Three Preceding Crops Increased the Yield of and Inhibited Clubroot Disease in Continuously Monocropped Chinese Cabbage by Regulating the Soil Properties and Rhizosphere Microbial Community

Crop rotation can improve soil properties and is one of the important measures to prevent soil-borne diseases. This study aimed to evaluate the effects of different preceding crops on clubroot disease in Chinese cabbage and soil microorganisms, to provide a theoretical basis for the ecological control of clubroot scientifically. In this experiment, soybeans, potato onions, and wheat were used as the preceding crops and compared with the local preceding crop garlic. The growth of the Chinese cabbage, disease occurrence, soil chemical properties and changes in microbial community structure were determined by using quantitative real-time polymerase chain reaction (PCR), soil microbial high-throughput sequencing and other methods. The results showed that the rotation of potato onion and wheat with Chinese cabbage could reduce the clubroot disease index of Chinese cabbage remarkably. Through Illumina Miseq sequencing, when three previous crops were harvested, the abundance and diversity of the bacteria increased obviously, while the fungi decreased. The relative abundance of the phylum Proteobacteria and Firmicutes was strikingly reduced, while that of Chloroflexi was significantly increased. These results show that three previous crops changed the structure of soil microorganisms, reduced the clubroot disease of Chinese cabbage, promoted growth, and suppressed disease. The ranked effect on promoting growth and inhibiting diseases was potato onion > wheat > soybean.


Introduction
Clubroot disease is a serious soil-borne disease caused by Plasmodiophora brassicae, threatening the production of cruciferous plants worldwide [1]. In China, about 3.2-4.0 hectares of cruciferous crops are affected by clubroot per year, leading to yield reductions even crop failures [2]. In the prevention and control of diseases, ecological methods are particularly important for the prevention and treatment of clubroot.
Planting previous crops is one of the cultivation techniques used to alleviate the damage to crops by soil pathogens, which can reduce the chance of soil-borne pathogens invading roots, creating unfavorable environments [3]. Different crops have different effects on the inhibition of different soil-borne diseases through mechanisms such as interrupting the cycle of pathogens, allelochemicals, antagonizing microorganisms, and increasing soil carbon content by root exudates and residues to increase soil microbial biomass and activity [4].The rotation of legumes and gramineous crops with cruciferous crops can alleviate the occurrence of cruciferous clubroot [5], the application of rice straw is able to stabilize soil bacterial community composition and inhibit clubroot disease [6], and wheat

Soil and Plant Preparation
An experiment was conducted in a field located in Baicheng Village, Acheng District, Harbin, China (45 • 41 N, 126 • 45 E) during 2018-2019. In this location, the farming pattern of planting garlic in spring and Chinese cabbage in autumn has been repeated for more than 30 years, and the incidence of clubroot has been severe in the past 10 years. The soil was sandy loam and contained pH 6.43, the value of electrical conductivity (EC) was 0.398 mS/cm, soil organic matter content (SOM) was 3.07%, alkali hydrolyzable nitrogen (AN) was 130.41 mg·kg −1 , available phosphorus (AP) was 125.25 mg·kg −1 , available potassium (AK) was 622.50 mg·kg −1 , and total nitrogen (TN) was 2.51%. The content of resting spores of P. brassicae in soil was 1.95 × 10 5 per gram.

Experimental Design
The experiment was conducted from April 2018 to October 2019 and repeated for two consecutive years. There were four treatments in the experiment, including soybean rotation, potato onion, wheat, and garlic rotation as control: (1) soybean Chinese cabbage (B); (2) potato onion Chinese cabbage (O); (3) wheat Chinese cabbage (W); (4) garlic Chinese cabbage (CK). The experiment was randomly designed with three replicates per treatment, and each treatment plot was five ridges (3 m × 7 m). Potassium sulfate compound fertilizer (15-15-15, 10 kg·hm −2 ) and diammonium hydrogen phosphate (10 kg·ha −2 ) were applied as Microorganisms 2022, 10, 799 3 of 17 base fertilizer before sowing. Potato onions, wheat, and garlic were planted from 5-10 April, and soybeans were planted on about 28 April. After the previous crop was harvested, Chinese cabbage was sowed on about 23 July, with a row spacing of 50 cm × 50 cm, and harvested on about 5 October.

Determination of Growth and Disease
When the Chinese cabbage was planted for 40 days, 9 Chinese cabbage plants were randomly selected from each plot, the biomass of dry above-ground parts was measured, and the rate and incidence of clubroot were investigated. Nine Chinese cabbage plants were randomly selected from each plot after heading to determine the yield, which was converted into a per-plot value. The clubroot disease classification, incidence and disease index calculations of Chinese cabbage were based on Sharma Kalpana et al. [18].

Soil Collection
Soil was collected from the upper soil layer (0-15 cm) and sieved (2 mm) for DNA extraction and high-throughput sequencing after the preceding crops were harvested.
To determine the concentration of the resting-spores of P. brassica and the changes in microorganisms when the previous crops were harvested, the physical and chemical properties were determined after drying naturally.

Determination of Soil Chemical Properties
The chemical properties of soil included soil pH, EC, organic matter, alkali hydrolyzed nitrogen, available phosphorus and available potassium content. The analysis of all indexes was based on Liu et al. [19].

DNA Extraction and Quantification PCR
All soil samples were stored at −80 • C. DNA was extracted from 250 mg of soil samples using a HiPure Soil DNA Kit according to the manufacturer's instructions.
For P. brassicae, the primer was based on Wallenhammar et al. [20]. The forward primer was AAACAACGAGTCAGCTTGAATGC, and the reverse primer was TTCGCGCACAAG-CACTTG. Each 20 µL of PCR reaction contained 9 µL of 2 × Real SYBR mixture, 0.2 µL of each primer (10 µM), 2.5 µL of template DNA, and 8.1 µL of ddH2O. The qPCR reaction conditions were as follows: 5 min at 95 • C for initial denaturation, 22 amplification cycles of 50 s at 95 • C for denaturation, 30 s at 65 • C for annealing, 1 min at 72 • C for an extension, 10 min at 72 • C for a final extension, and an amplified fragment length of about 230 bp.
The amplified products were purified with AMPure XP beads, quantified with the ABI StepOnePlus Real Time PCR system (Life Technologies, made in Carlsbad, CA, USA), and sequenced according to the PE250 mode pooling of Hiseq2500.

Analysis of Soil Microbial Diversity
The original data were analyzed by Turkey's HSD in SPSS 23.0 software at a p < 0.05 level. Pearson correlation analysis was also performed by SPSS 23.0. The bar diagram was prepared using Prism 8.00 software. The sequenced data were analyzed by Quantitative Insights Into Microbial Ecology (QIIME), Version 1.9.0. The community structures of bacteria and fungi were visualized by principal co-ordinates analysis (PCoA) with Bray-Curtis distance dissimilarity matrices to clarify the differences in the community compositions of bacteria and fungi in the different treatments. A beta diversity, RDA Mantel test was performed using the "vegan" package in the R environment (R Studio).

Effects of Different Preceding Crops on Growth of Chinese Cabbage
The yield of the Chinese cabbage in the three treatments increased by more than 20% compared with the CK. The yield and the dry weight on the ground of the Chinese cabbage in the treatments of potato onion Chinese cabbage and wheat Chinese cabbage were significantly higher than the CK over the two years. In addition, the dry weight on the ground of the Chinese cabbage in 2019 was generally lower than in 2018 (p < 0.05) ( Figure 1).
Insights Into Microbial Ecology (QIIME), Version 1.9.0. The community structures of bacteria and fungi were visualized by principal co-ordinates analysis (PCoA) with Bray-Curtis distance dissimilarity matrices to clarify the differences in the community compositions of bacteria and fungi in the different treatments. A beta diversity, RDA Mantel test was performed using the "vegan" package in the R environment (R Studio).

Effects of Different Preceding Crops on Growth of Chinese Cabbage
The yield of the Chinese cabbage in the three treatments increased by more than 20% compared with the CK. The yield and the dry weight on the ground of the Chinese cabbage in the treatments of potato onion Chinese cabbage and wheat Chinese cabbage were significantly higher than the CK over the two years. In addition, the dry weight on the ground of the Chinese cabbage in 2019 was generally lower than in 2018 (p < 0.05) ( Figure  1).

Effects of Different Preceding Crops on Incidence of Chinese Cabbage Clubroot Disease
In the two consecutive years, the disease index of clubroot disease in the three treatments was significantly lower than that of the control 40 days after the sowing of the Chinese cabbage; potato onion rotation had the lowest disease index (p < 0.05) ( Figure 2).

Effects of Different Preceding Crops on Soil Chemical Properties
The preceding crops significantly increased the pH of the soil and reduced the EC compared with the control (p < 0.05). The wheat rotation increased the soil total nitrogen

Effects of Different Preceding Crops on Incidence of Chinese Cabbage Clubroot Disease
In the two consecutive years, the disease index of clubroot disease in the three treatments was significantly lower than that of the control 40 days after the sowing of the Chinese cabbage; potato onion rotation had the lowest disease index (p < 0.05) ( Figure 2). Insights Into Microbial Ecology (QIIME), Version 1.9.0. The community structures of bacteria and fungi were visualized by principal co-ordinates analysis (PCoA) with Bray-Curtis distance dissimilarity matrices to clarify the differences in the community compositions of bacteria and fungi in the different treatments. A beta diversity, RDA Mantel test was performed using the "vegan" package in the R environment (R Studio).

Effects of Different Preceding Crops on Growth of Chinese Cabbage
The yield of the Chinese cabbage in the three treatments increased by more than 20% compared with the CK. The yield and the dry weight on the ground of the Chinese cabbage in the treatments of potato onion Chinese cabbage and wheat Chinese cabbage were significantly higher than the CK over the two years. In addition, the dry weight on the ground of the Chinese cabbage in 2019 was generally lower than in 2018 (p < 0.05) ( Figure  1).

Effects of Different Preceding Crops on Incidence of Chinese Cabbage Clubroot Disease
In the two consecutive years, the disease index of clubroot disease in the three treatments was significantly lower than that of the control 40 days after the sowing of the Chinese cabbage; potato onion rotation had the lowest disease index (p < 0.05) ( Figure 2).

Effects of Different Preceding Crops on Soil Chemical Properties
The preceding crops significantly increased the pH of the soil and reduced the EC compared with the control (p < 0.05). The wheat rotation increased the soil total nitrogen

Effects of Different Preceding Crops on Soil Chemical Properties
The preceding crops significantly increased the pH of the soil and reduced the EC compared with the control (p < 0.05). The wheat rotation increased the soil total nitrogen content and reduced the available potassium content obviously compared with the control and other treatments in 2018 (p < 0.05). In 2019, the preceding crops all reduced the available potassium content (p < 0.05); however, none of them affected the soil alkali hydrolyzable nitrogen or available phosphorus content, nor did they notably reduce the soil organic matter content (p > 0.05) ( Table 1).

Effects of Different Preceding Crops on Resting Spores of P. brassicae
Sixty days after the preceding crop sowing, the P. brassicae resting spore content was significantly lower than the control in 2018 and 2019. (p < 0.05) ( Figure 3). content and reduced the available potassium content obviously compared with the control and other treatments in 2018 (p < 0.05). In 2019, the preceding crops all reduced the available potassium content (p < 0.05); however, none of them affected the soil alkali hydrolyzable nitrogen or available phosphorus content, nor did they notably reduce the soil organic matter content (p > 0.05) ( Table 1).

Effects of Different Preceding Crops on Resting Spores of P. brassicae
Sixty days after the preceding crop sowing, the P. brassicae resting spore content was significantly lower than the control in 2018 and 2019. (p < 0.05) ( Figure 3).

Alpha and Beta Diversities of Bacterial and Fungal Communities in Soil
In this study, twelve samples of bacterial 16SRNA V3-V4 regions and twelve fungal ITS regions were subjected to Miseq sequencing. After flattening according to the minimum number of samples, a total of 2,209,500 sequences was obtained from the bacteria and 1,360,488 from the fungi.
The observable bacterial OTU number and Shannon index of the potato onion rotation treatment were significantly higher than the control and the other treatments after the preceding crops were harvested (p < 0.05), and the bacterial Shannon index of the three treatments was obviously higher than the control, indicating that the three treatments, especially the potato onion, could increase the number and diversity of bacterial communities compared with the control during planting ( Figure 4A). Compared with the control, the three treatments remarkably reduced the number of fungal OTUs in the soil (p < 0.05) ( Figure 4B).

Alpha and Beta Diversities of Bacterial and Fungal Communities in Soil
In this study, twelve samples of bacterial 16SRNA V3-V4 regions and twelve fungal ITS regions were subjected to Miseq sequencing. After flattening according to the minimum number of samples, a total of 2,209,500 sequences was obtained from the bacteria and 1,360,488 from the fungi.
The observable bacterial OTU number and Shannon index of the potato onion rotation treatment were significantly higher than the control and the other treatments after the preceding crops were harvested (p < 0.05), and the bacterial Shannon index of the three treatments was obviously higher than the control, indicating that the three treatments, especially the potato onion, could increase the number and diversity of bacterial communities compared with the control during planting ( Figure 4A). Compared with the control, the three treatments remarkably reduced the number of fungal OTUs in the soil (p < 0.05) ( Figure 4B).
For the bacterial communities, the PCoA revealed that the samples from the same treatment were grouped together, and the samples from the four treatments could be clearly distinguished ( Figure 4C); however, for the fungal communities, there was little difference between the four treatments ( Figure 4D). For the bacterial communities, the PCoA revealed that the samples from the same treatment were grouped together, and the samples from the four treatments could be clearly distinguished ( Figure 4C); however, for the fungal communities, there was little difference between the four treatments ( Figure 4D).

Compositions of Bacterial and Fungal Communities in Soil
The Miseq sequencing data that were classified at the 97% similarity level included 40 bacterial phyla and 7 fungal phyla. For the bacterial phylum community, there were 10 phyla with a relative abundance > 1%. Compared with the CK, the potato onion, soybean,

Compositions of Bacterial and Fungal Communities in Soil
The Miseq sequencing data that were classified at the 97% similarity level included 40 bacterial phyla and 7 fungal phyla. For the bacterial phylum community, there were 10 phyla with a relative abundance > 1%. Compared with the CK, the potato onion, soybean, and wheat rotation treatments showed a lower relative abundance of Proteobacteria and Firmicutes (p < 0.05), and a higher relative abundance of Chloroflexi (p < 0.05); the relative abundance of Acidobacteria in the potato onion and wheat treatments was significantly higher than the control (p < 0.05) ( Figure 5A and Supplementary Table S2). For the fungal phylum community, there were three phyla with a relative abundance > 0.1%. The dominant phyla were Ascomycota and Basidiomycota, with a total abundance > 85%. The relative abundance of Basidiomycota in the three rotation treatments was lower than in the control, albeit not significantly (p > 0.05) ( Figure 5B). and wheat rotation treatments showed a lower relative abundance of Proteobacteria and Firmicutes (p < 0.05), and a higher relative abundance of Chloroflexi (p < 0.05); the relative abundance of Acidobacteria in the potato onion and wheat treatments was significantly higher than the control (p < 0.05) ( Figure 5A and Supplementary Table S2). For the fungal phylum community, there were three phyla with a relative abundance > 0.1%. The dominant phyla were Ascomycota and Basidiomycota, with a total abundance > 85%. The relative abundance of Basidiomycota in the three rotation treatments was lower than in the control, albeit not significantly (p > 0.05) ( Figure 5B).

A B C D
Bacterial Fungal Among all the samples, the potato onion rotation had the highest number of unique OTUs (1572), and the control had the lowest number of unique OTUs (350) in bacteria ( Figure 5C). The control had the highest number of unique OTUs (216) and bean rotation had the lowest number of unique OTUs (78) in fungal ( Figure 5D).
As for classes level, 106 bacterial and 21 fungal taxa were detected. For the bacterial class communities, there were 16 classes with a relative abundance > 1%. Alphaproteobacteria and Saccharimonadia were the highest in the control, while Deltaproteobacteria and Acidimicrobiia were lower in the control than in the other treatments. The relative abundance of Actinobacteria was lower in the bean and wheat treatments than in the control. Compared with the control, bean rotation had a higher relative abundance of Thermoleophilia, while that of potato onion rotation was lower (p < 0.05) ( Figure 6A and Supplementary Table S4). For the fungal class community, there were five classes with a relative abundance > 1%. The abundance of Sordariomycetes was higher in the three Among all the samples, the potato onion rotation had the highest number of unique OTUs (1572), and the control had the lowest number of unique OTUs (350) in bacteria ( Figure 5C). The control had the highest number of unique OTUs (216) and bean rotation had the lowest number of unique OTUs (78) in fungal ( Figure 5D).
As for classes level, 106 bacterial and 21 fungal taxa were detected. For the bacterial class communities, there were 16 classes with a relative abundance > 1%. Alphaproteobacteria and Saccharimonadia were the highest in the control, while Deltaproteobacteria and Acidimicrobiia were lower in the control than in the other treatments. The relative abundance of Actinobacteria was lower in the bean and wheat treatments than in the control. Compared with the control, bean rotation had a higher relative abundance of Thermoleophilia, while that of potato onion rotation was lower (p < 0.05) ( Figure 6A and Supplementary Table S4). For the fungal class community, there were five classes with a relative abundance > 1%. The abundance of Sordariomycetes was higher in the three treatments than in the control, while those of Agaricomycetes, Eurotiomycetes, and Leotiomycetes were lower than in the control (p > 0.05) ( Figure 6B and Supplementary Table S5). treatments than in the control, while those of Agaricomycetes, Eurotiomycetes, and Leotiomycetes were lower than in the control (p > 0.05) ( Figure 6B and Supplementary Table  S5).  In total, 783 bacterial genera and 186 fungal genera were classified. For the bacterial genus community, there were 19 genera with a relative abundance > 0.5%. The dominant genera were Sphingomonas, Gemmatimonas, RB41, Pirellula, Lysobacter, and Gemmata spp.; Sphingomonas, Gemmatimonas, and Rhodanobacter spp. were higher in the control than in the other treatments, while Subgroup_10 and Arenimonas spp. were lowest in the control. The potato onion and wheat rotation had a higher RB41 relative abundance than the control; the bean and potato rotation had a higher Pirellula spp. relative abundance than the control. Compared with the control, the bean rotation had a higher Lysobacter spp. and lower Bryobacter and Luteimonas spp. relative abundance. The wheat rotation increase the relative abundance of Chthoniobacter spp. and reduced Nocardioides spp. and Luteimonas spp. compared with the control (p < 0.05) ( Table 2). For the fungal genus community, there were 10 genera with a relative abundance > 0.5%. The dominant genera were Fusarium and Cladorrhinum spp. Compared with the control, the bean rotation increased the relative abundance of Cladorrhinum significantly; the potato onion rotation and wheat rotation reduced the relative abundance of Conocybe remarkably (p < 0.05) ( Table 3).   In total, 783 bacterial genera and 186 fungal genera were classified. For the bacterial genus community, there were 19 genera with a relative abundance > 0.5%. The dominant genera were Sphingomonas, Gemmatimonas, RB41, Pirellula, Lysobacter, and Gemmata spp.; Sphingomonas, Gemmatimonas, and Rhodanobacter spp. were higher in the control than in the other treatments, while Subgroup_10 and Arenimonas spp. were lowest in the control. The potato onion and wheat rotation had a higher RB41 relative abundance than the control; the bean and potato rotation had a higher Pirellula spp. relative abundance than the control. Compared with the control, the bean rotation had a higher Lysobacter spp. and lower Bryobacter and Luteimonas spp. relative abundance. The wheat rotation increase the relative abundance of Chthoniobacter spp. and reduced Nocardioides spp. and Luteimonas spp. compared with the control (p < 0.05) ( Table 2). For the fungal genus community, there were 10 genera with a relative abundance > 0.5%. The dominant genera were Fusarium and Cladorrhinum spp. Compared with the control, the bean rotation increased the relative abundance of Cladorrhinum significantly; the potato onion rotation and wheat rotation reduced the relative abundance of Conocybe remarkably (p < 0.05) ( Table 3).

Relationships between Soil Microbial Communities and Soil Chemical Properties
A redundancy analysis (RDA) was used to analyze the relationship between the changes in the bacterial and fungal community structures and the environmental factors among the different treatments. The Mantel test results showed that changes in bacterial community structure were associated with the soil pH (r = 0.4342, p = 0.012), EC (r = 0.6642, p = 0.001), available K (r = 0.5469, p = 0.001), and available N (r = 0.4320, p = 0.011) ( Figure 7A), while the soil pH (r = 0.2979, p = 0.035) and available K (r = 0.3089, p = 0.013) were the dominant factors driving the changes in soil fungal community structure ( Figure 7B) (Supplementary Table S6).

Microbial Ecological Guilds in Soil
There are six types of primary bacterial functional layers, including metabolism, environmental information processing, genetic information processing, cellular processes, human diseases and organismal systems.
In the primary bacterial metabolic pathway, the control had the lowest relative abundance of human diseases. The relative abundance of metabolism was lowest in the bean rotation, while organismal systems and genetic information processing were higher than in the control. The bean and wheat rotations increased the relative abundance of cellular processes remarkably compared with the control (p < 0.05) (Figure 8

Microbial Ecological Guilds in Soil
There are six types of primary bacterial functional layers, including metabolism, environmental information processing, genetic information processing, cellular processes, human diseases and organismal systems.
In the primary bacterial metabolic pathway, the control had the lowest relative abundance of human diseases. The relative abundance of metabolism was lowest in the bean rotation, while organismal systems and genetic information processing were higher than in the control. The bean and wheat rotations increased the relative abundance of cellular processes remarkably compared with the control (p < 0.05) (Figure 8). In the fungi, seven primary functional layers were analyzed, including pathotrophsymbiotrophic fungi, pathotroph-saprotrophic fungi, saprotrophic-symbiotrophic fungi,

Microbial Ecological Guilds in Soil
There are six types of primary bacterial functional layers, including metabolism, environmental information processing, genetic information processing, cellular processes, human diseases and organismal systems.
In the primary bacterial metabolic pathway, the control had the lowest relative abundance of human diseases. The relative abundance of metabolism was lowest in the bean rotation, while organismal systems and genetic information processing were higher than in the control. The bean and wheat rotations increased the relative abundance of cellular processes remarkably compared with the control (p < 0.05) (Figure 8). In the fungi, seven primary functional layers were analyzed, including pathotrophsymbiotrophic fungi, pathotroph-saprotrophic fungi, saprotrophic-symbiotrophic fungi, In the fungi, seven primary functional layers were analyzed, including pathotrophsymbiotrophic fungi, pathotroph-saprotrophic fungi, saprotrophic-symbiotrophic fungi, symbiotrophic fungi, pathotroph, saprotrophic fungi and pathtroph-saprotrophic-symbiotrophic fungi.
In the primary fungal ecological function, the relative abundances of pathotroph and pathoph-saprotroph-symbiotroph were lower in the potato onion and wheat rotations than in the control; compared with the control, the bean rotation had a higher relative abundance of pathotroph-saprotroph, the potato onion rotation had a higher relative abundance of saprotroph, and wheat rotation had more symbiotroph (p < 0.05) (Figure 9).
In the primary fungal ecological function, the relative abundances of pathotroph and pathoph-saprotroph-symbiotroph were lower in the potato onion and wheat rotations than in the control; compared with the control, the bean rotation had a higher relative abundance of pathotroph-saprotroph, the potato onion rotation had a higher relative abundance of saprotroph, and wheat rotation had more symbiotroph (p < 0.05) (Figure 9).

Correlation Analysis between Resting Spores and Soil Microorganisms
A Pearson correlation analysis was conducted between the OTUs with a relative abundance > 0.5% and the resting spores of P. brassicae (p < 0.05).

Correlation Analysis between Resting Spores and Soil Microorganisms
A Pearson correlation analysis was conducted between the OTUs with a relative abundance > 0.5% and the resting spores of P. brassicae (p < 0.05).

Effects of Rotation on Growth of Chinese Cabbage
Previous studies have shown that rotation can promote plant growth and increase yield [21]. Our study found that, compared with the garlic rotation, the onion and wheat rotations increased the yield of the Chinese cabbage under the experimental conditions for two consecutive years. On the one hand, this was possibly related to the allelopathy of the preceding crops; for example, the allelochemicals secreted by wild mustard can promote the growth and increase the yield of cauliflower [22]. Root exudates also play a certain role in the growth of plants. The root exudates of potato onions promote the growth and improve the photosynthesis of tomatoes [23]. However, during the cultivation of cucumber/tomato, buckwheat/corn and sorghum/sesame in the same environment, the respective plants inhibit each other. On the other hand, environmental conditions more conductive to the growth of Chinese cabbage are created after preceding crops are planted and harvested. The growth of eggplant was significantly better when it was intercropped with corn than when it was alone [24]. Potato onion and wheat rotations can reduce the stress of P. brassicae on Chinese cabbage and alleviate the degree of hindrance of plant water and nutrient absorption, thereby promoting the growth of Chinese cabbage and increasing its yield.

Effects of Rotation on the Soil's Chemical Properties
Rotation can improve soil nutrients and increase the effectiveness of nutrient utilization, thereby promoting the growth of crops [25]. Different crops utilize different soil nutrients. Our study found that the soybean, potato onion, and wheat rotation treatments significantly reduced the EC value and potassium content, and increased the pH value of the soil. The content of potassium increased with the aggravation of clubroot, probably because the P. brassicae destroyed the cortexes and columns of the Chinese cabbage, hindering the absorption and utilization of nutrients in the soil by the roots. The suitable pH value for the Chinese cabbage was 6.5-7.0; rotation improved the soil pH, promoting the growth of the Chinese cabbage.

Effects of Rotation on Soil Microbial Communities
Soil microorganisms are important parts of soil fertility, participating in organic carbon decomposition, humus formation, and soil nutrient transformation and cycling. Higher plants can improve soil diversity by increasing soil carbon source diversity [26]. Generally, soil fertility is positively correlated with less fungal and more bacterial content in soil microbial community structures [27]. Soil community diversity comprehensively reflects the richness and evenness of communities; community richness is a measure of alpha diversity, representing the number of species in an ecosystem. Our study found that preceding soybean, potato onion, and wheat could increase the richness and diversity of soil bacteria and decrease the richness and diversity of fungi. Preceding crops provide nutrients to soil microorganisms through root exudates and plant residues [28]. Crop rotation can increase crop diversity and organic carbon input to a certain extent. Diversified carbon sources are able to support the growth and reproduction of diverse microbial groups, increasing soil bacterial diversity [29]. The abundances of potential pathogens and antagonistic microorganisms are lower in rotation soils, while those of potentially plantpromoting microorganisms are higher [30]. Xiong et al., sequenced the soil of long-term monoculture of vanilla, and found that the bacteria Actinomycota and Firmicutes were more abundant in disease-suppressive soil [31]. Through the comparison of species differences, our study found that the rotations of potato onion, soybean and wheat significantly reduced the relative abundance of Proteobacteria and Firmicutes. The comparison of the differences in the soil bacterial communities found that, compared with the control, the Gemmatimonas spp. remarkably decreased in the three treatments during the harvesting of the preceding crops. Gemmatimonas spp. is an oligotrophic flora that can promote the growth of and reduce disease in plants. Its lower abundance in the treatments was possibly due to the presence of richer soil nutrients during the cultivation of Chinese cabbage [32].

Effects of Rotation on the Clubroot of Chinese Cabbage
The occurrence of soil-borne diseases is a comprehensive manifestation of the interaction between crops and soil systems, involving changes in soil microbial community diversity, the accumulation of allelochemicals, imbalances in soil nutrients, and the degradation of soil physicochemical properties. These changes influence each other and work together, eventually leading to the occurrence of soil-borne diseases [33,34]. This experiment found that compared with the control, the three rotations all significantly reduced the content of the P. brassicae resting spores in the soil, as well as the incidence and disease index of the Chinese cabbage, during the two years. This was, probably because the physicochemical properties of the soil were altered by the planting of preceding crops, thereby reducing the incidence of clubroot. Soil moisture has a significant and persistent effect on clubroot pathogenesis. Under favorable temperature conditions, a soil moisture level of 60-70% is the most favorable for clubroot, and even with lower soil moisture, low levels of infection can occur. The incidence of clubroot in the field test was more significant because of the large amount of precipitation and high soil moisture. Studies have shown that rainfall is positively correlated with the severity of clubroot in Brassica vegetables, such as radish [35]. The suitable pH value for the survival of P. brassicae is 5.4-6.7 [36]. In neutral soil pH, the inhibition of spore germination is the main reason for the inhibition of clubroot [37]. Therefore, rotation increases the suitable pH value of P. brassicae, promoting its germination in soil, which leads to the death of zoospores without suitable live hosts, thereby reducing the content of soil P. brassicae and the occurrence of diseases.
The root exudates of non-host crops induce the germination of resting spores, resulting in the death of zoospores because of the absence of host plants. Many compounds in root exudates, such as carbohydrates, nucleosides, auxins, enzymes, and phenols, are capable of providing effective carbon and nitrogen sources for pathogens. Nitrogen, sodium and manganese can inhibit, while boron can stimulate the germination of resting spores. Sterols, especially sterols secreted by roots, can play a certain role in the germination of P. brassicae resting spores. Some hydrolyzates, such as trans-2-hydroxy-3-butene glucosinolate and 4-hydroxy-3-indolemethyl glucosinolate, can also stimulate the germination of resting spores [38]. The quantity and quality of root exudates depend on the plant species, the age of individual plants, and external factors, such as biotic or abiotic stress sources. Environmental factors, such as temperature, light, and soil moisture, also regulate the processes of root exudation [39]; for example, the root exudates of radish stimulate the germination of resting spores [40], while the root exudates of potato onions have an inhibitory effect on the germination of resting spores [41]. The next step should be to analyze the composition of the root exudates of previous crops and study their effect on the germination of dormant spores. A component analysis of the root exudates of preceding crops and their effects on the germination of resting spores should be carried out in further experiments.
The preceding crops caused changes in the soil microorganisms, which altered the ecological environment of the soil, enhancing the resistance of the plants to pathogenic bacteria [42,43]. Through high-throughput sequencing, it was found that the three preceding crops increased the richness and diversity of the soil bacteria and decreased the richness and diversity of the fungi after harvesting. The proteins related to the plant disease process are differentially expressed when plants are infected by pathogenic bacteria, which improves the resistance of crops to pathogenic bacteria [44]. Through the correlation analysis between the content of the P. brassicae resting spores and the soil microorganisms, it was found that the bacteria OTU 9 and OTU 39 were significantly negatively correlated with the content of the resting spores. OTU 9 belongs to Lysobacter whose relative abundance in the control was significantly lower than in the other treatments. Lysobacter can produce a large number of active secondary metabolites and extracellular enzymes, which can act on the cell walls of pathogenic bacteria to dissolve or inhibit them [45]. Lysobacter capsici AZ78 has toxic activity against the sporangia of Phytophthora infestans and Plasmopara viticola [46]; the metabolites of Lysobacter capsici YS1215 have root-knot nematode active substances [45]. Recent studies have demonstrated that Lysobacter can produce antifungal compounds that inhibit the reproduction of P. brassicae resting spores and alleviate clubroot disease [47]. OTU 1, OTU 3, OTU 8, OTU 17, and OTU 22 which all belong to Sphingomonas spp., were significantly positively correlated with the content of the resting spores, and their numbers were significantly decreased after the planting of the preceding crops. Sphingomonas is often used to protect plants against microbial disease as a biological control agent through substrate competition. This indicates that some species of Sphingomonas spp. might have a synergistic effect with P. brassicae; this needs to be verified in later studies. Sphingomonas strains protected Arabidopsis thaliana against leaf pathogenic Pseudomonas syringae in a controlled model system [48]. OTU 39 and OTU 92 belong to the Gemmatimonadaceae family, the genus was unclassified.
Among the fungi, OTU9, OTU15 and OTU 31 belonged to Ascomycota. Ascomycetes are mainly saprophytes, which can decompose organic matter into nutrients that can be absorbed by crops and decompose cellulose and lignin in soil [49]. OTU 27 belongs to Basidiomycota, which easily decomposes lignin with a high carbon-to-nitrogen ratio; increases in lignin content increase its relative abundance [50]. Based on this, we speculated that the Ascomycota and Basidiomycota were probably involved in promoting the germination of the resting spores of P. brassicae.

Conclusions
Overall, different rotations have different effects on plants and soil. Compared with the control, the rotation of the potato onion, bean and wheat treatments significantly reduced the clubroot disease index, promoted the growth of the Chinese cabbage, increased the yield of the Chinese cabbage, and improved soil chemistry. In addition, rotation decreased the content of the pathogenic bacteria in the soil, increased the richness and diversity of bacteria, and decreased the richness and diversity of fungi. This experiment shows that the rotation of non-cruciferous previous crops and cruciferous crops is an important strategy to improve the sustainable development of agriculture. Additional work is needed to explore the specific disease-inhibiting mechanism behind and the long-term effects of rotation on soil pathogens and other microorganisms.
Supplementary Materials: The following supporting information can be downloaded at: https: //www.mdpi.com/article/10.3390/microorganisms10040799/s1, Table S1: Diversity indices of soil microbial communities based on 16S rRNA and ITS genus were analyzed using Illumina MiSeq sequencing at a 97% sequence similarity; Table S2: Relative abundances of dominant bacterial phyla in soil; Table S3: Relative abundances of dominant fungal phyla in soil; Table S4: Relative abundances of dominant bacterial classes in soil; Table S5: Relative abundances of dominant fungal classes in soil; Table S6: Mantel test analysis of soil properties and bacterial and fungal community structures of the preceding crops during harvest.