*2.4. Physiological Response to Replant Disease Stresses in R. glutinosa*

The physiological indexes including root activity; superoxide dismutase (SOD), peroxidase (POD) and catalase (CAT) activities; and malondialdehyde (MDA) and hydrogen peroxide (H2O2) contents were investigated in *R. glutinosa* with different replant disease stress levels (Figure 4). In the acclimatization stage, there were no significant changes in the POD activities. The H2O<sup>2</sup> contents were significantly increased, and the others that used to eliminate toxicity of H2O<sup>2</sup> were decreased significantly. In contrast to 3 DAA (0 DAP), the root activities, SOD activities, POD activities and MDA contents in NP tended to increase consecutively from 3 DAP to 9 DAP, which reported the normal response of plantlets planted in healthy soil. In addition, root activity, SOD activity, CAT activity and H2O<sup>2</sup> contents in NP, 1/3TP, 2/3TP and TP at 3 DAP were significantly different from those at 3 DAA (0 DAP). Of which H2O<sup>2</sup> contents significantly decreased by 0.45 to 0.75 times, and root activity, SOD activity and CAT activity significantly increased by 0.36 to 2.75 times, 3.12 to 7.33 times, 2.76 to 4.26 times at 3 DAP, respectively. However, there were no gradient changes for the four physiological indexes at 3 DAP. Moreover, only the H2O<sup>2</sup> contents showed a gradient change at 6 DAP. Furthermore, there were significant positive correlations between the FO numbers and the CAT activity and the MDA content only at 6 DAP (Table 5). Overall, these results displayed that these physiological indexes acting downstream of life activities were affected by complex replant disease stresses and did not show significant correlation with the FO numbers until 6 DAP.

**Table 5.** Pearson correlations among the variation of the two microorganisms and the six physiological indexes under replant disease stresses during three and six days after transplanting.


\* *p* < 0.05; ∆ represents the variation in content.

four physiological indexes at 3 DAP. Moreover, only the H2O2 contents showed a gradient change at 6 DAP. Furthermore, there were significant positive correlations between the FO numbers and the CAT activity and the MDA content only at 6 DAP (Table 5). Overall, these results displayed that

**Figure 4.** The contents of physiological indexes in *R. glutinosa* roots during acclimatization stage and planting stage with different replant disease stress levels. 0 DAA, 3 DAA (0 DAP), NP, 1/3TP, 2/3TP and TP are gradient treatments. Two or four samples in each group are compared with 3 DAA (0 DAP) and NP respectively, and different lower-case letters indicate significant differences (*p* < 0.05; LSD). 3 DAP with NP, 1/3TP, 2/3TP and TP gradient treatments is compared with 3 DAA (0 DAP), and the asterisk indicate significant differences (*p* < 0.05; LSD). Data represented as the mean ± SD (*n*  = 3). DAA: Days after acclimatization. DAP: Days after planting. NP: Soil that was never planted **Figure 4.** The contents of physiological indexes in *R. glutinosa* roots during acclimatization stage and planting stage with different replant disease stress levels. 0 DAA, 3 DAA (0 DAP), NP, 1/3TP, 2/3TP and TP are gradient treatments. Two or four samples in each group are compared with 3 DAA (0 DAP) and NP respectively, and different lower-case letters indicate significant differences (*p* < 0.05; LSD). 3 DAP with NP, 1/3TP, 2/3TP and TP gradient treatments is compared with 3 DAA (0 DAP), and the asterisk indicate significant differences (*p* < 0.05; LSD). Data represented as the mean ± SD (*n* = 3). DAA: Days after acclimatization. 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.

with *R. glutinosa* for at least 10 years. TP: Soil that was consecutively planted with *R. glutinosa* in the

#### same soils for three years. **3. Discussion**

#### **Table 5.** Pearson correlations among the variation of the two microorganisms and the six *3.1. Replant Disease Promotes Proliferation of Fusarium oxysporum in R. glutinosa Rhizospheres Soil*

physiological indexes under replant disease stresses during three and six days after transplanting. ∆**Root Activity** ∆**SOD** ∆**POD** ∆**CAT** ∆**H2O2** ∆**MDA**  0~3 DAP ∆PS 0.0357 0.2189 0.3088 −0.1421 −0.3443 −0.3313 ∆FO 0.4389 −0.1874 0.2671 0.4416 0.1959 −0.1724 0~6 DAP ∆PS 0.1759 −0.0713 −0.0387 −0.2458 0.1394 −0.2020 ∆FO 0.2250 0.1574 0.2263 0.7464 \* −0.5732 0.7316 \* \* *p* < 0.05; **∆** represents the variation in content. Previous studies have revealed that *R. glutinosa* replant disease induced rhizosphere microbes' adverse chemotaxis [49,50], resulting in the increase of pathogenic FO abundance and the decrease of beneficial PS abundance, thus the identified PS and FO were usually used as a characteristic mark in the *R. glutinosa* replant disease study [10,28]. In this study, the PS numbers decreased and the FO numbers increased in 1/3TP, 2/3TP and TP soils from 0 to 9 DAP. Moreover, the significant positive correlations were presented between the addition levels of replant soils and the changes of the FO numbers and the FO/PS values. These results revealed that replant disease promoted FO proliferation in rhizospheres soils. Interestingly, the focus on maximum difference between the PS and FO numbers displayed that the inhibiting effect of NP soil on FO proliferation was stronger than the promoting effect of replanted soil on FO proliferation. Furthermore, the number of PS in NP soil was significantly lower than that in TP soil at 0 DAP, while the number of FO in NP soil was significantly greater than that in 1/3TP, 2/3TP and TP soils at 0 DAP. The changes were obviously different from that at 9 DAP. **3. Discussion** 

These results are different from those in the field [10], which might be related to the difference between the air-dried soil used in this experiment and the field soil after fallow cultivation in the literature [51]. A valuable clue was thus presented that soil may have a strong ability restored microbe balance in rhizosphere soil during the fallow stage. related to the difference between the air-dried soil used in this experiment and the field soil after fallow cultivation in the literature [51]. A valuable clue was thus presented that soil may have a strong ability restored microbe balance in rhizosphere soil during the fallow stage.

different from that at 9 DAP. These results are different from those in the field [10], which might be

*Int. J. Mol. Sci.* **2019**, *20*, x 9 of 20

Previous studies have revealed that *R. glutinosa* replant disease induced rhizosphere microbes' adverse chemotaxis [49,50], resulting in the increase of pathogenic FO abundance and the decrease of beneficial PS abundance, thus the identified PS and FO were usually used as a characteristic mark in the *R. glutinosa* replant disease study [10,28]. In this study, the PS numbers decreased and the FO numbers increased in 1/3TP, 2/3TP and TP soils from 0 to 9 DAP. Moreover, the significant positive correlations were presented between the addition levels of replant soils and the changes of the FO numbers and the FO/PS values. These results revealed that replant disease promoted FO proliferation in rhizospheres soils. Interestingly, the focus on maximum difference between the PS and FO numbers displayed that the inhibiting effect of NP soil on FO proliferation was stronger than the promoting effect of replanted soil on FO proliferation. Furthermore, the number of PS in NP soil

*3.1. Replant Disease Promotes Proliferation of Fusarium oxysporum in R. glutinosa Rhizospheres Soil* 

#### *3.2. NB-LRRs Failed to Respond Timely and E*ff*ectively to Pathogenic Fusarium oxysporum in Replanted R. glutinosa 3.2. NB-LRRs Failed to Respond Timely and Effectively to Pathogenic Fusarium oxysporum in Replanted R. glutinosa*

Pathogens almost always occupy extracellular niches [37]. Early studies had unraveled that *Fusarium oxysporum* could inhibit the release of ATP after invading the host plant [52], which is required for NB-LRR activation [37], but low doses toxins secreted by FO could induce the biosynthesis of phytoalexins in the host plants [53]. In this study, the upregulated expressions of *NB-LRRs* were mainly appeared at 3 DAP in TP and at 6 DAP in NP, which were consistent with the changes of the FO relative values (Figure 5). According to the original data associated with the relative values, we found that the upregulated expression of *NB-LRRs* was the strongest when the number of FO was at the lowest level (about 0.95 <sup>×</sup> <sup>10</sup><sup>8</sup> cell·g −1 soil), and gradually decreased with the increasing of FO number in six days after transplanting. These results exhibited that *NB-LRR* expressions have a typical regulars with "promotion in low concentration and suppression in high concentration" when countered FO invasion, which was consistent with Scott et al. (2019) [54]. Pathogens almost always occupy extracellular niches [37]. Early studies had unraveled that *Fusarium oxysporum* could inhibit the release of ATP after invading the host plant [52], which is required for NB-LRR activation [37], but low doses toxins secreted by FO could induce the biosynthesis of phytoalexins in the host plants [53]. In this study, the upregulated expressions of *NB-LRRs* were mainly appeared at 3 DAP in TP and at 6 DAP in NP, which were consistent with the changes of the FO relative values (Figure 5). According to the original data associated with the relative values, we found that the upregulated expression of *NB-LRRs* was the strongest when the number of FO was at the lowest level (about 0.95 × 108 cell·g−1 soil), and gradually decreased with the increasing of FO number in six days after transplanting. These results exhibited that *NB-LRR* expressions have a typical regulars with "promotion in low concentration and suppression in high concentration" when countered FO invasion, which was consistent with Scott et al. (2019) [54].

**Figure 5.** The heatmaps of the 35 *NB-LRR* expressions in *R. glutinosa* roots and the numbers of FO and PS and their ratios in *R. glutinosa* rhizosphere soils. (**A**) The expression profiles of 35 *NB-LRRs* in *R. glutinosa* roots over time. 3 DAA (0 DAP) is the control; (**B**) the changes in relative values of FO, PS and FO/PS over time. Calculation of the relative values: The cell numbers and the FO/PS values **Figure 5.** The heatmaps of the 35 *NB-LRR* expressions in *R. glutinosa* roots and the numbers of FO and PS and their ratios in *R. glutinosa* rhizosphere soils. (**A**) The expression profiles of 35 *NB-LRRs* in *R. glutinosa* roots over time. 3 DAA (0 DAP) is the control; (**B**) the changes in relative values of FO, PS and FO/PS over time. Calculation of the relative values: The cell numbers and the FO/PS values are first compared with that at 0 DAP (3 DAA), and then normalized (*Z*-score) for each microorganism respectively to display in the same color scale. DAA: Days after acclimatization. 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.

In antagonistic associations with microbes, plants have evolved to form two strategies of PTI and ETI for fighting microbial pathogens [37]. A well-known view is that immune receptor play a vital role in the recognition to pathogens [55,56], and constitutive downstream proteins are tightly controlled by both positive and negative regulators [39,57]. An integrated understanding for plant immune response is thereby in both the immune receptor and downstream signal transduction. Based on studies till date, there is mounting evidence that two possible interpretations were supported for the relationships of immune response and plant death. (i) Plant immunity has not been triggered [37,38]. (ii) Excessive immunity response often leads to inhibition of normal plant growth and even death [57–59]. In this study, the majority of *NB-LRRs* identified in replanted *R. glutinosa* roots were upregulated or downregulated in TP at 3 DAP in comparison to 0 DAP. At same time, there were significantly positive correlations found between 12 *NB-LRRs* and the FO numbers in NP, 1/3TP, 2/3TP and TP

during 0~3 DAP, but the FO numbers in 1/3TP, 2/3TP and TP were no different with those in NP at 3 DAP even significantly lower at 0 DAP. In addition, accompanied by the death of replanted *R. glutinosa* at 6 DAP, only 1 *NB-LRR* was upregulated (downregulated at 3 DAP) and 29 *NB-LRRs* were downregulated. Taking together these data showed that the stage of 0~3 DAP was the key stage for *NB-LRRs* to respond to replant disease stress, while the immune response was obviously inactivated to FO rather than excessive responses of NB-LRRs. Therefore, it was one of important reasons that *NB-LRRs* were not consecutively responsive to the FO proliferation at the transcriptional level in replanted *R. glutinosa* roots. These new findings provide insights into the response mechanism of *R. glutinosa* to replant disease.

One of the big gaps in our understanding of plant immunity is in the downstream signaling pathways after receptor protein activation [37]. Only two identified downstream signaling proteins of EDS1 and non-race-specific disease resistance 1 (NDR1) are required for signaling of all TIR-NB-LRRs and some CC-NB-LRRs, respectively [37]. For NB-LRR, the protein structure consists of a carboxy-terminal LRR domain for effector recognition, NB-ARC (nucleotide-binding adaptor shared by APAF-1, R proteins, and CED-4) and amino-terminal Toll/interleukin-1 receptor (TIR) or Coiled-coil (CC) domains for signal transduction to downstream proteins [41]. In this study, the six downregulated *NB-LRRs* (*RgNB5*, *RgNB14*, *RgNB26*, *RgNB29*, *RgNB34* and *RgNB35*) in TP at 3 DAP, were screened as candidates for responding to *R. glutinosa* replant disease. According to their functional conservation in these NB-LRRs, there were seven resistance in linkage group 1A (R1A), 2 resistance in linkage group 1B (R1B), 1 resistance to *Pseudomonas syringae* (RPS2), 1 target of AvrB operation 1 (TAO1) and 2 resistance to *powdery mildew* 8 (RPW8). Based on studies till date, EDS1 were required for downstream signaling of these identified NB-LRRs except for RPS2 (RgNB32) [60–63]. The results provide valuable clues for studying the signaling pathways that operate downstream of NB-LRR protein activation in replanted *R. glutinosa*.
