Agronomic Biofortiﬁcation of Garlic through Selenium and Arbuscular Mycorrhizal Fungi Application

: Garlic has a strong ability of selenium (Se) accumulation and is one of the best target crops for Se biofortiﬁcation. Arbuscular mycorrhizal fungi (AMF) inoculation might enhance the nutritional qualities and the absorption ability of exogenous Se in plants. However, little is known about the exogenous Se application and AMF inoculation on garlic. Here, we evaluated the effects of different concentrations of exogenous Se on the growth, nutritional quality, and selenium enrichment of garlic. The results demonstrated that signiﬁcantly higher Se content of garlic bulb was found in exogenous Se treated plants, and the Se accumulation was improved with the increasing of Se supply. Low application of exogenous Se appreciably improved the yield and the contents of soluble sugar and allicin in garlic bulbs, but the opposite was observed at high Se concentration. Furthermore, AMF inoculation signiﬁcantly reduced the inhibition effect of high concentration Se on garlic. AMF supply was effective in improving the growth and nutritional indicators of garlic, which promoted the exogenous Se utilization rate when combined with 10 mg/L exogenous Se treatment. The results will provide a more theoretical basis for the production of high-quality selenium enrichment garlic.


Introduction
Selenium (Se) is a rare element, one of the trace elements necessary for human and animal health, such as improving immune response-ability, preventing cancer and some other diseases, and an essential part of the antioxidant defense system. It has the functions of changing plant tissue active oxygen defense enzyme activity, regulating plant growth and metabolism, antagonizing heavy metals and regulating reproductive growth [1,2]. In the natural environment, Se exists in many forms (Se 2-, Se, Se 4+ , Se 6+ ) and cannot be directly absorbed and utilized by our bodies. In light cases, Se deficiency leads to malnutrition and severe cases could have blood heart disease, tumor, cancer and other diseases. Meanwhile, animals and plants can be used as the common source of Se supplements for people. The food chain of "soil-plant/animal-human" becomes the safest and most effective way to supply Se for us [3]. Therefore, it is imperative to develop and cultivate Se-rich foods to satisfy the dietary needs of our humans.
Among different forms of Se, the absorption rate of Se by plants depends on Se's chemical form. The main absorption forms are selenite (SeO 3 2− , HSeO 3 − or H 2 SeO 3 ), selenate (SeO 4 2− ). Compared with selenate, selenite can be more easily reduced to organic selenium, which can be absorbed by animals for utilization. Studies have shown that 98% of the plant bodies still existed in the form of selenate when the plants were treated with selenate for 1 h. While 92% Se was found in neutral substances and animals are more likely to absorb Se element from plant products when plants were cultured with selenite such as of this study will provide a theoretical basis and technical guidance for improving the Se utilization rate and promoting the production of selenium-rich garlic with high quality.

Materials and Methods
To reveal the influences of exogenous Se on garlic Se enrichment and quality, and to evaluate the effect of combined inoculation of AMF and Se on reducing garlic Se sensitivity and improving garlic quality, two experiments were performed, in which one is a field experiment (Trial 1) and another is a pot experiment (Trial 2). The two trials were respectively conducted at Wuquan Experiment Station and the Horticultural Experiment Station of Northwest A&F University in Yangling, Shaanxi Province, China.

Plant Materials and Growing Conditions
Four widely cultivated garlic cultivars (cv.), G024, G025, G103, and 2011-5 in China were used in this study. All of them are medium-late mature. cv. G024 is mainly used for bulb production with more substantial sized and red skin bulbs. The remaining three cultivars have white skin. Specifically, cv. G025 is typically used for scape and bulb production with an outstanding quality of scape and white skin bulbs with fewer and uniform cloves especially suitable for processing. All cultivars were used in Trial 1 and only cv. G024 that showed more sensitivity to Se concentration was used in Trial 2.
The region of Yangling has a temperate monsoon climate with an annual average temperature of 12-14 • C and a frost-free period of more than 200 days. Based on our measurement, the soil in Trail 1 has never been fertilized with selenium before and has the following characteristics: 7.67 of pH (1:1 soil:water), organic matter 1.7%, total nitrogen (N) 58.47 mg/kg, total phosphorus (P) 12.64 mg/kg, total potassium (K) 376 mg/kg, and total Se 0.14 mg/kg. The characteristics of the soil for trial 2 were observed: 7.67 of pH (1:1 soil:water), organic matter 1.3%, total N 24.28 mg/kg, total P 10.19 mg/kg, total K 221 mg/kg, and total Se 0.12 mg/kg, which was sifted with 2 mm mesh sieve and sterilized under 121 • C, 0.1 Mpa for 2 h.

AMF Propagation with Maize-Trap Culture
The full maize (Zea mays L.) seeds were soaked with 0.025 mol/L potassium permanganate for 6 min, washed with sterile water 3 times, then placed in a tray covered with gauze, and germinated in an incubator at 28 • C. Then the seedlings with the same growth vigor were selected and transplanted into the pots with a growth medium. The Glomus versiforme provided by the Vegetable physiology and biotechnology laboratory of Northwest A&F University was inoculated near the seedling roots. The plants were watered every day for the first 3 weeks after transplant, and then rehydrated every 3 days. Ten times diluted standard Hoagland nutrient solution was irrigated once a week. The plants were cultured under axenic conditions in a controlled greenhouse (culture temperature was 25/16 • C, the relative humidity was 70~75%, illumination time was 16 h/d) for 4 months. At last, the inoculum of the potting mixture containing spores, mycelium, and segmented mycorrhizal roots was obtained. Air dry, sift (2 mm), and store at 4 • C for use. The autoclaved substrate represented the non-inoculated and was prepared to use for non-AMF garlic plants. Trial 1 was a double-factor (variety × selenium concentrations) experiment performed in the way of a randomized complete block design with three replications. We spent two years performing the Se treatments on the garlic for the analysis of the effects on their growth. The uniform and healthy garlic cloves were selected and sowed in an open field with row space of 25 cm and plant space of 8 cm in September. Each replication per treatment included 150 garlic cloves (in three rows). Regular water and fertilizer management were performed during the growing period. The four cultivars (cv. G024, cv. G025, cv. G103, and cv. 2011-5) of garlic plants were treated at the differentiation stage of scale bud in March 2017. Different concentrations (0, 0.1, 1, 10, 100, and 1000 mg/L) of sodium selenite (Na 2 SeO 3 ) solution were sprayed with different processing times (once spraying on 23 April; twice spraying on 9 April and 23 April; three times spraying on 25 March, 9 April and 23 April) in 2017. Based on the study results in 2017, further treatments were executed. Two varieties (cv. G024, cv. G025) of garlic plants were applied with different concentrations (0, 5,10,15,20,40,60, and 80 mg/L) of Na 2 SeO 3 on 25 March and 1 April 2018. The chemical Na 2 SeO 3 (pure in 98%) used was Analytical grade and ordered from Shandong Xiya Chemical Co., LTD., Shandong, China. Twain 80 (30 drops/L) was added to the solution to improve its surfactivity. The garlic leaves were thoroughly wetted (about 6 mL per plant) at each time of treatment. The ground surface was covered with plastic film to avoid the Se solution dropping into the soil. The neighbor plants were separated with foam boards to avoid contamination.

Trial 2
Trial 2 was a pot experiment also with two variables, different concentrations of Se (0, 10, and 50 mg/L) and AMF treatment (inoculation and non-inoculation). In total, six treatments were named Se0-AMF, Se0+AMF, Se10-AMF, Se10+AMF, Se50-AMF, and Se50+AMF, respectively. For each treatment, six pots (26 cm × 26 cm × 20 cm) were used, and each pot was filled with 5 kg soil and 6 garlic cloves were planted. The plump and uniform cloves were selected and sterilized with 10% H 2 O 2 for 10 min before sowing. For AMF treatment, 20 g propagated mycorrhizal inoculants (about 2200 infective propagules per gram soil) were used in each pot. The prepared inoculum was applied near the garlic cv. G024 cloves (5 cm below) during the growing season. Non-AMF treatment plants received the same weight of non-mycorrhizae substrate (autoclaved inoculum). Sodium selenite solution of different concentrations was applied to the soil twice, 50 mL each time, after garlic sprouting.

Sampling and Growth Index Measurement
The garlic scape was cut and harvested from the bottom of the shoots (about 5 cm above the ground surface), the yield was weighed with an electronic balance and the diameter and length were measured with 60 randomly selected scape each experimental treatment (each replication harvest 20 scape, each treatment takes three repetitions). The weight, diameter, and height were measured with 45 randomly selected mature garlic bulbs each treatment after the harvest (each replication harvest 15 scape, each treatment takes three repetitions). Then those bulbs were divided into two groups, one stored at −80 • C in a refrigerator for determination of soluble protein and allicin, and another group dried to constant weight at 60 • C in an oven for measurement of Se and soluble sugar content. Each experiment had 3 technical replications.

Selenium Content Analysis
The 0.5 g oven-dried garlic cloves were digested at 160 • C with a 10 mL acid mixture (HNO 3 : HClO 4 , 4:1, v:v) until the digestion solution became clear. After cooling, 4 mL concentrated hydrochloric acid (6 mol/L) was added and bathed in boiling water for 30 min to evaporate the dissolved solution. HCl (10%) was used as the carrier solution, and the solution of 2% KBH 4 +0.5% NaOH was used as a reducing agent to measure the Se content by hydride atomic fluorescence spectrometry (HG-AFS) [30]. The standard substance used for Se content measuring was ordered from Sigma-Aldrich, USA. The Se determination system consists of an FUV-320 online digestion device, AFS-9780 dual-channel atomic fluorescence spectrometer, and Se unique hollow cathode lamp (Research Institute for Nonferrous Metals, Beijing, China).

Nutrient Acquisition Measurement
The soluble sugar content was measured using the anthrone colorimetric method [31,32]. The soluble protein content was determined using the coomassie brilliant blue method following Bradford [33]. Allicin was determined by High-performance liquid chromatography (HPLC) (Agilent Technologies, Santa Clara, CA, USA, Model 1260) following Baghalian [34].

Statistical Analysis
The collected data were processed by Microsoft Excel 2010. The values were calculated and compared by Tukey multiple range tests (p < 0.05). All datasets were tested for normal distribution and variance homogeneity. Normal distribution was analyzed by Shapiro-Wilk normality tests, and variance homogeneity was analyzed by Levene's test. Data were expressed as the mean value ± standard error (SE) for each treatment. The software of R (https://www.r-project.org/ (accessed on 22 June 2020) with Rstudio (https://rstudio.com/ (accessed on 31 October 2018) and GraphPad Prism 7 was used for statistical analysis and diagram drawing.

Wide-Ranging Gradients of Se Treatments on Garlic
To screen appropriate Se concentrations, spraying times, and garlic varieties, different treatments were combined to analyze their effect on garlic growth and quality (Figures S1-S5 and Tables S1-S4). The results showed that Se spraying significantly affected the growth of garlic. The fresh weight ( Figure S1) and diameter/height (Tables S1-S4) of garlic bulbs were improved under suitable Se treatments (<100 mg/L), however high usage Se concentration (1000 mg/L) caused the inhibition of garlic growth. Similarly, the content of soluble sugar ( Figure S2), protein ( Figure S3), allicin ( Figure S4) in garlic were also consistent with the growth situation. The Se content in garlic was improved with the increase in the exogenous Se concentration ( Figure S5). However, yellow leaf tips and flakelike white spots on the leaf surface ( Figure S5) were caused by excessive Se supply (1000 mg/L). The plants treated with 100 mg/L Se also showed slight yellowing of leaves. Although there was no significant difference in the spraying times (Figures S1-S4), the content of garlic soluble sugar ( Figure S2), protein ( Figure S3), and allicin ( Figure S4) by spraying twice was higher than that by spraying three times and once. The extremum of each nutrient index repeatedly appeared in 10 mg/L twice spraying of exogenous Se treatment group ( Figures S2-S4). The Se utilization rate in cv. G103 under 0.1 mg/L Se supply(12%) was the highest among all treatments, while the garlic with 1000 mg/L concentration of exogenous Se had had the lowest utilization rates (Table S6).
The results showed that the growth and nutrition indexes of garlic increased when the concentration was lower than 100 mg/L, and decreased when the concentration was higher than 100 mg/L. Spraying times had a significant effect on selenium content, but had no significant effect on bulb nutritional quality. Spraying once had the highest Se utilization efficiency and spraying twice gave the best nutritional quality of garlic. The garlic with spraying three times had the highest Se content and the lowest Se utilization efficiency.

Agronomic Parameters
The effects of Se concentrations, spraying times, and garlic varieties on the garlic growth and quality of garlic were investigated in the first year. The Se concentrations were the most significant factor affecting the selenium content and enrichment ability of bulbs, while garlic variety was the most striking factor affecting the growth quality of bulbs. Among the six Se concentrations in the preliminary screening, the application effect showed a parabolic trend, which firstly increased and then decreased with the increase in selenium concentration. However, due to the wide span of selenium application concentration, the optimal application concentration could not be accurately determined. It is necessary to carry out selenium concentration re-screening after determined the preliminary ruling on the provision of external Se for garlic growth. The preliminary test results showed that cv. G024 and cv. G025 were representative with excellent character and had significant differences in their own traits and Se sensitivity (the quality of cv. G024 treated with Se showed a more significant phenomenon of "low promotion and high inhibition" than the other three cultivars, and the Se content and nutrient composition of cv. G025 were double higher than those of other cultivars).
More accurate Se gradients (0, 5, 10, 15, 20, 40, 60, and 80 mg/L) were generated to explore the effect of garlic (cv. G024 and cv. G025) growth under Se supply. The supplement of exogenous Se in garlic bulb (Table 1) showed a noticeable consequence for garlic growth with a tendency of lower concentrations (≤10 mg/L) promoting and higher concentrations (≥40 mg/L) inhibiting, respectively. Compared with other treatments, the 10 mg/L Se always gave rise to the most massive increase in garlic bulbs growth of both varieties. The bulb diameter and bulb height were significantly increased under 10 mg/L Se supply, and the bulb weights of cultivars (cv.) G024 and G025 were 14.5% and 17.9% higher than their control (0 mg/L), respectively. The inhibitory effect of exogenous Se on growth appeared when the Se applied concentration reached 40 mg/L. As the concentration increased to 60 mg/L, the inhibitory impact became more obvious, but the differences were not significant compared to their control. Under the 80 mg/L Se concentration, garlic bulb weight, diameter, and height of both cultivars were significantly lower (p < 0.05) than their control. Moreover, compared with cv. G025, the growth inhibition of cv. G024 was more prominent at a high concentration of Se, which might suggest that cv. G024 was more sensitive to exogenous Se. The results indicated that moderate concentrations of exogenous Se significantly improved the growth of garlic.

Se Enrichment
For both cultivars, the Se content in all treated plant bulbs was higher than that of control (Figure 1), and increased with the increasing concentration of exogenous Se. When the concentration of exogenous Se exceeded 20 mg/L, the Se content in cv. G024 bulbs increased exponentially, and the values of different treatments were statistically significant (p < 0.01). The Se content of cv. G024 under 80 mg/L Se treatment (2.282 µg/g) was approximately 55 times higher than that of the control (0.041 µg/g). Se enrichment in cv. G025 was lower than that in cv. G024 under higher concentrations. For instance, the Se content of cv. G025 increased by about 43 times than the control at 80 mg/L concentration. The results implied that the Se enrichment capacity of garlic bulbs was mainly affected not only by exogenous Se but also by the garlic cultivar. Nevertheless, the Se content effect of exogenous Se on the selenium enrichment was consistent in the two cultivars. Overall, these results indicated that exogenous Se supply could improve the Se content in garlic bulbs and spraying a high concentration of exogenous Se has a stronger effect on selenium enrichment in garlic bulbs than that of low concentration. bulbs and spraying a high concentration of exogenous Se has a stronger effect on seleni enrichment in garlic bulbs than that of low concentration.

Nutritional Qualities
To clarify the effect of exogenous Se on the nutritional qualities of garlic bulbs, contents of allicin (Figure 2A,B) and soluble protein ( Figure 2C,D) were measured, a the results showed that lower concentrations of exogenous Se treatments could impro the content of allicin in garlic bulbs of both cultivars. For cv. G024, the allicin content u der 5 mg/L and 10 mg/L Se supply increased by 44.4% and 36.3% than the control, resp tively ( Figure 2A). While the higher concentrations Se treatments (≥15 mg/L) had no s nificance in improving the allicin content of bulbs (Figure 2A). For cv. G025, the cont of allicin was also increased in garlic bulbs treated with low Se (≤20 mg/L), but there w no statistically significant difference compared to control ( Figure 2B). Interestingly, higher concentrations (≥40 mg/L) showed significant inhibition for allicin accumulation bulbs.
The application of exogenous Se improved soluble protein contents in bulbs of b garlic cultivars ( Figure 2C,D). The content of soluble protein in cv. G024 and G025 reach the peak at 10 and 20 mg/L treatments, respectively. Subsequently, the increment of so ble protein content in cv. G024 and G025 showed a downward trend with the increas of exogenous Se concentrations. Compared with the control, the soluble protein conte of cv. G024 and G025 were increased by 38.6% and 14.5% under 10 mg/L Se treatme respectively. Although the contents of the soluble protein in cv. G024 and G025 dropp after the high concentration treatments, the values were still higher than the controls.
In general, appropriate exogenous Se improved the contents of allicin and solu protein in garlic bulbs. The lower Se supply could significantly enhance the nutrient cumulation in garlic bulbs, but higher concentrations showed a diminished effect.

Nutritional Qualities
To clarify the effect of exogenous Se on the nutritional qualities of garlic bulbs, the contents of allicin (Figure 2A,B) and soluble protein ( Figure 2C,D) were measured, and the results showed that lower concentrations of exogenous Se treatments could improve the content of allicin in garlic bulbs of both cultivars. For cv. G024, the allicin content under 5 mg/L and 10 mg/L Se supply increased by 44.4% and 36.3% than the control, respectively (Figure 2A). While the higher concentrations Se treatments (≥15 mg/L) had no significance in improving the allicin content of bulbs (Figure 2A). For cv. G025, the content of allicin was also increased in garlic bulbs treated with low Se (≤20 mg/L), but there was no statistically significant difference compared to control ( Figure 2B). Interestingly, the higher concentrations (≥40 mg/L) showed significant inhibition for allicin accumulation in bulbs.
The application of exogenous Se improved soluble protein contents in bulbs of both garlic cultivars ( Figure 2C,D). The content of soluble protein in cv. G024 and G025 reached the peak at 10 and 20 mg/L treatments, respectively. Subsequently, the increment of soluble protein content in cv. G024 and G025 showed a downward trend with the increasing of exogenous Se concentrations. Compared with the control, the soluble protein contents of cv. G024 and G025 were increased by 38.6% and 14.5% under 10 mg/L Se treatment, respectively. Although the contents of the soluble protein in cv. G024 and G025 dropped after the high concentration treatments, the values were still higher than the controls.
In general, appropriate exogenous Se improved the contents of allicin and soluble protein in garlic bulbs. The lower Se supply could significantly enhance the nutrient accumulation in garlic bulbs, but higher concentrations showed a diminished effect.

Plant Growth and Agronomic Parameters
We investigated if exogenous Se improved the yield and nutrient contents of garlic bulbs. However, high concentration treatment weakened the promoting effect and even affected the normal growth. To explore the method-solving phenomenon, the important role of AMF in promoting plant growth was used for this problem unraveling. Thus, exogenous Se, AMF, and their interaction effects on garlic growth characteristics were analyzed in cv. G024 garlic (Figure 3 and Table 2).
For the treatments without AMF, the Se10-AMF treatment slightly promoted both plant growth and bulb traits compared to Se0-AMF and Se50-AMF. The plant height of treatment Se50-AMF was lower than those of control (Se0-AMF). This was consistent with the results of Trial 1, that lower concentrations of exogenous Se stimulated garlic growth, while higher concentrations inhibited growth. The application of AMF improved the height and fresh weight of garlic plants compared to the treatments without AMF inoculation (Figure 3). Based on the measured growth parameters of Se0-AMF and Se0+AMF, AMF inoculation had a positive effect on garlic growth, especially the whole plant fresh weight and garlic bulb diameter and height, for which the differences were statistically significant. Even among the treatments Se10-AMF, Se10+AMF, Se50-AMF, and Se50+AMF, AMF inoculation also showed promotion of garlic growth, and significant differences were found in the diameter and length of scape between Se10-AMF and Se10+AMF. In addition, the whole plant fresh weight, bulb diameter and height, and space diameter and length of Se50-AMF were considerably lower than those of Se50+AMF ( Figure 3B and Table 2). Compared with Se50-AMF, the bulb weight, bulb diameter and height, and scape diameter and length of treatment Se50+AMF were significantly increased by 10.7%, 10.6%, 12.6%, 24.9%, and 17.9%, respectively ( Table 2). This indicated that the symbiotic relationship between AMF and garlic significantly alleviates the growth inhibition of garlic resulted from the exogenous Se with higher concentrations.

Plant Growth and Agronomic Parameters
We investigated if exogenous Se improved the yield and nutrient contents of garli bulbs. However, high concentration treatment weakened the promoting effect and even affected the normal growth. To explore the method-solving phenomenon, the importan role of AMF in promoting plant growth was used for this problem unraveling. Thus, ex

Nutritional Qualities
Without AMF inoculation, both Se treatments (Se10-AMF and Se50-AMF) significantly increased the soluble protein levels in garlic bulbs compared to the control (Se0-AMF) ( Figure 4A), which was similar to the results in Trial 1 ( Figure 2C). Soluble protein content was also enhanced by AMF inoculation, especially for the treatment of Se0+AMF. Higher concentration Se treatment (Se50-AMF) appeared a little inhibition ( Figure 4B) on soluble sugar content in garlic bulbs without AMF inoculation. Inoculation of AMF (Se50+AMF) favored the accumulation of soluble sugar in garlic bulbs. For the treatments without AMF, the Se10-AMF treatment slightly promoted both plant growth and bulb traits compared to Se0-AMF and Se50-AMF. The plant height of treatment Se50-AMF was lower than those of control (Se0-AMF). This was consistent with the results of Trial 1, that lower concentrations of exogenous Se stimulated garlic growth, while higher concentrations inhibited growth. The application of AMF improved the height and fresh weight of garlic plants compared to the treatments without AMF inoculation ( Figure 3). Based on the measured growth parameters of Se0-AMF and Se0+AMF, AMF inoculation had a positive effect on garlic growth, especially the whole plant fresh weight and garlic bulb diameter and height, for which the differences were statistically significant. Even among the treatments Se10-AMF, Se10+AMF, Se50-AMF, and Se50+AMF, AMF inoculation also showed promotion of garlic growth, and significant differences were found in the diameter and length of scape between Se10-AMF and Se10+AMF. In addition, the whole plant fresh weight, bulb diameter and height, and space diameter and length of Se50-AMF were considerably lower than those of Se50+AMF (Figure 3B and Table 2). Compared with Se50-AMF, the bulb weight, bulb diameter and height, and scape diameter and length of treatment Se50+AMF were significantly increased by 10.7%, 10.6%, 12.6%, 24.9%, and 17.9%, respectively ( Table 2). This indicated

Se Enrichment
The effects of exogenous Se and AMF on selenium enrichment of garlic bulbs were shown in Figure 4D. Regardless of inoculation or non-inoculation of AMF, Se content in garlic bulbs increased with the increasing concentration of exogenous Se. This was also agreed with the results of Trial 1. The highest Se content was observed at the treatment Se50-AMF, and the lowest Se content was recorded at the control group (Se0-AMF). Inoculation of AMF changed the Se enrichment capacity of garlic bulbs. It could be seen from the significant difference of Se content in garlic bulbs between inoculated and non-inoculated AMF treatments under a high concentration of exogenous Se (Se50-AMF vs. Se50+AMF). Moreover, higher Se contents were found in mycorrhizal garlic than nonmycorrhizal plants for the treatments of 0 mg/L and 10 mg/L exogenous Se, although the difference was not statistically significant. This phenomenon demonstrated that AMF significantly decreased Se transshipment and absorption under high concentration conditions of exogenous Se, but the garlic bulbs at 50 mg/L inoculated with AMF still had a higher Se content than lower Se treatments. Meanwhile, the lifting effect of exogenous Se and AMF interaction on Se enrichment was not significant under the low concentration conditions.

Effects of Exogenous Se on Garlic
Se is a trace element necessary for the health of both animals and plants, which has many forms in the natural environment. Of China's land, 51 percent is selenium-deficient soil and selenium in water, staple foods, vegetables, fruits and animal-based foods of these The effects of exogenous Se and AMF on the allicin content of garlic bulbs were shown in Figure 4C. Without AMF inoculation, the content of allicin significantly decreased with the increasing concentration of exogenous Se. This was also consistent with the results of Trial 1. The highest allicin content was observed at 10 mg/L exogenous Se without application of AMF (Se10-AMF), and the lowest allicin content was recorded with the treatment of Se50-AMF. Inoculation of AMF significantly enhanced the allicin levels for the treatments of 50 mg/L exogenous Se (50+AMF vs. 50-AMF). This promoting effect was not slightly visible under the controls (Se0+AMF vs. Se0-AMF) and was also not show up for the groups of 10 mg/L exogenous Se (Se10+AMF vs. Se10-AMF). This revealed that AMF could significantly alleviate the inhibition of high concentration exogenous Se on allicin accumulation in garlic bulbs.

Se Enrichment
The effects of exogenous Se and AMF on selenium enrichment of garlic bulbs were shown in Figure 4D. Regardless of inoculation or non-inoculation of AMF, Se content in garlic bulbs increased with the increasing concentration of exogenous Se. This was also agreed with the results of Trial 1. The highest Se content was observed at the treatment Se50-AMF, and the lowest Se content was recorded at the control group (Se0-AMF). Inoculation of AMF changed the Se enrichment capacity of garlic bulbs. It could be seen from the significant difference of Se content in garlic bulbs between inoculated and non-inoculated AMF treatments under a high concentration of exogenous Se (Se50-AMF vs. Se50+AMF). Moreover, higher Se contents were found in mycorrhizal garlic than non-mycorrhizal plants for the treatments of 0 mg/L and 10 mg/L exogenous Se, although the difference was not statistically significant. This phenomenon demonstrated that AMF significantly decreased Se transshipment and absorption under high concentration conditions of exogenous Se, but the garlic bulbs at 50 mg/L inoculated with AMF still had a higher Se content than lower Se treatments. Meanwhile, the lifting effect of exogenous Se and AMF interaction on Se enrichment was not significant under the low concentration conditions.

Effects of Exogenous Se on Garlic
Se is a trace element necessary for the health of both animals and plants, which has many forms in the natural environment. Of China's land, 51 percent is seleniumdeficient soil and selenium in water, staple foods, vegetables, fruits and animal-based foods of these areas are lower than other areas. According to WHO/FAO recommendations (26-34 µg/day), sixty percent of Chinese people do not have enough daily selenium intake [35]. Since people cannot directly absorb and utilize the natural inorganic Se, plants and animals, as the primary source of Se, are the best targets to meet our dietary needs. However, different plants have a different absorptive capacity for Se. Previous studies have shown that fruits generally contain a lower amount of Se compared to vegetables [36,37]. Se content in cereals ranges between 0.01 and 0.55 µg/g, changes from 0.001 to 0.17 µg/g FW in milk and dairy products [36], and is about 0.0071 µg/g in oil tea [37]. Our study showed that Se content in garlic was 0.041-0.071 µg/g, generally higher than many other plants, which indicated that garlic had a stronger Se accumulation capacity than other plants. This indicated that garlic might be a selenium-rich vegetable with strong Se accumulation ability and could be used as a target crop for Se biofortification.
As could be seen from the Se content of garlic bulbs (Figure 1), a significantly higher rate was achieved in garlic with higher concentrations of exogenous Se compared with lower Se supplement, indicating that the enrichment effect of high concentration Se treatment on garlic bulbs was better than that of low concentration. Although the increase in exogenous Se concentration improved the Se enrichment ability of garlic bulbs, the suitable concentration would not be blind in agricultural practice. Se can be called a double-edged sword, beneficial to plants at low levels but toxic at high levels [38]. An appropriate amount of Se could promote Brassica rapa L. growth and improve its quality [39]. In addition, some studies also showed that the application of exogenous Se at low concentrations resulted in chlorophyll enhancement and salt resistance [40]. Similarly, the results of our experiments showed that the application of Se had an appreciable impact on the growth and nutritional qualities of garlic bulbs. The influences of low and high concentrations of exogenous Se on the growth of garlic bulbs were different, displaying a trend of lower concentrations promoting growth and higher concentrations inhibiting growth. An amount of 10 mg/L was the optimal concentration of exogenous Se for garlic growth, while 80 mg/L inhibited the growth significantly (Table 1 and Figure 2). When the concentration of exogenous Se was lower than 10 mg/L, the growth and nutritional qualities of garlic bulbs were positively correlated with the applied concentrations. When the concentration of exogenous Se reached 40 mg/L, the promoting effect of exogenous Se on growth and nutritional indicators began to decline, and the inhibiting effect became increasingly evident with the increase in concentrations. Li et al. reported that when nano-selenium and selenite concentrations were less than 1 mg/L, the garlic seedlings in hydroponic culture showed no significant growth inhibition, but 10 mg/L concentration of selenite significantly inhibited the growth of garlic seedlings [41]. The demarcation of high and low concentrations is different from that of our study, which might be due to the different culture methods, research objects, and garlic genotypes. Moreover, the difference of Se content among vegetables is significant, which might mainly cause by the plant species, selenium application mode and rate, cultivation conditions, and soil characteristics [2].
Se content and enrichment capacity of garlic bulbs strongly differed between the two cultivars of garlic used in this study. The original Se content in garlic bulbs of cv. G024 was 0.041 µg/g, which was lower than that of cv. G025 (0.071 µg/g). Compared with cv. G025, lower concentrations Se promoted garlic bulb significant growth of cv. G024, and higher concentrations more significantly inhibited the growth of garlic bulbs (Table 1 and Figure 2). The reason for this phenomenon might be that different varieties of garlic had different susceptibility to Se. The garlic cv. G024 was more sensitive to exogenous Se, and the selenium enrichment capacity was also stronger than cv. G025. The difference of Se accumulation capacity among different accessions was also obtained in lettuce by Ramos et al. [42]. Therefore, to utilize more efficient exogenous Se, the factor of garlic cultivar should also be considered for choosing a suitable concentration in the production of high-quality selenium-rich garlic. It was worth mentioning that Se had a small range of healthy intakes as a trace element. Both excessive intake (>400 g/d) and insufficient intake (<30 g/d) would cause adverse effects on human health. According to the upper limit of the daily supplement of 400 g selenium poisoning standard ("Chinese dietary reference intakes (2017)"), garlic treated with 80 mg/L exogenous Se need to be eat 760 g/d cause poisoning. Obviously, it was safe under normal consumption conditions.

Effects of AMF Inoculation on Garlic
Based on the results of Trial 1, a high concentration of exogenous Se could improve the Se enrichment ability in garlic, but it inhibited the improvement of nutritional qualities. We were curious about whether we had a method to reduce the sensitivity of garlic to high concentration exogenous Se stress, eliminate the quality inhibition, produce highquality selenium-rich garlic, and further maximize the utilization rate of exogenous Se. Many studies have shown that the application of AMF could protect the plants from a variety of abiotic stresses such as cold [43], drought [44], salt, arsenic [45], and heavy metal stress [17,42]. Therefore, Trial 2 was conducted to investigate the effects of exogenous Se AMF inoculation on the growth, nutritional qualities, and selenium-enriched capacity of garlic. The results showed that the symbiotic relationship between AMF and garlic significantly alleviated the growth inhibition resulted in high concentration exogenous Se. Moreover, AMF inoculation changed the absorption capacity of exogenous Se and became the main factor in increasing the content of soluble protein and sugar. AMF coexisted with the root system of plants and formed a considerable mycelium network, in which a large number of bacteria established complex mycorrhizal interactions with AMF structures. Thus, the root-soil flora of plants inoculated with AMF showed higher bacterial diversity [7,31,46]. Furthermore, organic compounds released by AMF in the rhizosphere accelerated the microbial metabolism and nutrient cycling of soils [47]. Therefore, AMF promoting the absorption and utilization of soil nutrients might be a primary reason for the increase in soluble proteins and sugars in garlic plants. The microbial modification of the root area might also increase the root length, surface area, and the number of root hairs, then enhancing the plant availability of trace elements in the rhizosphere [15]. AMF promoting root hair growth, improving root structures, and then increasing plant biomasses had been verified in maize by Abdelhameed et al. [48]. This might explain why the garlic growth inoculated with AMF was faster and more orderly and robust than that of without AMF (Figure 3).
For exogenous Se treated garlic, the robust root structures resulted from AMF inoculation might contribute to the absorption and enrichment of nutrients in garlic under low concentration. At the same time, the improved robustness of plants increased the stress resistance of garlic plants and then reduced the exogenous Se sensitivity, thus retarding the harm caused by a high concentration of exogenous Se. Additionally, the phenomenon of AMF inoculation reducing the Se accumulation in garlic under the high concentration of exogenous Se might also be that AMF application reduced the upward transport and accumulation of exogenous Se in the host plants as a consequence of the enhanced binding of exogenous Se on mycelium and root surface.

Conclusions
Exogenous Se has a dose-dependent effect on the growth and nutritional qualities of garlic, namely lower concentration (≤10 mg/L) promotion and higher concentration (≥40 mg/L) inhibition. The Se enrichment ability was positively correlated with the applied concentrations, while higher application showed lower efficiency to parts of garlic nutritional qualities. Furthermore, AMF inoculation can reduce the harm of higher concentration Se on garlic. AMF inoculation combined with the application of appropriate exogenous Se will be an effective and promising method for high-quality selenium-rich garlic production.