*3.3. Lower Level of SA Biosynthesis Stimulated by Fusarium oxysporum Might Be Cloesly Related to the Formation of R. glutinosa Replant Disease*

Generally, the stress factors associated with replant disease were complex and multiple, including pathogens, nematode and abiotic stress [7]. To resist these stress factors, plant hormones are widely involved in resistance levels as important signaling molecules [64]. ABA is mainly associated with abiotic environmental stresses [44]. SA is typically involved in the defense against biotrophs [65]. JA and ET are generally thought to act together, and to play core roles in the defense against necrotrophs [66]. The relationship between the SA and JA-ET pathway is more antagonistic other than cooperative [44]. In this study, the four hormones were significantly activated under replant disease stresses compared with the control NP at 3 DAP, and presented each other significant positive correlation (*p* < 0.001). The cooperation for SA and JA-ET was consistent with some literatures, such as Wu et al. (2018) [67] and Adie et al. (2007) [68], which was different from the antagonistic relationship between SA and JA-ET. A reasonable explanation for the cooperation of ABA with SA was that ABA increased susceptibility to pathogens in some plant-pathogen interactions [45]. However, these inferences need to be further verified. Previous studies displayed that some plant hormones including ET and ABA involved in the formation mechanism of *R. glutinosa* replant disease and closely related to immune resistance [48,69]. Some studies have demonstrated that SA was involved in the defense against *Fusarium oxysporum* and associated with acteoside accumulation, one of the important pharmacodynamic component [46,47]. More importantly, there were significant negative correlations only between ∆FO and ∆SA with the gradient changes in the stress level of replant disease (Table 4). The results revealed that the SA synthesis, which may be inhibited by *Fusarium oxysporum*, was involved in the formation of replant disease in *R. glutinosa*.

Based on the above results, a possible depiction of the immune response and its potential crosstalk with microbes and plant hormones in replanted *R. glutinosa* was drawn, and it is shown in Figure 6. These findings provide insights into the formation of replant disease.

disease in *R. glutinosa*.

some literatures, such as Wu et al. (2018) [67] and Adie et al. (2007) [68], which was different from the antagonistic relationship between SA and JA-ET. A reasonable explanation for the cooperation of ABA with SA was that ABA increased susceptibility to pathogens in some plant-pathogen interactions [45]. However, these inferences need to be further verified. Previous studies displayed that some plant hormones including ET and ABA involved in the formation mechanism of *R. glutinosa* replant disease and closely related to immune resistance [48,69]. Some studies have demonstrated that SA was involved in the defense against *Fusarium oxysporum* and associated with acteoside accumulation, one of the important pharmacodynamic component [46,47]. More importantly, there were significant negative correlations only between **∆**FO and **∆**SA with the gradient changes in the stress level of replant disease (Table 4). The results revealed that the SA synthesis, which may be inhibited by *Fusarium oxysporum*, was involved in the formation of replant

Based on the above results, a possible depiction of the immune response and its potential

Figure 6. These findings provide insights into the formation of replant disease.

**Figure 6.** Schematic of effector-triggered immunity response mediated by consecutive monoculture stress in *R. glutinosa*. **Figure 6.** Schematic of effector-triggered immunity response mediated by consecutive monoculture stress in *R. glutinosa*.

#### **4. Materials and Methods 4. Materials and Methods**

#### *4.1. Plant Growth and Treatments 4.1. Plant Growth and Treatments*

To obtain aseptic plantlets, tuberous roots of *R. glutinosa* "Wen 85-5" were surface sterilized with 0.1% mercuric chloride solution for 17–20 min, washed five times with the sterile water, and then cultured in sterile bottles with two layers of damp gauze at the bottom. The shoots, approximately 1 cm long, were cut and cultured on hormone-free MS agar medium containing 30 g·L−1 sucrose and 10 g·L−1 agar [47]. The explants were cultured under controlled conditions (25 °C, 4000 lux, 14 h light/10 h dark photoperiod) in a growth chamber for 30 days. To obtain aseptic plantlets, tuberous roots of *R. glutinosa* "Wen 85-5" were surface sterilized with 0.1% mercuric chloride solution for 17–20 min, washed five times with the sterile water, and then cultured in sterile bottles with two layers of damp gauze at the bottom. The shoots, approximately 1 cm long, were cut and cultured on hormone-free MS agar medium containing 30 g·L −1 sucrose and 10 g·L <sup>−</sup><sup>1</sup> agar [47]. The explants were cultured under controlled conditions (25 ◦C, 4000 lux, 14 h light/10 h dark photoperiod) in a growth chamber for 30 days.

To enhance the quality of plantlets for transplantation, the aseptic plantlets of *R. glutinosa* with seven to eight leaves were adapted in a phytotron (28 ◦C, 10,000 lux, 14 h light/10 h dark photoperiod) for 12 h, followed by unscrewing the bottle caps with a small opening to adapt for 6 h, and then removing the cap to adapt for 30 h (Figure 7A). After carefully washing away the adherent medium on the roots, the plantlets were adapted in sterile water for one day and transplanted into plastic pots.

Pot experiments were performed under controlled conditions (28 ◦C, 10,000 lux, 14 h light/10 h dark photoperiod) at the Institute of GAP for Chinese Medicinal Materials, Fujian Agriculture and Forestry University. *R. glutinosa* plantlets after acclimatization were transplanted on 23 August 2018 and grown in plastic pots of 18 cm diameter and 15 cm height (1.38 kg soil per pot). Three plants were planted as three replicate sub-samples in each pot. Four treatments of replant disease levels were constructed by mixing two kinds of soils in different proportions. The soils were collected from the site where *R. glutinosa* had not been planted for at least 10 years (NP) and where *R. glutinosa* had been consecutively planted for three years (TP) in Wen County, Jiaozuo City, Henan Province, in the "geo-authentic" zone of *R. glutinosa* cultivation (34◦560 N, 112◦580 E). The air-dried soil samples were taken to the laboratory for this experiment. Four treatments thus included NP, 1/3TP (mixed by 2 NP plastic pots.

soils and 1 TP soil), 2/3TP (mixed by 1 NP soil and 2 TP soils), and TP, NP of which was used as the control (Figure 7B). taken to the laboratory for this experiment. Four treatments thus included NP, 1/3TP (mixed by 2 NP soils and 1 TP soil), 2/3TP (mixed by 1 NP soil and 2 TP soils), and TP, NP of which was used as the control (Figure 7B).

"geo-authentic" zone of *R. glutinosa* cultivation (34°56′N, 112°58′E). The air-dried soil samples were

*Int. J. Mol. Sci.* **2019**, *20*, x 12 of 20

and then removing the cap to adapt for 30 h (Figure 7A). After carefully washing away the adherent medium on the roots, the plantlets were adapted in sterile water for one day and transplanted into

Pot experiments were performed under controlled conditions (28 °C, 10,000 lux, 14 h light/10 h dark photoperiod) at the Institute of GAP for Chinese Medicinal Materials, Fujian Agriculture and Forestry University. *R. glutinosa* plantlets after acclimatization were transplanted on 23 August 2018 and grown in plastic pots of 18 cm diameter and 15 cm height (1.38 kg soil per pot). Three plants were planted as three replicate sub-samples in each pot. Four treatments of replant disease levels were constructed by mixing two kinds of soils in different proportions. The soils were collected from the site where *R. glutinosa* had not been planted for at least 10 years (NP) and where *R. glutinosa* had

**Figure 7.** The operation process for acclimatization and the phenotype changes of *R. glutinosa* in the experiment. Obvious irreversible injury occurred at 6 DAP until death at 9 DAP. (**A**) The six main steps of acclimatization and transplanting. Step 1: Closed adaptation for 12 h; Step 2: Small opening for 6 h; Step 3: Completely open for 30 h; Step 4: Washing the culture medium carefully with sterile pure water; Step 5: Plantlets were adjusted to sterile pure water for 24 h; Step 6: Transplanting three plants per pot; (**B**) the phenotype changes of *R. glutinosa* under different replant disease stresses within 9 DAP. In the pot stage, 1/3TP, 2/3TP and TP are compared with NP, and 3 DAP, 6 DAP and 9 DAP are compared with 0 DAP (3 DAA). DAP: Days after planting. NP: Soil that was never **Figure 7.** The operation process for acclimatization and the phenotype changes of *R. glutinosa* in the experiment. Obvious irreversible injury occurred at 6 DAP until death at 9 DAP. (**A**) The six main steps of acclimatization and transplanting. Step 1: Closed adaptation for 12 h; Step 2: Small opening for 6 h; Step 3: Completely open for 30 h; Step 4: Washing the culture medium carefully with sterile pure water; Step 5: Plantlets were adjusted to sterile pure water for 24 h; Step 6: Transplanting three plants per pot; (**B**) the phenotype changes of *R. glutinosa* under different replant disease stresses within 9 DAP. In the pot stage, 1/3TP, 2/3TP and TP are compared with NP, and 3 DAP, 6 DAP and 9 DAP are compared with 0 DAP (3 DAA). DAP: Days after planting. NP: Soil that was never planted with *R. glutinosa* for at least 10 years. TP: Soil that was consecutively planted with *R. glutinosa* in the same soils for three years.

#### planted with *R. glutinosa* for at least 10 years. TP: Soil that was consecutively planted with *R. glutinosa* in the same soils for three years. *4.2. The Collection of Fresh Root and Rhizosphere Soil Samples*

*4.2. The Collection of Fresh Root and Rhizosphere Soil Samples* On day zero, three after acclimatization (DAA), the fresh roots were collected after carefully washing with sterile water and drying with absorbent paper. The samples of 0 DAP and 3 DAA are the same. At 3, 6 and 9 DAP, the fresh roots and their rhizosphere soil were carefully collected as described in Wu et al. (2015) [10]. Briefly, the roots and the soil around the roots were carefully dug up using a sterilized fork spade and slightly shaken to remove loosely attached soil. The rhizosphere soil that was tightly attached to roots (1–3 mm zone around the root) was brushed off and collected. All of the collected samples were immediately frozen in liquid nitrogen and then stored at −80 °C for On day zero, three after acclimatization (DAA), the fresh roots were collected after carefully washing with sterile water and drying with absorbent paper. The samples of 0 DAP and 3 DAA are the same. At 3, 6 and 9 DAP, the fresh roots and their rhizosphere soil were carefully collected as described in Wu et al. (2015) [10]. Briefly, the roots and the soil around the roots were carefully dug up using asterilized fork spade and slightly shaken to remove loosely attached soil. The rhizosphere soil that was tightly attached to roots (1–3 mm zone around the root) was brushed off and collected. All of thecollected samples were immediately frozen in liquid nitrogen and then stored at <sup>−</sup><sup>80</sup> ◦C for further experiments for soil DNA extraction, absolute quantification of PS and FO, qRT-PCR of NB-LRR andmeasurement of plant hormones and physiological index.

## *4.3. The Extraction of Soil DNA and Its Method Comparison*

Approximately 5 g of soil of each sample was weighed for the extraction of soil DNA. Three extraction methods were compared according to the electrophoretic strips. Method I referred to the conventional cetyltrimethylammonium bromide (CTAB) method [70]. Methods II and III used different extraction methods based on the optimization for removing humic acid. PCR and gel electrophoresis were used to evaluate the different DNA extraction methods for PS and FO. For PCR, specific primers of PS (PS for: 50 -GGTCTGAGAGGATGATCAGT-30 , PS rev: 50 -TTAGCTCCACCTCGCGGC-30 ) and FO (ITS1-F: 50 -CTTGGTCATTTAGAGGAAGTAA-30 , AFP308R: 50 -CGAATTAACGCGAGTCCCAA-30 ) were synthesized with reference to Wu et al. (2015) [10] (SunYa Biotechnology Co., Ltd. Fuzhou, China). Conventional PCR was performed using a Thermo cycler instrument (ThermoFisher Scientific A24812, Waltham, MA, USA) to detect transcript abundance. Each 20-µL reaction contained 0.4 µM each primer, 0.5 U 2× Es taq MasterMix enzyme, cDNA and nuclease-free water. The amplification procedure was 95 ◦C for 2 min, 95 ◦C for 5 s, and 58 ◦C to anneal for 30 s, for 30 cycles. Horizontal gel electrophoresis (1% gel) was used to evaluate the effect of the extraction with a DL2000 DNA Marker (Takara, Japan) in 130 V, 200 mA (Liuyi DYY-12, Beijing, China). The results showed that the extraction effect of method III was relatively best (Supplementary Figure S1A).
