Biochar amendment controlled bacterial wilt through changing soil chemical properties and microbial community

Microbiological Research 231 (2020) 126373

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Biochar amendment controlled bacterial wilt through changing soil chemical properties and microbial community

T

Shu Chen, Gaofu Qi, Gaoqiang Ma, Xiuyun Zhao* College of Life Science and Technology, Huazhong Agricultural University, Wuhan, 430070, China

A R T I C LE I N FO

A B S T R A C T

Keywords: Bacterial wilt Ralstonia solanacearum Biochar Soil properties Microbial community

Long-term continuous cropping has led to epidemic of bacterial wilt disease in Southern China. Bacterial wilt disease is caused by Ralstonia solanacearum and difficult to control. In order to control bacterial wilt, rice hull biochar was applied to soil with different doses (0, 7.5, 15, 30 and 45 t ha-1) in a field trial. After three years, the influence of biochar on soil properties, incidence of bacterial wilt and microbial community were characterized. Biochar amendment significantly suppressed bacterial wilt through changing soil chemical properties and microbial composition. Compared with control, disease incidence and index of biochar amendments (7.5, 15, 30, and 45 t ha-1) significantly decreased. Disease incidence and index of biochar amendment (15 t ha-1) were the lowest. Compared to the unamended control, contents of soil organic matter in biochar amendments (15, 30 t ha1 ), available nitrogen in biochar amendment (15 t ha-1), and urease activity in biochar amendments (7.5, 15 t ha1 ) significantly increased. Biochar amendments (15, 30, and 45 t ha-1) increased the relative abundances of potential beneficial bacteria (Aeromicrobium, Bacillus, Bradyrhizobium, Burkholderia, Chlorochromatium, Chthoniobacter, Corynebacterium, Geobacillus, Leptospirillum, Marisediminicola, Microvirga, Pseudoxanthomonas, Telmatobacter). Biochar amendments (7.5, 30, and 45 t ha-1) reduced the relative abundances of denitrifying bacteria (Noviherbaspirillum, Reyranella, Thermus). Biochar amendments (7.5, 15, and 45 t ha-1) significantly decreased pathogen Ralstonia abundance. Overall, application of biochar effectively controlled bacterial wilt through sequestering more carbon and nitrogen, enriching specific beneficial bacteria and decreasing pathogen abundance. This study revealed the potential of biochar in control of bacterial wilt.

1. Introduction Bacterial wilt disease is caused by Ralstonia solanacearum and results in severe economic loss (Mansfield et al., 2012). R. solanacearum is pathogenic on more than 200 plant species belonging to 54 different families (e.g. banana, eggplant, peanut, potato, tobacco, tomato). R. solanacearum enters the plant through the roots and colonizes the vascular system and ultimately leads to whole plant wilting and death (Genin and Denny, 2012). R. solanacearum can survive in soil for many years in the absence of susceptible crop and can spread through water, rhizosphere contact and farming. So it is difficult to control bacterial wilt disease (Van Elsas et al., 2000). Long-term continuous cropping has led to epidemic of bacterial wilt in China. Bacterial wilt disease has been reported in 30 provinces of China, especially in southern provinces, and has caused great economic losses in the recent years. In China, R. solanacearum infected more than 90 plant species belonging to 39 botanical families (Jiang et al., 2017; Li et al., 2017). For instance, in the southern provinces of the Yangtze

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River, disease incidence of tomato bacterial wilt ranged from 10 % to 80 % (Wei et al., 2015). Potatoes planted in more than 10 provinces of China are infested by bacterial wilt with estimated yield losses ranging from 10 to 100% (Chen et al., 2005). Tobacco bacterial wilt occurs in 14 out of the 22 tobacco growing regions, with 15–75% disease incidence, and causes 50–100% yield reduction (Liu et al., 2017). Bacterial wilt affects about 800,000 ha of peanut land in China and causes 10–100% yield losses (Yu et al., 2011). Therefore, bacterial wilt disease is one of the most important diseases in China due to its wide distribution and cumulative losses on many crops, ornamental and medicinal plants. There are still no effective strategies to control bacterial wilt. Currently, antimicrobial substances produced by bacteria and chemical pesticides (e.g. zinc thiazole, bismerthiazol, and saisentong) are the main methods used to control bacterial wilt, which have limited efficacy and can’t effectively prevent the occurrence of bacterial wilt (Bai et al., 2016). In addition, long-term use of chemical pesticides results in heavily environmental pollution and induces the emergence of pesticide-resistant pathogens (Fujiwara et al., 2011). Thus, there is

Corresponding author. E-mail address: [email protected] (X. Zhao).

https://doi.org/10.1016/j.micres.2019.126373 Received 5 July 2019; Received in revised form 18 October 2019; Accepted 10 November 2019 Available online 11 November 2019 0944-5013/ © 2019 Elsevier GmbH. All rights reserved.

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an urgent need to find alternative environmentally friendly measures to effectively control bacterial wilt disease in China. Biochar is produced via pyrolysis of biomass wastes (e.g. crop residues, manure, wood wastes etc.) (Sohi et al., 2010). Rice hull is an agricultural waste annually accumulating for a large amount in Southern China, and the disposal of rice hulls poses significant public health and environmental risks. In this study, biochar was prepared by rice hull for its recycling use and suppressing bacterial wilt. Biochar contains multiple nutrient elements such as carbon, nitrogen, phosphorus, potassium, calcium and magnesium (Cao and Harris, 2010; Huang et al., 2013). Biochar addition has been shown to improve soil physical and biochemical properties, suppress plant disease, improve plant growth and stimulate soil microbial activity (Elad et al., 2010; Graber et al., 2010; Lehmann et al., 2011). Therefore, biochar is widely used as a soil amendment. Studies have evidenced the potential for biochar in improving soil fertility and crop productivity through enhancing soil organic carbon storage, nutrients and moisture availability, ameliorating acidic soils, and stimulating microbial activity and diversity (Lehmann and Joseph, 2009; Gwenzi et al., 2015; Ippolito et al., 2015). Biochar amendments alter soil microbial biomass, activity and community composition (Graber et al., 2010; Xu et al., 2014; Jaiswal et al., 2017). Most importantly, biochar reduces plant disease severity and induces plant resistance to disease possibly by increasing nutrient retention, alleviating soil acidity, altering microbial community as well as a better soil structure (Elad et al., 2010; Graber et al., 2014; Jaiswal et al., 2017). For example, biochar helps to control cucumber damping-off caused by Rhizoctonia solani (Jaiswal et al., 2014). Biochar amendments are effective in inhibiting bacterial wilt and significantly reduce the disease index (Lu et al., 2016; Zhang et al., 2017). Therefore, biochar amendment can serve as a management tool for suppressing plant diseases (Lehmann and Joseph, 2009; Jaiswal et al., 2017). However, most trials about biochar application have been carried out in the laboratory over short time periods. The long-term effects (i.e. > 12 months) of biochar application on soil microbial community, soil biochemical properties and bacterial wilt had been poorly understood in field experiment (Jones et al., 2012). Effect of biochar on inhibiting plant disease varied with different doses of biochar applied (Jaiswal et al., 2014, 2015; Copley et al., 2015; Huang et al., 2015). In this study, action of different doses of biochar amendment treatments was compared in order to find the optimal dose of biochar for control of bacterial wilt disease. The experiment was set up in a bacterial wiltinfected tobacco field in Enshi State of Southern China. After amending biochar for three years, effect of different doses of biochar on incidence of bacterial wilt and soil properties and microbial communities were investigated. We hypothesized that application of biochar would alter soil properties and microbial abundance, with an increase in some beneficial bacteria, which would lead to a decrease in bacterial wilt incidence. Different doses of biochar would have different control efficiency on bacterial wilt disease. These study would find the optimal dose of biochar for effectively inhibit bacterial wilt.

the stretching vibration of -C = O carboxyl, and 1061 cm-1 representing phenolic hydroxyl group Ar−OH. SEM image showed that there were many microspores (< 1 μm in diameter) and small pores (1−5 μm in diameter) on the surface of biochar. 2.2. Biochar amendment field trials The field trial was established at Xuanen County, Hubei Province, China (29°97′N, 109°39′E). The replicated (n = 3) trial plots (10 m × 4 m) were laid out in a randomized block design in an existing flat agricultural field where tobaccos have been continuously planted during the last 20 years, and the outbreak of tobacco bacterial wilt disease was recorded in recent years. In March 2015, the site was ploughed, and biochar was spread on the surface soil at different rates of 0 (control, BC1), 7.5 (BC2), 15 (BC3), 30 (BC4) and 45 (BC5) t ha-1, respectively. The biochar was then harrowed into the topsoil (0−30 cm). In May 2015, 2016, and 2017, total 60 tobacco seedlings at six-leave stage were transplanted into each plot following standard farming practices, respectively. 2.3. Soils sampling and bacterial wilt recording Soil samples were collected in August 2017 (90 d after transplanting). At this time, bacterial wilt disease was at its peak. In each plot, soil sample was taken from 10 different sites, 100 g of soil was collected from each site, then mixed together to form one composite sample. The soil samples were used for analyzing soil chemical properties and enzymatic activities and microbial community. At the same time, tobacco height, stem circumference, the incidence and severity of bacterial wilt (n = 60 plants per plot) was recorded. Disease incidence was calculated by the percentage of diseased tobaccos. Disease index was evaluated using a disease score method: 0= no symptom; 1= less than a half of tobacco leaves are wilted; 3= one half to two-thirds of leaves are wilted; 5 = more than two thirds of leaves are wilted; 7 = all leaves are wilted; and 9 = stems collapsed or tobaccos are dead. Disease index was calculated using the formula (Yi et al., 2007): Disease index = [∑(r × N) / (n × R)] × 100 Where r is the disease severity, N is the number of infected tobaccos with a rating of r, n is the total number of tobaccos tested, and R is the value of the highest disease severity in each plot. 2.4. Analysis of soil chemical properties and enzyme activities Soil pH was determined using a 1:2.5 (w/v) soil : distilled water ratio. Contents of available potassium (AK), available phosphorus (AP), alkaline nitrogen (AN), and soil organic matter (SOM) were determined as the methods described previously (Wang et al., 2017). Activities of soil phosphatase, urease, invertase, and catalase activity were determined as the methods described previously (Tabatabai and Bremner, 1969; Sinha, 1972; Kandeler and Gerber, 1988; Schinner and Wvon, 1990).

2. Materials and methods 2.1. Biochar preparation

2.5. Assay of microbial community Biochar was produced by the Nanjing Institute of Soil Science, Chinese Academy of Sciences, China. Briefly, dry rice hull was pyrolysed at 400 °C for 3 h under oxygen-limited conditions. The physiochemical characteristics of the biochar were detected as follows: pH 9.2, total nitrogen 13.5 g/kg, total carbon 630 g/kg, total phosphate 4.5 g/kg, total potassium 21.5 g/kg, and ash content 140 g/kg. Fourier transform infrared spectroscopy (FTIR) and scanning electron microscope (SEM) images were shown in Figure S1. FTIR analysis showed biochar with the following characteristic absorption peaks: 3338 cm-1 representing the stretching vibration of −OH, 1592 cm-1 representing

Soil DNA was extracted from 0.4 g of each soil sample following the instructions of FastDNA Spin Kit (MP Biomedicals, USA). The extracted DNA was used as template to amplify the bacterial 16S ribosomal RNA (rRNA) gene. Hypervariable V3 and V4 regions of 16S rRNA gene were amplified by forward primer 338 F (5′-ACTCCTACGGGAGGCAGCA-3′) and reverse primer 806R (5′-GGACTACHVGGGTWTCTAAT-3′) (Wang et al., 2017). Amplicons were sequenced on Illumina Miseq platform (Illumina, San Diego, USA) at SHBIO Technology (Shanghai, China). Raw sequences were processed with QIIME quality filter (Sun et al., 2

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treatments and the control. Compared to the control, the urease activity in biochar amendments (BC2 and BC3) significantly (P < 0.05) increased (Table 2), suggesting amendment with 7.5−15 t ha-1 biochar could increase soil urease activity. No significant difference was observed for invertase and catalase activities among biochar treatments and the control. Phosphatase activity in BC5 was significantly (P < 0.05) lower than that in BC1, BC2, BC3 and BC4, suggesting that amendment with excessive biochar (45 t ha-1) decreased phosphatase activity.

2014), then analyzed via UPARSE pipeline (Caporaso et al., 2010). Paired-end clean reads were merged using FLASH (V1.2.11) according to the relationship of the overlap between the paired-end reads. Sequences were assigned to each sample based on their unique barcode and primer using Mothur software (V1.35.1), after which the barcodes and primers were removed and got the effective clean tags. Sequences analysis was performed by usearch software (V10). Sequences with ≥97 % similarity were assigned to the same operational taxonomic unit (OTU). An OTU is thought to possibly represent a species. Alpha diversity is applied for analyzing complexity of bacterial specie diversity of a sample including observed species, Chao1, Shannon and Simpson index, which were calculated with QIIME (V1.9.1) and displayed with R software (V2.15.3) (Caporaso et al., 2011). Beta diversity analysis was used to evaluate differences between samples of bacterial specie complexity. Sample cluster analysis was performed as an UPGMA (Unweighted Pair-group Method with Arithmetic Means) method to interpret the distance matrix using average linkage and was conducted by upgma_cluster.py script in QIIME software based on the unweighted unifrac distance matrix (Lozupone et al., 2006).

3.3. Influence of biochar amendment on bacterial communities At three years after biochar amendment, no significant difference in the observed species, Chao1, Shannon, and Simpson index was found between biochar treatments and control (Table S1), suggesting that biochar had no effect on bacterial α-diversity. 5186 OTUs (53.1 %) were shared by all soil samples (Fig. 1A). Each biochar treatment possessed its unique bacterial species. BC3 had the most unique OTUs (1578 OTUs), followed by BC4 (1241 OTUs), suggesting that 15−30 t ha-1 biochar could increase the number of unique soil bacterial species. UPGMA analysis showed that BC1 and BC5 were clustered together; while BC2 and BC4 were clustered together (Fig. 1B). BC3 sample was different from other samples, suggesting that the bacterial community structure of BC3 was changed most.

2.6. Statistical analysis Treatment differences in soil and tobacco properties, alpha diversity and microbial abundance were compared by one-way analysis of variance (ANOVA) with Tukey pair-wise comparisons (P < 0.05, P < 0.01). Correlation between disease index and microbial abundance was analyzed by Pearson correlation analysis with SPSS 20 software (Wang et al., 2017).

3.4. Abundance of different phyla Total 43 bacterial phyla were found in the soil samples. Actinobacteria, Chloroflexi and Proteobacteria were the most abundant phyla and together occupied 70 % of bacterial abundance (Fig. 1B). Biochar amendments significantly (P < 0.05) changed the relative proportions of seven bacterial phyla. Compared to control, relative abundances of Armatimonadetes, Firmicutes, Saccharibacteria and Verrucomicrobia in biochar amendment (BC3), Cyanobacteria and Verrucomicrobia in biochar amendment (BC4), Planctomycetes in biochar amendment (BC5) significantly (P < 0.05) increased (Table 3), Gemmatimonadetes in biochar amendments (BC3 and BC4) significantly (P < 0.05) decreased. These results suggested that biochar amendments changed bacterial community composition, which was related to the biochar doses.

3. Results 3.1. Disease severity index of bacterial wilt The disease incidence and indices of biochar treatments (BC2, BC3, BC4, and BC5) were all significantly (P < 0.05) lower than those of control (Table 1). Among all treatments, disease index and incidence of BC3 was the lowest, the disease incidence and the severity of disease was decreased by 58.72 % and 69.81 %, respectively when compared to the control. These results indicated that biochar amendment could potentially suppress bacterial wilt disease, and 15 t ha-1 of biochar showed the best control effect on bacterial wilt. No significant difference in tobacco height and stem girth was found among biochar treatments and control.

3.5. Abundance of bacterial genera The biochar treatments significantly enriched potential beneficial bacteria such as Aeromicrobium, Bacillus, Bradyrhizobium, Burkholderia, Chlorochromatium, Chthoniobacter, Corynebacterium, Geobacillus, Leptospirillum, Marisediminicola, Microvirga, Pseudoxanthomonas and Telmatobacter in soil (Fig. 2). Compared to the control, relative abundances of Aeromicrobium, Bacillus, Bradyrhizobium, Chthoniobacter, Corynebacterium, Geobacillus, Microvirga and Pseudoxanthomonas in BC3, Chthoniobacter and Marisediminicola in BC4, Burkholderia, Chlorochromatium, Leptospirillum and Telmatobacter in BC5 significantly (P < 0.05 or P < 0.01) increased. In contrast, biochar treatment caused the significant (P < 0.05) decrease of the relative abundances of denitrifying bacteria (e.g.

3.2. Influences of biochar amendment on soil properties Biochar amendment significantly (P < 0.05) affected soil nutrient status. Compared to the control, the concentration of alkaline nitrogen in biochar amendment (BC3) increased by 16 %, soil organic matter in biochar amendments BC3 and BC4 increased by 46.7 % and 46.8 %, respectively (Table 2). These results suggested that amendment with 15−30 t ha-1 biochar could increase the soil nitrogen and organic matter contents. No significant difference was observed for available phosphorus and potassium contents and soil pH among biochar Table 1 Influence of biochar on tobacco properties and bacterial wilt. Tobacco properties

BC1

BC2

BC3

BC4

BC5

Plant height (cm) Stem girth (mm) Disease incidence (%) Disease index

95.56 ± 4.44 a 9.33 ± 0.29 a 20.18 ± 0.40 a 3.08 ± 0.12 a

93.44 ± 6.55 a 9.33 ± 0.29 a 10.09 ± 0.29 c 1.12 ± 0.23 c

93.00 ± 4.18 a 9.17 ± 1.04 a 8.33 ± 0.66 d 0.93 ± 0.13 c

96.78 ± 6.99 a 8.83 ± 0.29 a 10.09 ± 1.06 c 1.12 ± 0.20 c

96.11 ± 3.56 a 9.00 ± 0.50 a 14.47 ± 0.23 b 2.00 ± 0.30 b

All data are represented as the means ± SE. Values with different letters in the same row are significantly (P < 0.05) different. 3

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Table 2 Influence of biochar on soil chemical properties and enzymatic activities. Soil properties

BC1

BC2

BC3

BC4

BC5

AN (mg/kg) AK (mg/kg) AP (mg/kg) SOM (g/kg) pH Urease (mg/g) Invertase (mg/g) Phosphatase (mg/g) Catalase (mg/g)

120.25 ± 6.28 b 464.73 ± 111.25 a 44.45 ± 7.38 a 25.41 ± 5.29 b 5.20 ± 0.45 a 0.39 ± 0.05 b 14.71 ± 5.59 a 1.05 ± 0.02 a 5.11 ± 0.58 a

121.11 ± 12.79 b 520.60 ± 24.92 a 63.41 ± 12.70 a 32.00 ± 4.33 ab 5.24 ± 0.37 a 0.57 ± 0.09 a 18.59 ± 2.81 a 1.03 ± 0.06 a 4.91 ± 0.46 a

139.56 ± 4.75 a 616.39 ± 169.93 a 71.01 ± 42.21 a 37.28 ± 6.19 a 5.43 ± 0.46 a 0.68 ± 0.11 a 16.94 ± 5.98 a 1.05 ± 0.02 a 4.81 ± 0.60 a

127.50 ± 9.93 ab 553.86 ± 198.76 a 53.17 ± 31.62 a 37.31 ± 1.71 a 5.48 ± 0.35 a 0.35 ± 0.07 b 19.17 ± 4.55 a 1.08 ± 0.18 a 4.89 ± 0.39 a

112.38 ± 4.67 b 588.45 ± 69.59 a 53.85 ± 13.68 a 30.21 ± 9.14 ab 5.08 ± 0.40 a 0.35 ± 0.03 b 15.60 ± 0.79 a 0.84 ± 0.03 b 4.28 ± 0.75 a

All data are represented as the means ± SE. Values with different letters in the same row are significantly (P < 0.05) different.

Fig. 1. Analysis of bacterial community. (A) Venn showed the shared and unshared OTUs among biochar treatments and control; (B) UPGMA clustering tree of bacterial community in different treatments.

abundances of Gemmatimonadetes, Ralstonia, Reyranella and Thermus were significantly (P < 0.05) positively correlated with the disease index. Thereby, a higher relative abundance of these microbes in soils is possible to promote the outbreak of bacterial wilt.

Noviherbaspirillum, Reyranella, Thermus) and Ralstonia (pathogen of bacterial wilt). Compared to the control, relative abundances of Noviherbaspirillum in BC5, Reyranella in BC2, Thermus in biochar treatments (BC2 and BC4), Ralstonia in biochar treatments (BC2, BC3 and BC5) significantly (P < 0.05) decreased. Compared to the control, the abundance of Ralstonia in biochar treatments (BC2, BC3, BC4 and BC5) decreased by 56.7 %, 69.9 %, 46.3 % and 68.8 %, respectively.

4. Discussion This study found that rice hull biochar had potential for controlling bacterial wilt disease. Biochar amendments significantly suppressed tobacco bacterial wilt in the field trial through changing soil chemical properties and microbial abundances. The lowest disease incidence and index was observed in biochar treatment BC3, followed by BC2 and BC4, suggesting that 30 t ha-1 biochar had the most positive effect on disease suppression, and appropriate biochar doses (7.5−30 t ha-1) could more effectively suppress bacterial wilt disease, which was

3.6. Correlation between bacterial abundance and severity of bacterial wilt The relative abundances of Armatimonadetes, Bacillus, Chthoniobacter and Saccharibacteria were significantly (P < 0.05) negatively correlated with the disease index of bacterial wilt (Fig. 3). Thereby, a higher abundance of these bacteria in biochar treatments might be helpful for inhibiting bacterial wilt. In contrast, the relative 4

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Table 3 Abundance of bacterial phyla in biochar amended and unamended soils. Taxonomy

BC1

BC2

BC3

BC4

BC5

Acidobacteria Actinobacteria Armatimonadetes Bacteroidetes Chloroflexi Cyanobacteria Chlorobi Chlamydiae Deinococcus-Thermus Elusimicrobia Firmicutes Gemmatimonadetes Latescibacteria Microgenomates Nitrospirae Parcubacteria Planctomycetes Proteobacteria Saccharibacteria Thermotogae Verrucomicrobia

5.65 ± 2.99 a 35.19 ± 9.30 a 0.18 ± 0.13 b 2.62 ± 0.60 a 18.21 ± 12.05 a 0.34 ± 0.30 b 0.05 ± 0.03 a 0.01 ± 0.00 a 0.06 ± 0.05 a 0.03 ± 0.01 a 1.57 ± 0.24 b 5.76 ± 2.00 a 0.27 ± 0.24 a 0.06 ± 0.05 a 0.55 ± 0.24 a 0.02 ± 0.02 a 0.19 ± 0.15 b 25.37 ± 6.36 a 3.12 ± 2.27 b 0.07 ± 0.07 a 0.05 ± 0.05 b

5.85 ± 1.4 a 34.17 ± 5.08 a 0.32 ± 0.10 ab 2.31 ± 0.27 a 20.78 ± 6.98 a 0.67 ± 0.30 ab 0.05 ± 0.01 a 0.01 ± 0.01 a 0.02 ± 0.01 a 0.03 ± 0.02 a 1.31 ± 0.38 b 4.42 ± 0.35 ab 0.23 ± 0.03 a 0.09 ± 0.03 a 0.42 ± 0.16 a 0.05 ± 0.05 a 0.45 ± 0.16 ab 23.93 ± 3.39 a 4.13 ± 1.03 ab 0.04 ± 0.02 a 0.11 ± 0.03 ab

8.88 ± 5.33 a 27.55 ± 11.34 a 0.41 ± 0.09 a 2.73 ± 1.02 a 21.16 ± 9.19 a 0.52 ± 0.07 ab 0.05 ± 0.01 a 0.01 ± 0.01 a 0.02 ± 0.02 a 0.05 ± 0.05 a 2.32 ± 0.32 a 3.79 ± 0.52 b 0.56 ± 0.65 a 0.07 ± 0.04 a 0.82 ± 0.88 a 0.04 ± 0.02 a 0.49 ± 0.24 ab 22.87 ± 5.61 a 6.33 ± 1.93 a 0.06 ± 0.07 a 0.23 ± 0.08 a

7.28 ± 3.11 a 30.81 ± 8.29 a 0.34 ± 0.12 ab 2.16 ± 0.31 a 23.01 ± 8.23 a 1.04 ± 0.71 a 0.06 ± 0.03 a 0.02 ± 0.02 a 0.01 ± 0.01 a 0.03 ± 0.01 a 1.61 ± 0.58 b 3.53 ± 0.43 b 0.47 ± 0.34 a 0.08 ± 0.04 a 0.57 ± 0.26 a 0.03 ± 0 a 0.48 ± 0.16 ab 22.35 ± 4.68 a 4.80 ± 2.03 ab 0.04 ± 0.03 a 0.25 ± 0.19 a

7.21 ± 2.34 a 33.72 ± 2.97 a 0.18 ± 0.05 b 2.33 ± 0.10 a 17.27 ± 1.28 a 0.54 ± 0.05 ab 0.04 ± 0.02 a 0.01 ± 0.00 a 0.02 ± 0.03 a 0.04 ± 0.01 a 1.39 ± 0.16 b 4.87 ± 0.77 ab 0.25 ± 0.25 a 0.12 ± 0.02 a 0.43 ± 0.23 a 0.06 ± 0.04 a 0.53 ± 0.17 a 26.30 ± 0.35 a 3.63 ± 1.22 ab 0.04 ± 0.03 a 0.10 ± 0.06 ab

All data are represented as the means ± SE. Values with different letters in the same row are significantly (P < 0.05) different.

Several studies have demonstrated that biochar amendment makes soil nutrients more available (Rodrigues et al., 2007; Rondon et al., 2007; Hernandez-Soriano et al., 2016). Biochar retains more nitrogen in soils through enhancing nitrogen fixation, ammonia/ammonium retention, reducing N2O emissions and nitrate leaching (Rodrigues et al., 2007; Rondon et al., 2007). In this study, we found that biochar application significantly increased content of alkaline nitrogen and urease activity. Increase of urease activity enhances the availability of nitrogen

consistent with previous studies indicating that relatively low concentrations of biochar suppressed plant diseases, higher concentrations of biochar were mostly ineffective or even accelerated plant diseases (Jaiswal et al., 2014, 2015; Copley et al., 2015; Huang et al., 2015). As previously reported, plant resistance to disease was frequently dependent on biochar dose applied, plant disease responses eventually show an inverted U-shaped dose/response curve if dose of biochar is further increased (Jaiswal et al., 2014, 2015).

Fig. 2. Heat map showed the relative abundances of bacterial genera in different treatments. Red color represented higher abundance, blue color represented lower abundance. 5

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Fig. 3. Correlation between bacterial genera abundances and disease index of bacterial wilt.

humus (Kimetu and Lehmann, 2010). Soil organic matter is an important indicator of soil fertility and an important source of microbial nutrients (Cookson et al., 2005). In addition, we supposed that the higher soil nitrogen and carbon content and urease activity could increase activity of beneficial microorganisms against pathogen such as R. solanacearum (Bailey and Lazarovits, 2003; Yin et al., 2011; Zhang et al., 2017). It was speculated that biochar application changed soil properties that indirectly suppressed pathogen growth by promoting antagonistic microorganisms. Our results suggested that biochar might stimulate soil microbial activity and affect bacterial abundances via increasing soil carbon and nitrogen, which was consistent with the previous report (Gomez et al., 2014). The abundances of potential beneficial bacteria, which have the functions of cellulose-degradation, N-cycling, P-solubilization and pathogen suppression, were improved in biochar amended soils. For

to meet the need of plant growth. In BC3 treatment, biochar amendment increased soil nitrogen level, which was consistent with previous study (Xu et al., 2014). Biochar has been showed reducing levels of denitrification via improving soil aeration (Yanai et al., 2007). We supposed that biochar inhibited denitrifying bacteria and N2O-reducing bacteria. Biochar is a product with high carbon content, which may increase the soil organic matter content after application to soil. Many studies have showed that biochar improves soil fertility and crop yields (Shaaban et al., 2018). This study demonstrated that the biochar significantly increased the content of organic matter in the soil (Table 2), which verified the results of previous studies (Liang et al., 2014; Yin et al., 2014). Biochar can adsorb and promote soil organic molecule polymerization to form organic matter (Van Zwieten et al., 2010). In addition, decomposition of biochar contributes to the development of 6

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(Kolton et al., 2017). We supposed that biochar made from different organic wastes might show different effect on soil microorganisms. On the other hand, we found that different doses of biochar showed different effects on soil properties and microbial abundances. In this study, 15−30 t ha-1 biochar had a better effect on improving the soil properties and microbial abundances and controlling bacterial wilt. Excessive biochar (45 t ha-1 in BC5) could not enhance the enzymatic activity and soil microbial growth, possibly because large amount of biochar contains excessive aldehydes and ketones that are not suitable for microbial growth (Chen et al., 2011). This was consistent with the reports that lower doses of biochar stimulated positive effects but higher doses of biochar caused toxicity and inhibition (Jaiswal et al., 2014; Kammann and Graber, 2015). We suggested that applying 7.5−30 t ha-1 biochar to soil. 30 t ha-1 of biochar was the optimum application rate for control bacterial wilt. In conclusions, the results presented here indicated that biochar additions to soil significantly reduced the disease incidence and severity of bacterial wilt. The disease incidence and index in biochar amendments (7.5−45 t ha-1) significantly decreased when compared with control. Amending 15 t ha-1 of biochar showed the best control effect on bacterial wilt disease. This study provided an alternative potential method to control bacterial wilt. Biochar amendment increased soil organic matter, nitrogen and urease activity. Biochar amendment enriched potential beneficial bacteria involved in carbon, nitrogen and phosphorus cycling and bacteria producing anti-microbial compounds. Biochar also decreased the abundances of denitrifying bacteria and plant pathogen when compared with control. All these changes in soil properties and microorganisms might contribute to the control action of biochar on bacterial wilt disease. However, biochar couldn’t fully inhibit bacterial wilt, in BC3 treatment, 8.33 % of tobacco plants was infected by R. solanacearum. Combination of biochar amendment and biocontrol agent might further improve the control effect. On the other hand, the effect of biochar on microbial function genes is still unknown.

example, biochar application significantly increased the abundance of Aeromicrobium, Armatimonadetes, Bacillus, Bradyrhizobium, Burkholderia, Chthoniobacter, Corynebacterium, Geobacillus, Leptospirillum, Marisediminicola, Microvirga, Pseudoxanthomonas, Saccharibacteria and Telmatobacter. These potential beneficial bacteria have positive effects on soil nutrients cycling. For instance, Chthoniobacter, Geobacillus, Pseudoxanthomonas and Telmatobacter can degrade hemicellulose, lignocellulose and cellulose (Sangwan et al., 2004; Pankratov et al., 2012; Kumar et al., 2015; Daas et al., 2018). Corynebacterium glutamicum degrades and assimilates a rich spectrum of aromatic compounds including lignin-derived aromatic compounds (Shen et al., 2012). Bradyrhizobium, Burkholderia, Leptospirillum and Microvirga are nitrogenfixing bacteria, which can increase the nitrogen contents by N2 fixation (Parro and Moreno-Paz, 2004; Suárez-Moreno et al., 2008; Radl et al., 2014; Osei et al., 2018). In soils, huge reserves of inorganic or organic phosphorus are present in immobilized or unavailable form. Bacillus and Burkholderia are phosphate-mobilizing bacteria, which have the potential to mineralize and solubilize organic and inorganic phosphorus in soil (Panhwar et al., 2014). These potential beneficial bacteria can improve soil nutrients availability for plant uptake through N fixation, P mobilization and carbon degradation. Aeromicrobium, Bacillus, Burkholderia and Marisediminicola have the potential of control plant disease by producing anti-microbial molecules (Reeves et al., 2004; Wang and Liang, 2014; Rojas-Rojas et al., 2018). For example, Bacillus amyloliquefaciens effectively controlled bacterial wilt by producing antimicrobial substances and inducing plant resistance (Tan et al., 2013; Wang and Liang, 2014). We speculated that a higher abundance of Bacillus in biochar treatment (BC3) could more effectively inhibit R. solanacearum, which was confirmed by the lowest disease incidence and index in BC3 treatment, and the negative correlation between abundance of Bacillus and disease index. Arthrobacter pascens can promote plant growth by producing indole aectic acid (Li et al., 2018). Arthrobacter is involved in decomposition of aromatic compounds, which are rich in biochar (O’Loughlin et al., 1999). Chthonomonas calidirosea belonging to phylum Armatimonadetes, as a saccharide scavenger, can utilize a wide range of carbohydrates in soil (Lee et al., 2014). Saccharibacteria species utilize and degrade the complex carbon sources (cellulose, hemicellulose, pectin, starch and 1,3-β-glucan) from plant root exudates and cell wall (Starr et al., 2018). We supposed that possibly Armatimonadetes and Saccharibacteria can compete with R. solanacearum for carbon source to suppress pathogen growth. These results revealed that biochar enriched more potential beneficial bacteria, which participated in nutrients cycling and suppressed plant soil-borne pathogens. These findings explained the reason for higher soil nutrients (C and N) supplying capacity and crop resistance to disease of biochar treatments. The genera with decreased abundance by biochar treatments were reported as denitrifying bacteria (Noviherbaspirillum, Reyranella, Thermus), N2O-reducing bacteria (Gemmatimonadetes) and plant pathogen (Ralstonia). Ralstonia is the pathogen of bacterial wilt, suggesting that biochar could inhibit the growth of pathogen Ralstonia in soils. This study proved that biochar had the potential to control soilborne disease. Denitrification is a stepwise, anaerobic microbial process reducing nitrate to N2, N2O and NO, which can decrease soil nitrogen content (Seitzinger et al., 2006). Species of Noviherbaspirillum, Reyranella and Thermus are able to reduce nitrate and nitrite then to NO and N2O (Kim et al., 2013; Alvarez et al., 2014; Ishii et al., 2017). Gemmatimonas aurantiaca belonging to Gemmatimonadetes phylum can reduce N2O to N2 (Park et al., 2017). We supposed that biochar amendment possibly decreased soil nitrogen losses by reducing the abundance of denitrifying bacteria. Besides, the abundances of Gemmatimonadetes, Reyranella and Thermus were positively correlated with the disease index, suggesting that decreasing denitrifying bacteria by biochar could help to control bacterial wilt. In this study, biochar amendment did not elicit augmentation of bacterial diversity, which was not consistent with the previous studies

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