological activities of garlic (\u003cem\u003eAllium sativum\u003c/em\u003e L.): A Review. \u003cem\u003eNutrients \u003c/em\u003e12(3): 872. https://doi.org/10.3390/nu12030872\u003c/li\u003e\n\u003cli\u003eBurchett S, Niven S, Fuller M (2006) The effect of cold-acclimation on the water relations and freezing tolerance \u003cem\u003eHordeum vulgare\u003c/em\u003e L. \u003cem\u003eCryo Letters\u003c/em\u003e 27(5):295\u0026ndash;303\u003c/li\u003e\n\u003cli\u003eC\u0026aacute;rdenas-Navarro R, Adamowicz S, Robin P (1999) Nitrate accumulation in plants: a role for water. \u003cem\u003eJ Exp Bot \u003c/em\u003e73(13): 613-624. https://doi.org/10.1093/jxb/50.334.613\u003c/li\u003e\n\u003cli\u003eChanchan M, Thapa P, Hore J (2018) Effect of biofertilizers with graded levels of nitrogen and phosphorus on growth and yield of garlic (\u003cem\u003eAllium sativum\u003c/em\u003e L.). \u003cem\u003eRes Crop\u003c/em\u003e 19(1):127. https://doi.org/10.5958/2348-7542.2018.00021.9\u003c/li\u003e\n\u003cli\u003eChen J, Zhang Y, Tan Y, Zhang M, Zhu L, Xu G, Fan X (2016) Agronomic nitrogen‐use efficiency of rice can be increased by driving \u003cem\u003eOsNRT2.1\u003c/em\u003e expression with the \u003cem\u003eOsNAR2.1\u003c/em\u003e promoter. \u003cem\u003ePlant Biotechnol J\u003c/em\u003e 14(8):1705-1715. https://doi.org/10.1111/pbi.12531\u003c/li\u003e\n\u003cli\u003eDechorgnat J, Nguyen CT, Armengaud P, Jossier M, Diatloff E, Filleur S, Daniel-Vedele F (2011) From the soil to the seeds: the long journey of nitrate in plants. \u003cem\u003eJ Exp Bot\u003c/em\u003e 62(4):1349\u0026ndash;1359. https://doi.org/10.1093/jxb/erq409\u003c/li\u003e\n\u003cli\u003eDevi BSR, Kim YJ, Selvi SK, Gayathri S, Altanzul K, Parvin S, Yang DU, Lee OR, Lee S, Yang DC (2012) Influence of potassium nitrate on antioxidant level and secondary metabolite genes under cold stress in \u003cem\u003ePanax ginseng\u003c/em\u003e. \u003cem\u003eRuss\u003c/em\u003e \u003cem\u003eJ Plant Physiol\u003c/em\u003e 59, 318\u0026ndash;325. https://doi.org/10.1134/S1021443712030041\u003c/li\u003e\n\u003cli\u003eDhir B (2021) Nanofertilizers and their applications. 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Plant Sci 289:110270. https://doi.org/10.1016/j.plantsci.2019.110270\u003c/li\u003e\n\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":true,"highlight":"","institution":"","isAcceptedByJournal":false,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true},"keywords":"Allium sativum, Nitrogen, Nitrate, Microbial biofertilizer, Nanofertilizer ","lastPublishedDoi":"10.21203/rs.3.rs-2063527/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-2063527/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003e\u003cstrong\u003ePurpose: \u003c/strong\u003eThe purpose of this study was to assess the effect of microbial biofertilizer and nanofertilizer on the growth, allicin content and elemental content of garlic in China.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eMethod: \u003c/strong\u003eWe conducted field trial of microbial biofertilizer and germanium-containing controlled release nanofertilizer along two consecutive winter periods of 2020/2021 and 2021/2022 in a major garlic production area at Pizhou city to determine the effect of treatment in comparison to the local fertilization practice.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eResult: \u003c/strong\u003eThe results revealed that the application of microbial biofertilizer and germanium-containing controlled release nanofertilizer increased the bulb yield by approximately 6-28% and 14%, respectively, compared with local fertilization practice. Meanwhile, the application of microbial biofertilizer consistently increased the allicin content of garlic in year 2021 and 2022. Furthermore, we also observed a strong positive correlation between shoot nitrate concentration at spring garlic stage and the final garlic bulb yield, implying that the accumulation of nitrate content in garlic at spring garlic stage maybe beneficial to biomass accumulation through either nutritional effect and increased chilling tolerance.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eConclusion: \u003c/strong\u003eTaken together, our results revealed that the application of microbial biofertilizer and germanium-containing controlled release nanofertilizer can increase growth and nitrogen use efficiency of garlic in our experiment.\u003c/p\u003e","manuscriptTitle":"Study on the influence of different fertilization regimes on the yield and bulb quality of garlic (Allium Sativum L.)","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2023-03-16 18:51:42","doi":"10.21203/rs.3.rs-2063527/v1","editorialEvents":[{"type":"communityComments","content":0}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"7fc88e16-2ee2-48b3-a689-9987d85dc122","owner":[],"postedDate":"March 16th, 2023","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"posted","subjectAreas":[],"tags":[],"updatedAt":"2023-09-25T17:10:04+00:00","versionOfRecord":[],"versionCreatedAt":"2023-03-16 18:51:42","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-2063527","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-2063527","identity":"rs-2063527","version":["v1"]},"buildId":"WrCJVZZCHTDjtuVLN7oU0","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}