YANG Xiao-xiang , HUANG Xiao-qin , WU Wen-xian XlANG Yun-jia DU Lei ZHANG Lei , LlU Yong

1 Institute of Plant Protection, Sichuan Academy of Agricultural Sciences, Chengdu 610066, P.R.China

2 Key Laboratory of Integrated Pest Management on Crops in Southwest, Ministry of Agriculture and Rural Affairs, Chengdu 610066, P.R.China

Abstract Clubroot disease, caused by Plasmodiophora brassicae, is one of the most destructive soil-borne diseases in cruciferous crops worldwide. New strategies are urgently needed to control this disease, as no effective disease-resistant varieties or chemical control agents exist. Previously, we found that the incidence rate and disease index of clubroot in oilseed rape decreased by 50 and 40%, respectively, when oilseed rape was planted after soybean. In order to understand how different rotation patterns affect the occurrence of clubroot in oilseed rape, high-throughput sequencing was used to analyze the rhizosphere microbial community of oilseed rape planted after leguminous (soybean, clover), gramineous (rice, maize) and cruciferous (oilseed rape, Chinese cabbage) crops. Results showed that planting soybeans before oilseed rape significantly increased the population density of microbes that could inhibit P. brassicae (e.g., Sphingomonas, Bacillus, Streptomyces and Trichoderma). Conversely, consecutive cultivation of cruciferous crops significantly accumulated plant pathogens,including P. brassicae, Olpidium and Colletotrichum (P＜0.05). These results will help to develop the most effective rotation pattern for reducing clubroot damage.

Keywords: oilseed rape, clubroot, Gramineae, Leguminosae, Cruciferae, rhizosphere soil

1. lntroduction

Clubroot is a serious global disease in cruciferous crops that is caused by the soil-borne protist Plasmodiophora brassicae (Hwang et al. 2012; Chen et al. 2016). Clubroot itself severely disrupts the host root by forming finger, bar or spherical galls in host roots and restricting the uptake of water and nutrients from the soil into host plants (He et al. 2019). The disease causes substantial damage to quality and yield, accounting for up to 10-15% loss in global cruciferous crop production (Dixon 2009; Li et al. 2013); it is a major threat to the cruciferous crop production industry in China (Chai et al. 2010). The resting spores of P. brassicae can survive up to 20 years in the soil (Kageyama and Asano 2009). Some methods for managing clubroot- such as increasing the soil pH by liming or killing microorganisms with agrochemicals - may damage the soil ecosystem,which can cause environmental pollution and create food quality problems (Murakami et al. 2002; Kowata-Dresch and May-De Mio 2012). Therefore, it is an urgent to find alternative strategies for controlling clubroot in cruciferous crops.

The plant rhizosphere is an important soil ecological environment for plant-microorganism interactions (Luo et al.2017). The microorganisms in rhizosphere soil play a very important role in plant growth and disease occurrence(Zhang et al. 2015; Jian et al. 2018; Li and Liu 2019).The species, composition and diversity of rhizospheric microorganisms are greatly affected by different plant root exudates (Bais et al. 2006; Zhou et al. 2011). Among the enormous numbers of rhizosphere microorganisms,deleterious rhizozosphere strains are closely related to reducing production during continuous cropping. They inhibit plant growth by secreting plant toxins, competing nutrients and inhibiting mycorrhizal functions (Nehl et al.1996). Various growth-promoting rhizobacteria in the soil benefit plant growth by increasing nutrient supply to plants,inducing resistance and suppressing plant pathogens (Cui et al. 2019). Therefore, adopting reasonable crop rotation systems and making full use of the biocontrol potential of these beneficial microorganisms in agricultural ecosystems will help to reduce the impact of pesticides and fertilizers,lower the severity of plant diseases and insect pests,promote the growth of plants, and realize the safe, efficient and sustainable development of agriculture.

Previously, the occurrence of clubroot disease in Xindu,Pengzhou, Wenjiang and Guanghan of Sichuan Province in China was investigated by continuous observation for four years. The results showed that different crop rotation patterns had different effects on the incidence of clubroot in oilseed rape. When oilseed rape was planted after soybean (Soybean-Oilseed rape rotation), the incidence of clubroot in oilseed rape was significantly reduced, as compared to Oilseed rape-Oilseed rape and Rice-Oilseed rape rotation patterns. In order to identify the microbiological mechanism affecting the rotation pattern and incidence of clubroot disease, high-throughput sequencing was used to analyze the rhizosphere microbial community structure of oilseed rape planted after leguminous (soybean, clover),gramineous (rice, maize) and cruciferous (oilseed rape,Chinese cabbage) crops. The results from this study will help with developing the most effective rotation pattern for reducing the damage from clubroot disease.

2. Materials and methods

2.1. Experimental site and soil sampling

The experimental area is located in Pengzhou (30.98°N,103.93°E), a city located in the northern part of the subtropical humid climate zone of Sichuan basin, China.The average annual temperature is 15.9°C, and the annual average precipitation is about 867 mm. The cultivated soil is sandy loam, loose in structure and rich in organic matter;soil nutrients are relatively uniform. Eighteen plots, each 10 square meters, were established. Soybean, clover, rice,maize, oilseed rape and Chinese cabbage were planted in three plots, respectively, through the completely randomized block design.

Preceding crops (soybean, clover, corn, rice, oilseed rape and cabbage) were planted for 90 days, cutted and oilseed rape was planted with conventional plowing. Soil samples were collected from the rhizosphere of ten randomly selected oilseed rape in each plot 90 days after planting. Genomic DNA was extracted from 250 mg of rhizosphere soil using the Power Soil Kit (MolBio, United States), according to the manufacturer's instruction.

2.2. lnvestigation of clubroot disease in oilseed rape

The incidence and disease index of clubroot were investigated 90 days after planting with the following formula:

2.3. Microscopic analysis of resting spores

Soil samples were collected and air dried, and 20 mL of ddH2O was added to 500 mg of air-dried soil and mixed evenly with a blender for 1 min. The solution was filtered through 8-layers of cheese cloth and the filtrate transferred to a 15 mL centrifuge tube and centrifuged at 3 900×g for 15 min. The supernatant was discarded, and 6 mL of a 50% (w/v) sucrose solution was added to the pellet.

After 2 min of agitation, the suspension was centrifuged at 1 700×g for 5 min. The supernatant was transferred into a 50 mL centrifuge tube, mixed thoroughly with 45 mL of ddH2O and centrifuged at 3 900×g for 15 min. The supernatant was discarded and 5 mL of ddH2O added to the pellet and centrifuged at 3 900×g for 15 min. The final resting spore pellet was mixed with 2 mL of ddH2O and analyzed under a light microscope (400×) using a haemocytometer.

2.4. High-throughput amplicon sequencing and data processing

The 16S rRNA gene and ITS fragments were amplified using specific primers (515F (5′-GTGCCAGCMGCCGCGG-3′)and 806R (5′-GGACTACNVGGGTWTCTAA-3′)for 16S rRNA (Wagneret al. 2014), ITS5-1737(5′-GGAAGTAAAAGTCGTAACAAGG-3′) and ITS2-2043R(5′-GCTGCGTTCTTCATCGATGC-3′) for ITS (Luet al.2013)) tagged with a barcode. The 30 μL PCR reactions contained 15 μL of Phusion?High-Fidelity PCR Master Mix (New England Biolabs, United States), 0.2 μmol L-1of forward and reverse primers and approximately 10 ng of template DNA. The cycling conditions were 98°C for 1 min;30 cycles of 98°C for 15 s, 50°C for 30 s and 72°C for 30 s;and 72°C for 5 min. An equal volume of 1× loading buffer(containing SYBR green) was mixed with the PCR products and verified by 2% agarose gel electrophoresis. Amplicons were verified by 2% agarose gel electrophoresis, and PCR products were mixed in equidensity ratios and recovered using a gel recovery kit (QIAGEN, Germany). Sequencing libraries were generated using the TruSeq?DNA PCR-Free Sample Preparation Kit (Illumina, United States), according to the manufacturer's recommendations. The library was quantified by Qubit and Q-PCR and sequenced on an Illumina HiSeq2500 PE250 platform by Beijing Novogene Bioinformatics Technology Co. Ltd. (China). The raw datas for 16S rRNA and ITS were stored in FASTQ format and deposited in the Sequence Read Archive (http://www.ncbi.nlm.nih.gov/sra/) under accession numbers SRP187389 and SRP187391, respectively.

The raw sequence datas were de-multiplexed, qualityfiltered and processed using FLASH (V1.2.7, http://ccb.jhu.edu/software/FLASH/) (Magoc and Salzberg 2011),as previously described in Zhaoet al(2017). The highthroughput amplicon sequencing were used to generate operational taxonomic units (OTUs) at 97% sequence similarity with UPARSE (Uparse v7.0.1001, http://drive5.com/uparse/) (Edgar 2013). The observed-species,abundance-based coverage estimator (ACE), Chao1,Shannon and goods-coverage were calculated with QIIME Software (version 1.7.0). The rarefaction curve corresponding to OTUs observed at different sequencing depths was examined using R Software (version 2.15.3) to determine whether the depth was reasonable.

3. Results

3.1. Effects of different rotation patterns on the occurrence of clubroot in oilseed rape

The effects of different rotation patterns on the occurrence of clubroot were investigated 90 days after oilseed rape was planted. Results showed that the incidence rate of clubroot was highest in Rice-Oilseed rape, Oilseed rape-Oilseed rape and Cabbage-Oilseed rape rotation patterns.However, when oilseed rape was planted after soybean,clover and maize, the incidence rate and disease index of clubroot decreased significantly (Table 1). Planting soybeans, in particular, before oilseed rape was most efficient in controlling clubroot, with the incidence rate and disease index decreasing by 50 and 40%, respectively.

3.2. Concentration of P. brassicae resting spores in the soil

Table 1 shows the density ofP.brassicaeresting spores in the soil analyzed under the microscope after each crop rotation pattern. A higher density ofP.brassicaeresting spores (＞6.0×106spores g-1soil) was found when oilseed rape was planted after oilseed rape or cabbage, while the lowest density (＜2.0×106spores g-1soil) was found when soybeans were planted before oilseed rape. These results reveal that a Soybean-Oilseed rape rotation could effectively reduce the resting spores ofP.brassicae.

3.3. Abundance and diversity of bacteria and fungi in rhizosphere soil of oilseed rape

In order to identify the possible microbiological mechanism that is affecting the order of crops on clubroot disease, highthroughput amplicon sequencing was used to analyze therhizosphere microbial community structure of oilseed rape planted after leguminous (soybean, clover), gramineous (rice,maize) and cruciferous (oilseed rape, Chinese cabbage)crops. The rarefaction curves of bacteria and fungi tended to flatten with increasing sequencing data. Sequencing coverage ranged from 99.13 to 99.80% (Table 2; Fig. 1),indicating the sequencing depth was sufficient, the data volume reasonable, which could truly reflect the an accurate community composition of rhizosphere soil microorganism can be reflected. The bacterial OTUs in the rhizosphere of oilseed rape planted after soybean (Soybean-Oilseed rape, 3 667), oilseed rape (Oilseed rape-Oilseed rape,3 572), maize (Maize-Oilseed rape, 3 561) and rice (Rice-Oilseed rape, 3 524) were significantly higher than that of Chinese cabbage (Cabbage-Oilseed rape, 3 325) and clover(Clover-Oilseed rape, 3 224). There was no significant difference in the fungal OTUs of oilseed rape among the different rotation patterns.

Table 1 The effects of crop rotation patterns on the occurrence of clubroot and concentration of Plasmodiophora brassicae resting spores in soil

The ACE, Chao1 and Shannon index can reflex the abundance and diversity of microorganism. The abundance and diversity of bacteria in the rhizosphere of Soybean-Oilseed rape were the highest, followed by Oilseed rape-Oilseed rape, Rice-Oilseed rape, Maize-Oilseed rape and Cabbage-Oilseed rape. There were no significant differences in the fungal ACE, Chao1 and Shannon indices for the different rotation patterns (Table 2). These results showed that the diversity of fungi was less affected by crop rotation, while the abundance and diversity of bacteria in rhizosphere soil decreased when oilseed rape was planted after clover.

Table 2 The abundance and diversity of bacteria and fungi in the rhizosphere soil of each crop rotation pattern1)

Fig. 1 Rarefaction curve analysis in each rhizosphere soil of bacteria (A) and fungi (B). Data are mean±SD (n=3).

3.4. Composition of bacterial community in rhizosphere

Fig. 2 shows that the 10 most abundant bacterial phyla based on 16S rRNA sequence reads of all samples were Proteobacteria (39.29-48.32%), Chloroflexi (5.90-12.59%), Bacteroidetes (9.65-11.98%), Actinobacteria(6.05-10.00%), Gemmatimonadetes (6.37-7.83%),Acidobacteria (5.07-5.89%), Firmicutes (2.25-3.78%),Verrucomicrobia (1.33-2.55%), Planctomycetes (1.00-1.89%) and Oxyphotobacteria (0.13-0.87%). Bacteroidetes,Gemmatimonadetes, Actinobacteria, Firmicutes,Verrucomicrobia and Planctomycetes showed no significant differences in the rhizosphere of oilseed rape among the different rotation patterns. Chloroflexi and Actinobacteria were significantly more abundant in the rhizosphere of Soybean-Oilseed rape, while Proteobacteria was the lowest. Oxyphothe obacteria was lowest after planting cruciferous crops.

Fig. 2 Relative abundances at the phylum level of bacteria (A) and fungi (B). Pro, Proteobacteria; Chl, Chloroflexi; Bac, Bacteroidetes;Act, Actinobacteria; Gem, Gemmatimonadetes; Aci, Acidobacteria; Fir, Firmicutes; Ver, Verrucomicrobia; Pla, Planctomycetes;Oxy, Oxyphotobacteria; Mor, Mortierellomycota; Asc, Ascomycota; Muc, Mucoromycota; Olp, Olpidiomycota; Bas, Basidiomycota;Roz, Rozellomycota; Glo, Glomeromycota. Data are mean±SD (n=3). Different letters indicate significant differences among treatments at P＜0.05.

At the genus level (Fig. 3), Sphingomonas, Pseudomonas,Rhodanobacter, unidentified_Gammaproteobacteria and Gemmatimonas were the dominant genera (average relative abundances＞1%), followed by Flavobacterium, unidentified_Anaerolineae,Lysobacter,HaliangiumandThermomonas.Sphingomonaswas the most abundant, accounting for 5.24-12.13% of the total reads in all samples. The bacterial abundance differed among all rhizosphere soils for all bacteria except unidentified_Gammaproteobacteria andGemmatimonas.Sphingomonaswas significantly more abundant than all other samples when planted after soybeans and clover.

Fig. 3 Relative abundances at the genus level of bacteria (A) and fungi (B). Sph, Sphingomonas; Pse, Pseudomonas; Rho,Rhodanobacter; Gam, unidentified_Gammaproteobacteria; Gem, Gemmatimonas; Fla, Flavobacterium; Ana, unidentified_Anaerolineae; Lys, Lysobacter; Hal, Haliangium; The, Thermomonas; Mor, Mortierella; Fus, Fusarium; Xer, Xeromyces; Olp,Olpidium; Act, Actinomucor; Pol, Polythrincium; Tra, Trapelia; Mor, unidentified_Mortierellomycota; Plu, Pluteus; Tal,Talaromyces.Data are mean±SD (n=3). Different letters indicate significant differences among treatments at P＜0.05.

3.5. Composition of fungal community in rhizosphere

At the phylum level (Fig. 2), Mortierellomycota (37.65-54.38%), Ascomycota (9.41-18.45%), Mucoromycota (2.09-6.29%), Olpidiomycota (0.51-5.82%) and Basidiomycota(0.79-1.28%) were dominant. Olpidiomycota did not change significantly among the different rhizosphere soil samples.Mortierellomycota was significantly more abundant in Rice-Oilseed rape and Ascomycota in Soybean or Clover-Oilseed rape, while Mucoromycota and Basidiomycota were significantly lower in Clover or Oilseed rape-Oilseed rape.

At the genus level (Fig. 3), the 10 most abundant fungal genera wereMortierella,Fusarium,Xeromyces,Polythrincium, Olpidium, Actinomucor, Trapelia, unidentified_Mortierellomycota, Pluteus and Talaromyces. Olpidium was three times more abundant in the rhizosphere of oilseed rape after planting cruciferous crops, but lowest in Soybean-Oilseed rape. Polythrincium was significantly higher in the rhizosphere of oilseed rape planted after leguminous plants.

3.6. Bacteria and fungi classification statistics based on its function

Among the highly abundant bacteria and fungi, nine bacterial and four fungal genera have been attributed to controlling biological functions or promoting plant growth,two bacterial and eight fungal genera to causing plant disease and two bacterial and two fungal genera beneficial to plant growth (Fig. 4). Bacterial and fungal genera with biocontrol and biopromoting functions were much greater in Soybean-Oilseed rape, while plant disease causing genera were lowest in all samples. On the contrary, when cruciferous crops were planted, biocontrol and biopromoting genera were the lowest and disease inducing highest in the rhizosphere soil of oilseed rape.

4. Discussion

Clubroot disease is rampant in China, seriously affecting the industrial development of cruciferous crops. Studies have proven that oilseed rape planted after some non-cruciferous plants (e.g., oat and ryegrass) could reduce clubroot disease in cruciferous crops (Friberg et al. 2006). However, the mechanism controlling the presence of clubroot in plants and the transmission of P. brassicae is largely unclear. And it is clearly impractical to plant oat or ryegrass in areas that mainly produce cruciferous crops in China. Previously, we found that clubroot disease in cruciferous crops was reduced when soybeans were planted beforehand. As soybean is an important cash crop in China, controlling clubroot disease by rotating soybean and oilseed rape is a feasible option.

Fig. 4 Bacteria and fungi classification statistics based on its function. I, biological control or plant growth promotion; II, disease inducing; III, others.

Rhizosphere soil microorganisms are important for plant growth and disease occurrence. The species of rhizosphere soil microorganism are greatly affected by plant root exudates. High-throughput sequencing was used to analyze the rhizosphere microbes of oilseed rape to help understand the microbiological mechanism behind different preceding crops and the occurrence of clubroot in oilseed rape. The ultimate goal is to identify microbial resources that can control this disease.

Results showed that the rhizosphere soil in all samples had the same dominant phylum groups. The most abundant phyla were Proteobacteria, Chloroflexi,Bacteroidetes, Actinobacteria, Mortierellomycota,Ascomycota, Mucoromycota and Olpidiomycota. Soybean-Oilseed rape contained many microbial genera in the rhizosphere soil with biocontrol and biopromoting potential(e.g., Sphingomonas, Flavobacterium, Clonostachys,Streptomyces, Chitinophage, Rhizobium, Bradyrhizobium,Trichoderma and Glomus) (Zhao et al. 2017). One specific microbe, Sphingomonas, is often used as a biological control agent to protect plants against fungal (Wachowska et al. 2013) and bacterial disease (Innerebner et al. 2011)through substrate competition. Sphingomonas can also produce plant growth-stimulating factors (Enya et al.2007). Moreover, we found in our unpublished article that Sphingomonas strain SPH24 produced phytosphingosine,which has a remarkable inhibitory effect on clubroot disease.Studies have also found that Bacillus and Streptomyces have excellent biocontrol potential against clubroot. Bacillus XF-1 had strong inhibitory effects on both the survival and germination of resting spores of P. brassicae (He et al.2019). Streptomyces strain 3-10 can produce anti-clubroot active substances in oilseed rape (Shakeel et al. 2016).Trichoderma, a biocontrolling fungi, can be used not only for preventing and treating plant diseases caused by various filamentous fungi, but also for controlling clubroot (Cheah et al. 2000).

Soybean-Oilseed rape had more AM fungi and Rhizobium in the rhizosphere soil than other rhizosphere soils. AM fungi can promote plant growth and plant absorption of phosphorus (Bona et al. 2015), manage soil-borne diseases and induce plant disease resistance, while Rhizobium can alleviate the symptoms of clubroot disease (Zhao et al.2017). Additionally, as the cell wall of P. brassicae resting spore is rich in chitin, all beneficial microorganisms with chitinase production (e.g., Chitinophaga) can become potential antagonistic microorganisms of P. brassicae (Jin et al. 2006). However, the rhizosphere soil of cruciferous crops not only had a lower population density of beneficial microorganisms, but it also had significantly higher plant pathogens, including P. brassicae. Planting gramineous crops as the preceding crop reduced the disease index of clubroot disease in oilseed rape when compared with planting cruciferous crops. However, microorganisms with anti-clubroot potential in the rhizosphere soil of gramineous crops-oilseed rape were relatively less than Soybean-Oilseed rape. As the root resting spores of P. brassicae can survive in the soil for many years and spread with water,the gramineous-cruciferous crop rotation system- or the classic paddy-upland rotation (Rice-Oilseed rape rotation)- would not help manage clubroot disease in cruciferous crop. On the contrary, this rotation system may lead to the resting spores of P. brassicae spreading with irrigation water.

Soybeans and clover as preceding plants may help reduce the incidence of clubroot. It is not practical to plant clover in the main cruciferous producing areas of China.However, soybean is an important economic crop, making the Soybean-Oilseed rape rotation system feasible for managing clubroot disease in oilseed rape.

5. Conclusion

High-throughput sequencing was used to explain the microbiological mechanism behind how the preceding crop influenced clubroot disease in oilseed rape. Results showed that soybeans as the preceding crop can significantly promote the presence of beneficial microorganisms, while cruciferous crops significantly accumulate plant pathogens.Future studies with isolate the beneficial microorganisms in the soybean rhizosphere soil that help to control clubroot.

Acknowledgements

This work was supported by the National Key Research and Development Program of China (2017YFD0200600),the Financial Innovation Capacity Enhancement Project in Sichuan Province, China (2019QNJJ-011), and the National Modern Agricultural Industry Technology System of Sichuan Rape Innovation Team, China (2019-2023).