*2.6. Dynamic Variations in Flavonoid DAMs*

In order to measure the dynamic variations of nine flavonoid DAMs in M and NM at different growth stages, the extracts were detected using HPLC-MS/MS. The optimized parameters for each analyte are shown in Table 2. Figure 6 showed the TIC diagrams of nine standards in the positive and negative ion mode. The method validation including linearity, limit of detection (LOD), limit of quantification (LOQ), stability, precision, and repeatability is shown in Table 3 with an *R*<sup>2</sup> higher than 0.9901, LOD ranging from 0.488 ng/mL to 15.63 ng/mL, LOQ ranging from 0.977 ng/mL to 31.25 ng/mL, the relative standard deviations (RSDs) of the stability ranging from 1.94% to 4.22%, the RSDs of the precision ranging from 1.43% to 4.49%, and the RSDs of the repeatability in the range of 1.74% to 4.93%. Additionally, the recovery rate was determined by adding 50%, 100% and 150% of the standard content of the sample. The average recovery rate of nine standards was from 86.03% to 99.54% with RSD between 1.09% and 4.55% (Table S2). These data indicated the reliability of the method.


**Ionization Mode**

**Retention** **Time (min)**

> ESI+

 ESI+

 ESI+

 ESI+

 ESI−

 ESI−

 ESI−

 ESI−

 ESI−

4.56

3.51

3.53

4.67

3.44

3.69

3.84

3.43

3.50

 ion spray

*Int. J. Mol. Sci.* **2020**, *21*, 564

DP de-clustering potential; EP entrance potential; CXP collision cell exit potential; CE collision energy; PI product ions; q for quantification; i for identification; CUR curtain gas; ISvoltage; TEM temperature; GS1 ion source gas 1; GS2 ion source gas2; CUR = 35 psi; TEM = 500 ◦C; both GS1 and GS2 = 55 psi.

9

kaempferol-3-O-glucoside

 592.9

 284.9

 255

−50

−40

−10

−15

−4500



**Figure 6.** TIC diagrams of nine flavonoid standards in the positive and negative ion mode. (**A**) the positive ion mode; A1 represents the chromatograms of narcissin (retention time = 3.51 min); A2 is the ones of isorhamnetin-3-O-beta-d-glucoside (retention time = 3.53 min); A3 is the ones of nobiletin (retention time = 4.56 min); A4 is the ones of tangeretin (retention time = 4.67 min); (**B**) the negative ion mode; B1 represents the chromatograms of quercetin-7-O-glucoside (retention time = 3.43 min); B2 is the ones of rutin (retention time = 3.44 min); B3 is the ones of kaempferol-3-O-glucoside (retention time = 3.50 min); B4 is the ones of quercetin (retention time = 3.69 min); B5 is the ones of isorhamnetin (retention time = 3.84 min).

For M and NM growth for six months, the tangeretin content was inhibited, while the content of the other 8 flavonoid DAMs was significantly upregulated which was consistent with the results of the metabolome and showed that the metabolome data were reliable. The levels of four metabolites (i.e., isorhamnetin-3-o-beta-d-glucoside, rutin, isorhamnetin, and kaempferol-3-O-glucoside) gradually increased across the growth time. For narcissin, quercetin, and quercetin-7-O-glucoside, the content tended to increase at first, then decrease and then increase again. While the tangeretin content reached the peak of accumulation at day 0 and the fourth month. Flavonoid accumulation in the plantlets showed significant difference between the fungal and no-fungal inoculations. The AR2 had a positive effect on narcissin, rutin, quercetin, and quercetin-7-O-glucoside content in *A. roxburghii* growth for one month, while it inhibited the accumulation of isorhamnetin-3-O-beta-d-glucoside, isorhamnetin, and kaempferol-3-O-glucoside and had no significant effect on nobiletin and tangeretin. Compared with the control group, significantly higher amount of narcissin, rutin and quercetin-7-O-glucoside accumulated in mycorrhizal plantlets from the first month to the sixth month. Up until the sixth month, AR2 could significantly promote the accumulation of nobiletin, but there was no significant difference at other times. The detailed results are shown in Figure 7. In conclusion, AR2 could significantly affect the accumulation of different flavonoids in *A. roxburghii*.

**Figure 7.** Dynamic variations of 9 flavonoids in the mycorrhizal and non-mycorrhizal *A. roxburghii* growth 0 month to 6 months. NM and M represents non-mycorrhizal *A. roxburghii* and mycorrhizal *A. roxburghii*, respectively. Each value is the mean of three replicates, and error bars indicate standard deviations. Statistical analysis of the data was performed by independent samples *t*-test using the SPSS 22.0 software (IBM, Chicago, IL, USA). \* and \*\* above the columns are significantly different at *p* ≤ 0.05 and *p* ≤ 0.01, respectively.
