*2.1. Changes in the Numbers of Pseudomonas spp. and Fusarium oxysporum in Rhizosphere Soils of Replant Disease R. glutinosa*

To understand the changes in the beneficial PS and pathogenic FO in rhizospheres of replanted *R. glutinosa*, the numbers of them were showed in Figure 1. The numbers of PS were 3.87 <sup>×</sup> <sup>10</sup>8~5.99 <sup>×</sup> 10<sup>8</sup> (cell·g −1 soil), the numbers of FO were 0.71 <sup>×</sup> <sup>10</sup>8~2.68 <sup>×</sup> <sup>10</sup><sup>8</sup> (cell·g −1 soil), and the ratio of FO to PS were 0.16~0.55. The maximum difference ranged between 0 DAP and 9 DAP, where PS was in TP soil and FO was in NP soil (Figure 1A,B). Moreover, only the numbers of FO decreased significantly over time in control NP (Figure 1E). The inhibiting effect of NP soil on FO proliferation was thus stronger than the promoting effect of replanted soil on FO proliferation. During 0~9 DAP, the FO numbers and ratio of FO to PS in TP soil were significantly lower than that in NP soil at 0 DAP, and the reversing results with significant difference were at 9 DAP (Figure 1B,C). The PS numbers were significantly lower only in NP soil than that in TP soil at 0 DAP, and the reversed trends with no significant difference were at 9 DAP (Figure 1A). Furthermore, there were the significant positive correlations between the addition level of replant soils and the changes of the FO numbers and the FO/PS values, and between the FO numbers and the FO/PS values (Tables 1 and 2). The results evidenced that *R. glutinosa* replant disease (1/3TP, 2/3TP and TP) could significantly promote the FO proliferation in rhizospheres soils. Interestingly, at 0 DAP, the PS numbers in 1/3TP, 2/3TP and TP soil were 1.04, 1.11 and 1.21 times than that in NP soil respectively, and the FO numbers in NP soil were 1.39, 1.44 and 1.83 times than that in 1/3TP, 2/3TP and TP soil respectively. The changes were obviously different from that at 9 DAP. The results suggested that the soil had a strong ability that could restore balances among microbial communities, and this means that the appropriate ration between FO and PS in rhizosphere soil presented in *R. glutinosa* grows healthly, during fallow stage.

*Int. J. Mol. Sci.* **2019**, *20*, x 4 of 20

**Figure 1.** The influences of different replant disease stress levels on the numbers and ratio of FO to PS in *R. glutinosa* rhizosphere soils over time. NP, 1/3TP, 2/3TP and TP are the gradient treatments of replant disease stresses. Four samples in each group are compared with NP (**A**–**C**) and 0 DAP (**D**– **F**) respectively, and different lower-case letters indicate significant differences (*p* < 0.05; LSD). Data represented as the mean ± SD (*n* = 3). DAP: Days after planting. PS: *Pseudomonas* spp. FO: *Fusarium oxysporum*. 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. **Figure 1.** The influences of different replant disease stress levels on the numbers and ratio of FO to PS in *R. glutinosa* rhizosphere soils over time. NP, 1/3TP, 2/3TP and TP are the gradient treatments of replant disease stresses. Four samples in each group are compared with NP (**A**–**C**) and 0 DAP (**D**–**F**) respectively, and different lower-case letters indicate significant differences (*p* < 0.05; LSD). Data represented as the mean ± SD (*n* = 3). DAP: Days after planting. PS: *Pseudomonas* spp. FO: *Fusarium oxysporum*. 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.

**Table 1.** Pearson correlations among the different replant disease stress levels and the changes in **Table 1.** Pearson correlations among the different replant disease stress levels and the changes in amplitude of FO/PS and the numbers of PS and FO within 3 DAP.


\* *p* < 0.05, \*\* *p* < 0.01; **∆** represents the change from 0 to 3 DAP. **Table 2.** Pearson correlations among the FO/PS and the numbers of PS and FO within 9 DAP.


#### *2.2. NB-LRR Lists Response to Replant Disease Stresses in R. glutinosa 2.2. NB-LRR Lists Response to Replant Disease Stresses in R. glutinosa*

To reveal the responding mode of *R. glutinosa NB-LRRs* during replant disease formation, the expression levels of *NB-LRRs* were determined by qRT-PCR. Of 35 expressed *NB-LRRs* in acclimatization stage, seven were upregulated and 11 were downregulated (Figure 2). In contrast to NP, 22 *NB-LRRs* were upregulated and six *NB-LRRs* were downregulated in TP at 3 DAP, which To reveal the responding mode of *R. glutinosa NB-LRRs* during replant disease formation, the expression levels of *NB-LRRs* were determined by qRT-PCR. Of 35 expressed *NB-LRRs* in acclimatization stage, seven were upregulated and 11 were downregulated (Figure 2). In contrast to NP, 22 *NB-LRRs* were upregulated and six *NB-LRRs* were downregulated in TP at 3 DAP, which

accounted for 80% of the 35 *NB-LRRs*. The stage from 0 to 3 DAP was thus the key stage to *NB-LRRs*

accounted for 80% of the 35 *NB-LRRs*. The stage from 0 to 3 DAP was thus the key stage to *NB-LRRs* that responded to replant disease stress. In addition, only 1 *NB-LRR* was upregulated and 29 *NB-LRRs* were downregulated at 6 DAP, indicating that replant disease interference to the expression of *NB-LRRs*. In 3 DAP, the Pearson correlation analyses displayed that 35 *NB-LRRs* had no significant correlation with the PS numbers. The significant positive correlation was only between 12 *NB-LRRs* and the FO numbers in NP, 1/3TP, 2/3TP (Table 3). However, these *NB-LRRs* that have significantly positive correlation with the FO numbers, have ineffectively prevented the death of *R. glutinosa* plants at 6 DAP. The results displayed that the 12 upregulated *NB-LRRs* might not respond to the pathogenic FO in replanted *R. glutinosa*. Therefore, 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. In addition, *RgNB14* and *RgNB26* of the 6 *NB-LRRs* were upregulated expression, and the remaining 4 *NB-LRRs* were downregulated or presented a decreasing trend at expression level in acclimatization stage. The two groups of *NB-LRRs* represented whether they responded to abiotic stress or not. Noticeably, the expression levels of *RgNB5* and *RgNB29* were continuously downregulated with addition levels of replant soils at 3 DAP.

**Table 3.** Pearson correlations among the expression of 35 NB-LRRs and the variation of PS and FO numbers from 0 DAP to 3 DAP.


\* *p* < 0.05; \*\* *p* < 0.01.

at 3 DAP.

that responded to replant disease stress. In addition, only 1 *NB-LRR* was upregulated and 29 *NB-LRRs* were downregulated at 6 DAP, indicating that replant disease interference to the expression of *NB-LRRs*. In 3 DAP, the Pearson correlation analyses displayed that 35 *NB-LRRs* had no significant correlation with the PS numbers. The significant positive correlation was only between 12 *NB-LRRs* and the FO numbers in NP, 1/3TP, 2/3TP (Table 3). However, these *NB-LRRs*  that have significantly positive correlation with the FO numbers, have ineffectively prevented the death of *R. glutinosa* plants at 6 DAP. The results displayed that the 12 upregulated *NB-LRRs* might not respond to the pathogenic FO in replanted *R. glutinosa*. Therefore, 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. In addition, *RgNB14* and *RgNB26* of the 6 *NB-LRRs* were upregulated expression, and the remaining 4 *NB-LRRs* were downregulated or presented a decreasing trend at expression level in acclimatization stage. The two groups of

**Figure 2.** Nucleotide binding-leucine-rich repeats (*NB-LRRs*) expression in *R. glutinosa* roots during acclimatization and planting phase with different replant disease stress levels. The ratios of 0 DAA, 3 DAA (0 DAP), NP, 1/3TP, 2/3TP and TP gradient treatments to 3 DAA (0 DAP) are calculated and shown. Two or four samples in each group are compared with 3 DAA and NP respectively, and different lower-case letters 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.

### *2.3. Plant Hormones Response to Replant Disease Stresses in R. glutinosa*

Four plant hormones including ABA, ET, JA and SA were measured, and their contents in 1/3 TP, 2/3TP and TP treatments fluctuated obviously from 0 DAA to 9 DAP (Figure 3). In the acclimatization stage (from 0 DAA to 3 DAA), the contents of plant hormones decreased significantly except for SA. The sensitive stages for rapid alteration of hormones was from 0 to 3 DAP based on the comparison

between 3 DAP and 3 DAA (0 DAP). At 3 DAP, the four hormones were significantly activated under replant disease stresses compared with the control NP by 0.56 to 1.53 times for ABA, 0.59 to 2.02 times for JA, 0.50 to 1.50 times for ET and 0.37 to 1.19 times for SA (*p* < 0.05), which showed that the four plant hormones responded strongly to replant disease stress levels. From 0 to 3 DAP, the four hormone contents in 1/3TP, 2/3TP and TP have significantly increased at 3 DAP compared with that at 0 DAP, excepting SA treated with TP soils. Of which, ABA contents increased by 65.26% to 173.64%, JA by 60.19% to 203.50%, ET by 65.26% to 173.84% and SA by 3.91% to 66.39% under replant disease stresses, but only increased by 8.50%, 0.33%, 9.73% and −24.72% in NP, respectively (*p* < 0.05). Notably, there were significant negative correlations between FO and SA (*p* < 0.05) and significant positive correlations among the four hormones (*p* < 0.01) (Table 4). The results showed that the FO in replant disease rhizosphere soil inhibited the SA biosynthesis in root. Taking together these changes showed that the complex replant disease stress, including biotic and abiotic stress, promoted the four hormones release, but FO inhibited SA biosynthesis, resulting in *R. glutinosa* death at 6 DAP. *Int. J. Mol. Sci.* **2019**, *20*, x 7 of 20

**Figure 3.** The four plant hormone contents 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 **Figure 3.** The four plant hormone contents 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.

same soils for three years. **Table 4.** Pearson correlations among the variation of the PS, FO, jasmonic acid (JA), abscisic acid (ABA), ethylene (ET) and salicylic acid (SA) contents under replant disease stresses from 0 to 3 DAP.


∆ABA 0.9650 \*\* 0.9543 \*\* \* *p* < 0.05, \*\* *p* < 0.001; ∆ represents the variation in content.

∆ET 0.9009 \*\* \* *p* < 0.05, \*\* *p* < 0.001; **∆** represents the variation in content.

the acclimatization stage, there were no significant changes in the POD activities. The H2O2 contents were significantly increased, and the others that used to eliminate toxicity of H2O2 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 H2O2 contents in NP, 1/3TP, 2/3TP and TP at 3 DAP were significantly different from those at 3 DAA (0 DAP). Of which H2O2 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

The physiological indexes including root activity; superoxide dismutase (SOD), peroxidase

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