Improvement of Straw Changed Soil Microbial Flora Composition and Suppressed Chinese Cabbage ( Brassica rapa L. ssp. pekinensis ) Clubroot Disease

: Straw incorporation is known as an environmentally friendly agricultural practice that can effectively enhance soil nutrient contents and crop yields; its potential to suppress soil-borne disease has also been reported in recent years. Here, we perform a ﬁeld experiment for two consecutive years (2017–2018) to evaluate the effectiveness of maize ( Zea mays ), rice ( Oryzae sativa L.) and wheat ( Triticum aestivum L.) straws incorporation in alleviating Chinese cabbage ( Brassica rapa L. ssp. pekinensis ) clubroot disease caused by Plasmodiophora brassicae Woronin. Microbial composition in Chinese cabbage rhizosphere and soil P. brassicae abundance were estimated by high-throughput amplicon sequencing and quantitative polymerase chain reaction (qPCR). Results showed that, during the two-year ﬁeld experimental cycle, all three straw amendments promoted Chinese cabbage plant growth, inhibited clubroot disease and increased the alpha diversity of the bacterial community in Chinese cabbage rhizosphere. Rice and wheat straws also increased the alpha diversity of the fungal community. These straws diversiﬁed the composition of the Chinese cabbage rhizosphere microbial community. All three straws promoted Cryptococcus carnescens ; both rice and wheat straws stimulated Lysobacter sp.; maize straw boosted Sphingomonas sp. and wheat straw increased Talaromyces sp. These microbial taxa are either considered to have positive inﬂuences on plant growth or potential biocontrol effects. In addition, straw amendments also increased soil pH, electrical conductivity, available nitrogen and available potassium contents in both years of the ﬁeld experiment. Taken together, we concluded that these three gramineous straw amendments ameliorated Chinese cabbage rhizosphere microorganisms, inhibited clubroot disease and promoted the growth of Chinese cabbage, and that rice straw worked best amongst the three. This study could potentially provide a new tactic of massive grain crop straw utilization and a direction in dealing with clubroot disease.


Introduction
Clubroot, caused by Plasmodiophora brassicae, is a soil-borne disease which has been a severe threat to cruciferous production worldwide since the 19th century [1].In China, approximately 3.2-4.0ha cruciferous crops suffered annually, leading to a major yield loss or even total crop failure [2].P. brassicae is very well-fitted for successful life with wellprotected, and apparently long-lived, resting spores [3].Once deposited, those spores can persist in the soil for over 20 years, thus making the field no longer suitable for cruciferous cropping [4].Furthermore, the commonly applied continuous monocropping practice critically aggravated the incidence of clubroot disease [5].Previous studies showed that the vitality of P. brassicae resting spores are hugely boosted in acidic pH soil (5.5-6.5)[6][7][8].In addition, soil organic matter and the content of calcium, silicon and boron were reported to have an impact on the viability of the resting spores as well [9][10][11].
Several practices have been put forward to alleviate clubroot, yet none are currently satisfactory.Chemicals agents such as cyazofamid, chlorothalonil, carbendazol and fluazinam could lead to unpredictable, long-lasting negative ecological impacts.Resistance breeding is not always effective due to variation in physiological race [12,13].Studies have also been carried out in biocontrol agents against P. brassicae such as Bacillus and Streptomyces, which have been reported to have suppression effects, while the instability of soil colonization became an obstacle for the application of these antagonizing microorganisms [14][15][16][17][18].Even so, plant-microbial interaction is still considered a promising scenario to suppress soil-borne disease and improve plant fitness [19,20].
Crop straw was often regarded as a by-product during agricultural production with insufficient utilization [21].However, crop straw is a considerably underestimated organic carbon source with potential soil ecological functions [22].During the process of straw returning, the decomposition of straw in soil influences the dynamic aggregation process of soil organic matter and other nutrients [23][24][25].It is reported that straw incorporation contributed to soil nitrogen content and availability [26,27].Soil available potassium content was also observed to increase in rice and oil seed rape cropping systems, according to Sui et al. and Zhu et al. [28,29].The enhancement of soil organic carbon content was also widely reported in diverse cropping systems with straw incorporation adopted [30][31][32].In these cases, straw returning is an effective method to enhance agricultural sustainability [33].Moreover, returning straw to the cropping field can alter soil microorganisms [34][35][36], of which some alterations have proven to be beneficial to plant fitness [37][38][39][40].It has also been reported in recent years that straw incorporation has a positive effect on mitigating soil sickness and controlling soil-borne disease [41].Research indicated that wheat straw incorporation alleviated Fusarium wilt of watermelon (Citrullus lanatus (Thunb.)Matsum.& Nakai); root and stem rot of cucumber (Cucumis sativus L.) could be inhibited by lettuce (Lactuca sativa L. var.capitata) soil amendment; and Jerusalem artichoke (Helianthus tuberosus L.) straw suppressed tomato (Solanum lycopersicum L.) root-knot nematodes disease [42][43][44].However, the effect of grain crop straws on clubroot disease remains unclear so far.Therefore, we aimed to test the effect of straw amendment on the plant performance and clubroot disease incidence of Chinese cabbage (Brassica rapa L. ssp.pekinensis) through a two-year consecutive field experiment.We hypothesized that straw amendment could alter soil properties and shift the soil microbial community, hence improving plant performance and suppressing clubroot disease of Chinese cabbage.To test our hypothesis, we (i) determined the effect of straw amendments on the inoculum density of P. brassicae in Chinese cabbage continuous cropping field; (ii) studied the alteration in the rhizosphere microbiome of Chinese cabbage plants upon straw incorporation; (iii) tested the soil properties that closely related to the life cycle of P. brassicae (Table 1).
Table 1.Effects of straw amendments on soil chemical properties including soil organic matter (SOM), available nitrogen (AN), available phosphorus (AP), available potassium (AK), pH and electrical conductivity (EC).CK, M, R and W represent the treatments of control, maize, rice and wheat straw incorporation, respectively.Different letters indicate significant differences among treatments based on Tukey's HSD test (p < 0.05).

Study Site
The experiment site was located in Acheng, Harbin, China (126 • 59 E, 45 • 29 N), where Chinese cabbage has been continuously cultivated for two decades and there is a serious infestation of P. brassicae.The soil of the field was classified as black soil (Mollisol), containing 37.60 g•kg −1 of organic matter, 169.22 mg•kg −1 of available nitrogen (ammonium and nitrate), 102.25 mg•kg −1 of available phosphorus, 156.81 mg•kg −1 of available potassium, pH of 6.15 and electrical conductivity (EC) of 0.26 mS•cm −1 .

Chinese Cabbage Seedling and Crop Straws Preparation
The Chinese cabbage cultivar chosen for this study was Gailiangdongbai 1 (susceptible to P. brassicae), which was provided by the Cabbage Breeding Laboratory of Northeast Agricultural University (Harbin, China).Straws of maize (Zea mays), rice (Oryza sativa L.) and wheat (Triticum aestivum L.) were selected as the experiment materials.Those straws were collected from conventional production fields, naturally dried in shade and mechanical crushed into 1-3 cm pieces for further use.

Field Experimental Design
The two-consecutive-year study was conducted in 2017 and 2018 from mid-July to early October, following the local cropping routine for Chinese cabbage.The experimental field plots were arranged through randomized block design.Four treatments were employed, including three straw application groups and a control group with no straw addition.Among them, maize straw application treatment was symbolized as (M), rice straw application group as (R), wheat straw application group as (W) and (CK) representing the control group.Therefore, twelve plots were selected with three replicates for each treatment, each experimental plot (240 cm × 400 cm) with 60 × 60 cm plant spacing.In straw-addition groups, three kinds of straws were mixed with 0-20 cm top soil layer respectively (2.4 kg each plot) before sowing.A compound fertilizer (N, 18%; P 2 O 5 , 46%; 450 kg•ha −1 ) was applied for fertilization before the plantation and urea (375 kg•ha −1 ) was applied in late August according to local recommendations.Irrigation in the cultivation process was timed on the basis of the rainfall situation.

Measurement of Clubroot Disease Incidence and the Plant Biomass
After 30 days of sowing, ten Chinese cabbage plants were collected randomly from each of the twelve experimental plots for clubroot disease investigation.The disease severity was measured by the following calculation method [14,45] The remaining Chinese cabbage plants were harvested 60 days after sowing, and plants of each treatment in each replicate were oven-dried at 60 °C to constant weight for biomass measurement.

Soil Sampling and Chemical Properties Analysis
Triplicate rhizosphere soils were collected 30 days after sowing from 10 plants of each replicate and were mixed to make a composite sample as previously described [46].Therefore, there were three composite samples for each treatment.Soils tightly adhering to the roots were considered rhizosphere soils.Soil samples were sieved through 2 mm mesh to remove large stones and plant residues.One part of these soils was stored at −80 °C for microbial determination, the other part was naturally dried in shade for chemical analysis.Soil organic matter was extracted by K 2 Cr 2 O 7 and H 2 SO 4 and titrated with FeSO 4 •7H 2 O. Soil available nitrogen, available phosphorus and available potassium were measured with a continuous flow analyzer (San++, Skalar Analytical B.V., Breda, The Netherlands).Soil pH and electrical conductivity were determined with a pH meter and conductivity meter after shaking the soil-water suspension at a soil/water ratio of 1:5 for 30 min [47,48].

Soil DNA Extraction and Quantitative PCR Analysis
Total rhizosphere soil DNA was extracted with PowerSoil DNA Isolation Kit (MO BIO Laboratories Inc., Carlsbad, CA, USA) from 0.25 g soil following the manufacturer's protocol.The absolute P. brassicae abundance in the Chinese cabbage rhizosphere soil was determined by quantitative PCR assays with the primer set PbF/PbR, which targets the internal transcribed spacer (ITS) region of P. brassicae [49].Quantitative PCR assays were conducted with a qTOWER 3 real-time PCR system (Analytik Jena, Jena, Germany).Detailed methods as previously described [50].

Amplicon Sequencing and Data Processing
Soil total community composition was analyzed with Illumina MiSeq sequencing as previously described [51].The bacterial and fungal communities were analyzed by sequencing of the V4-V5 regions of bacterial 16S rRNA genes and fungal ITS1 region, using the primer set 515F/907R [52] and ITS1F/ITS2 [53].The forward and reverse primers had a 6-bp barcode unique to identify each sample.Soil DNA samples were amplified in triplicate (including two negative control reactions) and the amplicons were pooled as previously suggested [54].Purified amplicons were pooled in equimolar and paired-end sequences (2 × 300 bp) on an Illumina Miseq platform at Majorbio Bio-Pharm Technology Co., Ltd., Shanghai, China.
The de-multiplexed and quality-filtered control of sequence reads were conducted with Quantitative Insights Into Microbial Ecology (QIIME) 1.7.0 [55].The cluster of operational taxonomic units (OTUs) was generated at the 97% sequence similarity threshold with Usearch (version 7.0) [56].The representative sequence of each OTU was taxonomically classified with the Ribosomal Database Project (RDP) classifier based on the SILVA database, with a confidence threshold value of 0.8 [57,58].The raw data was deposited in the NCBI Sequence Read Archive database with the submission accession Number SRP126988.

Statistical Analysis
The alpha diversity indices were generated in QIIME [55].The beta diversities were analyzed and visualized using principal coordinates analysis (PCoA) based on the Bray-Curtis distance, using Vegan and ggplot2 packages in the R project (v 4.1.0).The Spearman correlation coefficient was calculated to determine the relationship among soil factors, abundance of P. brassicae and relative abundance of microbes.The correlation between soil properties and soil microbial communities was analyzed by redundancy analysis (RDA).Data were analyzed with one-way analysis of variance (ANOVA) following Tukey's honestly significant difference (HSD) test.The data means were considered significantly different at p < 0.05.

Chinese Cabbage Plant Performance and Clubroot Disease Incidence
Chinese cabbage treated with straw showed better performances in seedling survival rate and plant growth (Tukey's HSD test, p < 0.05) (Figure 1a).Moreover, the disease rate and disease index were lower in the straw amendment groups compared with the control.Real-time PCR results showed lower abundance of rhizosphere P. brassicae in those straw treatments than that of the control group.(Figure 1b).An obvious promoting effect of straw amendments on Chinese cabbage root fitness can also be observed during the field experimental period (Figure 1c).

Soil Chemical Properties
In straw amendment groups, major increases can be found in soil organic matter and soil available potassium.The value of pH and electrical conductivity were higher in the soils amended with rice and maize straw, whereas soil available phosphorus did not differ among control and treatments amended with straws (Table 1) The calculated LSD values showing the differences among treatments were listed thereafter (Table 2).Illumina Miseq sequencing generated 199,392 and 355,668 quality sequences of bacteria and fungi, respectively, across all the soil samples.Good's coverage reflects the captured diversity, with averages of 97.15% and 99.70% for bacterial and fungal communities, respectively.The rarefaction curves of all samples tended to reach the saturation plateau and Good's coverage was identified at 97% similarity (Figure S1a,b).Therefore, the number of sequences was sufficient to represent the diversity of Chinese cabbage rhizosphere microbial communities.
For alpha diversity of bacteria, the Shannon indices of all straw treatments were higher than the control, and the number of observed OTUs of wheat straw treatment was higher than control.(ANOVA, p < 0.05) (Figure 2a).For alpha diversity of fungi, higher Shannon indices could be seen in the treatments amended with rice and wheat compared with control.(ANOVA, p < 0.05) (Figure 2b).These results indicated that straw amendment could increase the diversity of bacterial and fungal communities in Chinese cabbage rhizosphere soil.
Principal coordinates analysis (PCoA) indicated that the rhizosphere bacterial communities of those three straw amendments were distinguished from the control; non-parametric multivariate statistical tests analyses demonstrated that straw amendments altered soil bacterial community composition among treatments (Adonis, R 2 = 0.651, p = 0.001) (Figure 2c).In addition, soil fungal community composition was also distinguished among treatments, and the differences were significant (Adonis, R 2 = 0.714, p = 0.001) (Figure 2d).

Microbial and Fungal Communities' Composition in Chinese Cabbage Rhizosphere
For bacteria, a total of 14,875 partial bacterial 16S rRNA gene sequences were obtained per sample.The sequences were cluster and map, as the standard of more than 97% of sequence similarity and 23,138 OTUs were clustered.Overall, 35 phyla, 75 classes and more than 528 genera were detected.Proteobacteria, Acidobacteria, Bacteroidetes, Actinobacteria and Chloroflexi were the dominant bacterial phyla, with average relative abundances higher than 5% across all samples (Figure 3a, Table S2).Compared to the control, the relative abundance of Proteobacteria significantly decreased, while Acidobacteria and Planctomycetes significantly increased in the soils amended with rice and wheat straws (p < 0.05).Differential abundance analysis with the limma approach showed that 101, 75 and 71 OTUs were enriched in maize, rice and wheat straw treatments, respectively, while 70, 89 and 77 OTUs were depleted compared with the control.It's noteworthy that OTU1695, belonging to Lysobacter sp., is enriched in both rice and wheat straw treatments (Figure 3c).

Microbial and Fungal Communities' Composition in Chinese Cabbage Rhizosphere
For bacteria, a total of 14,875 partial bacterial 16S rRNA gene sequences were obtained per sample.The sequences were cluster and map, as the standard of more than 97% of sequence similarity and 23,138 OTUs were clustered.Overall, 35 phyla, 75 classes and more than 528 genera were detected.Proteobacteria, Acidobacteria, Bacteroidetes, Actinobacteria and Chloroflexi were the dominant bacterial phyla, with average relative abundances higher than 5% across all samples (Figure 3a, Table S2).Compared to the control, the relative abundance of Proteobacteria significantly decreased, while Acidobacteria and Planctomycetes significantly increased in the soils amended with rice and wheat straws (p < 0.05).Differential abundance analysis with the limma approach showed that 101, 75 and 71 OTUs were enriched in maize, rice and wheat straw treatments, respectively, while 70, 89 and 77 OTUs were depleted compared with the control.It's noteworthy that OTU1695, belonging to Lysobacter sp., is enriched in both rice and wheat straw treatments (Figure 3c).For the fungal community, Illumina Miseq sequencing generated 437,197 high-quality fungal sequences with an average read length of 266 bp, and the number of sequences per sample ranged from 30,280 to 43,781.A total of 6190 operational taxonomic units (OTUs) were identified at 97% similarity.In total, 8 phyla, 26 classes, 246 genera and 980 OTUs were detected in all soil samples.Ascomycota was the most abundant phylum in each of the four treatments (43.27-60.02%)(Figure 3b, Table S2).Higher relative abundance of Basidiomycota and lower Zygomycota abundance can be observed in straw amendment treatments compared with the control (ANOVA, p < 0.05).Differential abundance analysis also found that, compared to the control, 41, 47 and 46 OTUs were enriched in maize, rice and wheat straw treatments, respectively, while the abundance of 40, 20 and 9 OTUs were decreased.Notably, OTU947, recognized as Cryptococcus carnescens, is found to be enriched in all three straw-amended treatments (Figure 3d).

Correlation Analysis
A correlation analysis was conducted between Chinese cabbage rhizosphere dominant bacterial and fungal phyla (relative abundance > 0.5%) and clubroot disease index using Spearman's correlation test (p < 0.05) (Figure 4).The results showed that nine phyla of microorganism were significantly related to the clubroot disease, including P. brassicae, among which Proteobacteria, Bacteroidetes, Zygomycota and P. brassicae were positively correlated, and Acidobacteria, Chloroflexi, Planctomycetes, Latescibacteria and Ascomycota were negatively correlated with the disease index (p < 0.05).Analysis was also performed with Spearman's correlation test (p < 0.05) to investigate the correlation between the abundance of P. brassicae and the dominant bacterial and fungal OTUs (relative abundance > 0.5%) in Chinese cabbage rhizosphere, identifying 10 bacterial OTUs and 9 fungal OTUs that were significantly correlated.Among them, three bacterial and five fungal OTUs were positively related with P. brassicae; seven bacterial and four fungal OTUs were related negatively (Tables S3 and S4).

Discussion
Soil-borne disease has always been a major problem of crop production, and causes considerable losses.It becomes even more difficult to deal with due to conventional longterm monocropping strategies [59].Accordingly, clubroot disease is ge ing worse in the nearby area of our field experimental site.In this study, we combined local cultivation practices and put forward the field experiment to test the effect of straw amendment on clubroot disease.We found that all three straw amendments improved the plant perfor-

Discussion
Soil-borne disease has always been a major problem of crop production, and causes considerable losses.It becomes even more difficult to deal with due to conventional longterm monocropping strategies [59].Accordingly, clubroot disease is getting worse in the nearby area of our field experimental site.In this study, we combined local cultivation practices and put forward the field experiment to test the effect of straw amendment on clubroot disease.We found that all three straw amendments improved the plant performance of Chinese cabbage and alleviated the incidence of clubroot.Our findings validate the previous viewpoint that straw amendments could very much suppress some of the soil-borne diseases and promote plant growth.For example, wheat straw and rice straw amendments inhibited Fusarium wilt disease in watermelon and banana (Musa spp.), respectively [41,43,60], and several straw amendments reduced root knot nematode severity in tomato [42].

Straw Incorporation Ameliorated Soil Properties and Contributed to Plant Performance and Mitigation of Clubroot Disease in Chinese Cabbage
As a widely adopted agricultural practice, straw incorporation is reported to be beneficial to the soil nutrient conditions [61][62][63].Our research observed an increase of soil total organic matter and available potassium contents during the two consecutive years of the field experiment, which is consistent with the previous study [64,65].Previous studies suggested that straw incorporation could contribute to plant performance; for instance, Brennan et al. reported an increased crop yield in winter wheat after straw incorporation [66], and comparable result could also be observed in maize and rice cropping with straw incorporation adopted [67][68][69][70].Our study also showed the growth-enhancement effect in the straw incorporation cropping system, which is in line with the previous studies.However, it is worth mentioning that the plant-growth promotion effect in this research could have resulted from the alleviation of the severe clubroot disease that significantly holds back the growth of Chinese cabbage.This could also explain the abnormal relevance between the root biomass and shoot biomass in this study that greater root biomass in the control group does not represent better plant performance, but indicates proliferating gall tissue with almost no ability of nutrient uptake, instead.In that case, additional studies are essential to further test the growth-promotion effect in Chinese cabbage with proper cropping soil.
Moreover, it has been reported that adding crop straw to soil can increase the pH value of the soil, thus ameliorating acidic soil; a similar effect was shown in each of the two experimental cycles in this study [71][72][73][74].Soil pH is considered a crucial factor that influences the viability of P. brassicae.It is generally acknowledged that the favorable edaphic environment for P. brassicae is acidic soil, and optimal pH for the occurrence and development of clubroot disease is 5.5~6.5 [75,76].Correspondingly, our study showed that soil pH significantly increased after amendment with all three kinds of straw in both years (>6.5), which makes the edaphic environment unsuitable for the life cycle of P. brassicae, which can explain the dramatic decline in the soil P. brassicae inoculum density and the incidence of clubroot disease in this study to some extent.Besides, our study spotted that soil organic matter contents, soil available potassium contents and soil electrical conductivity were also negatively correlated with the abundance of P. brassicae in Chinese cabbage rhizosphere along with soil pH values (Table S1).

Straw Amendment Altered Soil Microbial Communities and Contributed to the Suppression of Clubroot Disease
According to our Illumina MiSeq sequencing results, all three straw amendments increased the diversity of the bacterial community in the Chinese cabbage rhizosphere compared to the control group, with higher Shannon indices and number of OTUs observed (Figure 2a); rice and wheat straws also increased the diversity of the fungal community (Figure 2b), which corresponds with the idea that returning straw to the field could promote the richness and diversity of soil microbiota [24,77].It has been proven that higher soil microbial diversity plays a vital role in the ability of the community to resist pathogens and maintain stability [78,79].Consequently, the increase of bacterial community diversity in the Chinese cabbage rhizosphere may contribute to the reduced severity of clubroot in the treatments amended with straws in the current study.
According to the PCoA analysis of rhizosphere bacterial and fungal communities based on the Bray-Curtis dissimilarity matrix, a clear separation was displayed in bacterial communities among each treatment, demonstrating that the composition of Chinese cabbage rhizosphere's bacterial communities was distinctly shifted in all three straw amendment treatments (Figure 2c), which is consistent with the previous studies [80].Interestingly, only the rice straw amendment group showed a clear separation from the control group in fungal community (Figure 2d), which could probably explain the better disease-suppression effect of rice straw treatment than the other two gramineous straw amendments, according to previous studies [81][82][83].
Corresponding with previous studies [84,85], the current study also indicated differently enriched taxa in rhizosphere microbiomes between straw treatments and the control.Acidobacteria, Chloroflexi, Planctomycetes and Latescibacteria were the bacteria phyla; Ascomycota was the fungal phylum enriched in straw treatments compared to the control (relative abundances > 1.0%) (Figure 3a,b and Figure 4).In particular, several fungal taxa previously proven to be closely associated with straw decomposition, namely Aspergillus, Trichoderma and Coprinus, were enriched in the three straw amendment treatments in comparison with the control group [86][87][88] (Table S5).Furthermore, we also found a higher relative abundance of several pre-proven plant-beneficial microbes or biocontrol agents in the straw treatments, including Pedobacter [89], Halomonas [90], Steroidobacter [91], Rhizophlyctis [92], Preussia [93], Cryptococcus carnescens [94], Talaromyces [95], etc. (Figure 3c,d).Plant-beneficial microbes can inhibit plant-growth pathogens and induce systematic resistance reactions in plants, thus cutting down the severity of plant disease [96,97].Consequently, it is a potentially reasonable explanation to combine straw amendment with disease reduction, and in vitro experiments are necessary in order to further investigate the interactions between certain soil microbial taxa and assemblies.
Soil microbes could immobilize and buffer nitrogen, phosphorus and potassium, contributing to soil granular structure formation and the carbon cycle [98].Our redundancy analysis (RDA) showed the relationship between the soil microbial communities and environmental variables.The results indicated that the soil microbial communities of rice and wheat straw treatments were more affected by soil pH, whereas an obvious correlation was found between soil organic matter and the soil microbial communities of maize treatments, while available nitrogen is the main factor for the control group (Figure S2).Altogether, straw decomposition and soil microbe alteration work together on the soil ecological environment, and the dynamic balance needs to be investigated in further study.

Straw Amendment Reduced the Inoculum Density of P. brassicae
The inoculum density of P. brassicae is assumed to be a direct reflection of clubroot severity [99].Quantitative PCR (qPCR) analysis has become a routine approach to estimate P. brassicae spore inoculum load in soil [100].Clubroot incidence could be observed on highly susceptible cruciferous cultivars under the density of 10 3 resting spores per gram soil, and 10 6 resting spores per gram soil is generally considered as another threshold of inoculum density [101,102].Under a density of 10 6 spores per gram soil, almost all cruciferous plant will get infected, and merely differentiation in the degree of the disease could be observed [103,104].In our field experiment, the inoculum load is 10 7 resting spores per gram soil before the straw amending practice.Each of the three straw amendments significantly reduced the inoculum density (Tukey's HSD test, p < 0.05).In particular, the rice straw amendment reduced the concentration of P. brassicae dramatically to 10 5 resting spores per gram soil.The count of the disease index was more or less in line with the qPCR result.Previous studies revealed the potential allelopathic effect of straws to alter the soil microorganisms [105,106], and even partially simulated crop rotation effects [2].Therefore, it is valid to consider that the decline of P. brassicae spores' density in soil in this field experiment resulted from certain allelochemicals in the straws or metabolites during the process of straw decomposition in the soil, which needs to be further investigated.

Conclusions
In summary, our study demonstrated that the application of crop straws as soil amendments improved the rhizosphere microbiome in Chinese cabbage and decreased the abundance of P. brassicae in particular.Moreover, straw incorporation also contributed to the soil's properties.Those combined factors may contribute to the growth-promotion and clubroot disease-suppression effect of straw application in this study.Taken together, our study puts forward a new tactic for clubroot disease management and provides a practical strategy of grain crop straw utilization in vegetable cropping, which could be an innovative application for gramineous straws in clubroot disease management and may become an enhancement to agricultural sustainability.However, long-term, replicated field experiments are still needed to further validate field performance, and further studies should be conducted to discover the specific mechanism between the rhizosphere microbiome and the occurrence of clubroot disease.It is also worthy to focus on the interaction between the pathogen P. brassicae and certain species diversely enriched in straw treatments, from which biological control agents against P. brassicae could potentially be discovered.

:
Disease Incidence (%) = Number of infected plants/ Total number of investigated plants × 100 Disease Index = (∑ (Number of infected plants at each disease stage × Relative value))/ (Total number of plants under investigation × Highest incidence of disease) × 100

Figure 1 .Figure 1 .
Figure 1.Plant performance and clubroot disease incidence in response to straw application.CK, M, R and W represent the treatments of no straw control, maize, rice and wheat straw amendment, Figure 1.Plant performance and clubroot disease incidence in response to straw application.CK, M, R and W represent the treatments of no straw control, maize, rice and wheat straw amendment, respectively.(a) Chinese cabbage plant dry biomass (shoot and root) and seedling survival rate after 60 days of sowing.(b) Clubroot disease incidence, disease index and P. brassicae abundance in Chinese cabbage rhizosphere soil.(c) Chinese cabbage root infection during the field experimental period.Different letters indicate significant differences among treatments based on Tukey's HSD test (p < 0.05).

Figure 2 .
Figure 2. Diversity and structure of Chinese cabbage rhizosphere bacterial and fungal communities under the incorporation of three gramineous crop straws.(a)The number of OTUs and α-diversity of bacterial and (b) fungal communities; (c) β-diversity of bacterial and (d) fungal communities.CK, M, R and W represent the treatments of control group, maize, rice and wheat straw treatments, respectively.Different le ers indicate significant differences among treatments based on Tukey's HSD test (p < 0.05).

Figure 2 .
Figure 2. Diversity and structure of Chinese cabbage rhizosphere bacterial and fungal communities under the incorporation of three gramineous crop straws.(a)The number of OTUs and α-diversity of bacterial and (b) fungal communities; (c) β-diversity of bacterial and (d) fungal communities.CK, M, R and W represent the treatments of control group, maize, rice and wheat straw treatments, respectively.Different letters indicate significant differences among treatments based on Tukey's HSD test (p < 0.05).

Figure 3 .
Figure 3. Effects of three gramineous straw amendments on the composition of bacterial and fungal communities in Chinese cabbage rhizosphere.(a) Relative abundances of dominant bacterial phyla and (b) fungal phyla in Chinese cabbage rhizosphere.Volcano plots demonstrating differentially enriched (c) bacteria and (d) fungi OTUs between straw amended soils and control.Bacterial and fungal phyla with average relative abundances >1.0% at least were shown.CK, M, R and W represent the treatments of control, maize, rice and wheat straw application, respectively.

Figure 3 .
Figure 3. Effects of three gramineous straw amendments on the composition of bacterial and fungal communities in Chinese cabbage rhizosphere.(a) Relative abundances of dominant bacterial phyla and (b) fungal phyla in Chinese cabbage rhizosphere.Volcano plots demonstrating differentially enriched (c) bacteria and (d) fungi OTUs between straw amended soils and control.Bacterial and fungal phyla with average relative abundances >1.0% at least were shown.CK, M, R and W represent the treatments of control, maize, rice and wheat straw application, respectively.

Table 2 .
Calculated LSD value of soil properties based on LSD test, including soil organic matter (SOM), available nitrogen (AN), available phosphorus (AP), available potassium (AK), pH and electrical conductivity (EC).CK, M, R and W represent the treatments of control, maize, rice and wheat straw incorporation, respectively.