Watermelon planting is capable to restructure the soil ...

Applied Soil Ecology xxx (xxxx) xxx–xxx

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Watermelon planting is capable to restructure the soil microbiome that regulated by reductive soil disinfestation Liangliang Liua, Shuhua Chena, Jun Zhaoa,b,c,d,e, Xing Zhoua, Baoying Wanga, Yunlong Lia, ⁎ Guoqiang Zhengf, Jinbo Zhanga,b,c,d,e,f, Zucong Caia,b,c,d,e, Xinqi Huanga,b,c,d,e, a

School of Geography Science, Nanjing Normal University, Nanjing 210023, China Jiangsu Center for Collaborative Innovation in Geographical Information Resource Development and Application, Nanjing 210023, China c Key Laboratory of Virtual Geographical Environment (Nanjing Normal University), Ministry of Education, Nanjing 210023, China d Jiangsu Provincial Key Laboratory of Materials Cycling and Pollution Control, Nanjing Normal University, Nanjing 210023, China e State Key Laboratory Cultivation Base of Geographical Environment Evolution (Jiangsu Province), Nanjing 210023, China f Jiangsu Biyuntian Agriculture and Forest Technology Co., Ltd, Zhenjiang 212362, China b

A R T I C LE I N FO

A B S T R A C T

Keywords: Reductive soil disinfestation Watermelon Fusarium wilt Fusarium oxysporum Microbial communities

Reductive soil disinfestation (RSD) is an effective method to suppress many types of soil-borne diseases in various crops. However, the changes of soil-borne pathogens and microbial communities after crop cultivation in the RSD-treated soils are poorly understood. In the present study, pot experiment was conducted to evaluate the impacts of RSD treatment, with the incorporation of sugar fermentation liquor, on soil Fusarium oxysporum populations, microbial activities, and microbial communities at both after RSD treatment and watermelon planting. The results showed that RSD treatment could considerably inhibit the F. oxysporum and Fusarium wilt disease, promote the growth of watermelon, and enhance microbial activity. However, the pathogen of F. oxysporum resurged quickly after the watermelon cultivation. Although RSD treatment could significantly alter soil microbial communities, the watermelon cultivation also had the capacity to restructure the soil microbial communities to a similar status with control. Collectively, RSD treatment could considerably reduce soil conduciveness to Fusarium wilt and this conduciveness was quickly resurged after watermelon planting. Thus, the changes of F. oxysporum and soil microbial community after watermelon cultivation were likely due to the root exudates released by watermelon that had enough powerful driving force to select similar taxa by filtering the microbial seed bank.

1. Introduction Watermelon (Citrullus lanatus (Thunb.) Matsum. et Nakai) widely planted and consumed around the world is a popular and important fruit. To meet the growing consumption demand worldwide, monocultures become the major cropping system for watermelon production recently. However, watermelon subjected to consecutive monoculture is susceptible to Fusarium wilt caused by the soil-borne fungus of Fusarium oxysporum f. sp. niveum that leads to the huge production and economic losses annually (Hopkins and Elmstrom, 1984; Ren et al., 2008). Once the soil has been infected by F. oxysporum, it is virtually impossible to remove this pathogen, with survival up to 10 years due to the strong resistant ability to environmental stress (Miguel et al., 2004) To suppress this soil-borne disease, applying soil chemical fumigants, such as methyl bromide, has been considered as the most effective measure for a long time (Cebolla et al., 2000). However, the long-

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term use of chemical fumigants leads to the environmental and food quality problems. Particularly, since 2004, the use of methyl bromide has been banned under the Montreal protocol because of its ozonedepleting traits (Gamliel et al., 2000). Therefore, developing biological and environmentally friendly approaches have been urgent to prevent this disease in the last two decades. For example, Ren et al. (2008) demonstrated that intercropping with aerobic rice significantly alleviated Fusarium wilt of watermelon and altered the rhizosphere microbial communities through the production of rice root exudates. Application of bioorganic fertilizer containing the antagonistic microorganisms also effectively suppressed this disease and promoted watermelon production (Ling et al., 2010; Wu et al., 2009). Nonetheless, the suppressive efficiencies of these approaches were not consistent and stable under fluctuating environmental conditions, such as temperature, soil moisture, crop season, etc. (Wei et al., 2011). Reductive soil disinfestation (RSD), alternatively named as

Corresponding author at: School of Geography Science, Nanjing Normal University, Nanjing 210023, China. E-mail address: [email protected] (X. Huang).

https://doi.org/10.1016/j.apsoil.2018.05.004 Received 24 November 2017; Received in revised form 29 March 2018; Accepted 4 May 2018 0929-1393/ © 2018 Elsevier B.V. All rights reserved.

Please cite this article as: Liu, L., Applied Soil Ecology (2018), https://doi.org/10.1016/j.apsoil.2018.05.004


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conform the field soil moisture condition. After the 20 days incubation, all of the soils were naturally drained, sieved (8 mm) and thoroughly mixed. The soil samples (about 30 g for each pot) were collected as three biological replicates per treatment by pooling 10 pots into one to minimize the community variation within each replicate. The collected soils were respectively stored at 4 °C and −80 °C for further analysis. After sampling, the per kilogram soil was thoroughly incorporated with 1 g (w/w) inorganic compound fertilizer (N:P:K = 16:16:16) and then one watermelon seedling with three leaves was planted into each pot and cultivated for 90 days with the average day and night temperate at 30 °C and 15 °C, respectively. During this period, 50 mg N kg−1 soil of urea was applied to each pot every ten days. After 40 days growth, the plants disease index and shoot length were recorded every ten days and the plants were collected to investigate the root length and biomass at the end of cultivation according to Cao et al. (2016). The soil samples were also collected with the strategy described above for further analysis (hereby defined as CK-C and RSD-C, respectively). The plant disease index was determined by the following formula: disease index = Σ (individual disease score)/(3 × total number of plants) × 100%. Individual disease scores are as follows: 0 = healthy, 1 = less than 1/2 vascular bundle skin infected, 2 = more than 1/2 vascular bundle skin infected, 3 = leaves wilted.

anaerobic soil disinfestation (ASD) or biological soil disinfestation (BSD), firstly and independently developed in the Netherlands (Blok et al., 2000) and Japan (Shinmura, 2000) in 2000, has been proven to successfully control multiple soil-borne diseases in various crops by effectively suppressing their causal pathogens, such as Ralstonia solanacearum, Fusarium oxysporum f. spp. cubense, spinaciae, lycopersici, Verticillium dahliae, etc. (Huang et al., 2015a; Messiha et al., 2007; Mowlick et al., 2013a; Shinmura, 2004). Furthermore, RSD only requires a moderate temperature and short duration time with a relatively simple field operation procedure. Thus, RSD has been attracted considerable attention and widely applied as an alternative to chemical fumigations and biological control in Japan, USA, and the Netherlands (Goud et al., 2004; Momma et al., 2013; Shennan et al., 2014) It is well known that the soil microbial community play vital roles in maintaining soil health and suppressing plant diseases (Qiu et al., 2012), whereas plants also have the capacity to drive and shape the microbiome via releasing a broad variety of chemical compounds (Haichar et al., 2008). In addition, increasing researches indicated that RSD could significantly alter soil microbial communities which were mainly dominated by microbiome conducive to non-pathogens, such as Clostridia, Bacillus, pseudomonas, etc (Huang et al., 2015b, 2016; Mowlick et al., 2013a). However, the responses of soil microbial communities regulated by RSD to plant cultivation are still unclear. Therefore, two hypotheses were proposed here: 1) the microbial communities in the RSD-regulated and control soils become much more different after plant growth because the communities differences are exacerbated under the influence of root exudates; 2) the microbial communities in the RSD-regulated and control soils tend to be similar after plant growth because certain root exudates are capable to select some similar specific soil microbiome. To test these hypotheses, pot experiment using homogenized soil with controlled temperature, moisture, and nutrient contents was conducted to evaluate the changes in soil microbial populations, microbial activities, and microbial communities at both after RSD and watermelon cultivation via Miseq sequencing approach coupled with quantitative PCR.

The TOCs in the soil and SF were measured by wet digestion with H2SO4–K2Cr2O7 (Bremner and Jenkinson, 1960), and TNs were determined using semi-micro-Kjeldahl digestion (Bremner, 1960). The EOC in the SF was detected according to a previous study (Liu et al., 2016). Soil pH was measured using an S220K pH meter (Mettler-Toledo International Inc., Shanghai, China) with the ratio of 1:2.5 (soil/water, w/v). Total soil microbial activity was measured by fluorescein diacetate (FDA) hydrolysis according to Adam and Duncan (2001) and expressed as μg fluorescein released by per gram of dry weight soil per hour.

2. Materials and methods

2.4. DNA extraction and real-time quantification assay

2.1. Soil and organic material

Soil DNA was extracted from 0.5 g soil using BioFast Soil Genomic Isolation kit (Bioer Technology Co., Ltd) according to the manufacturer’s instructions. The extracted DNA was then quality-controlled and quantified by a Nanodrop ND-1000 spectrophotometer (Thermo, Waltman, MA, USA). All DNA extracts were subsequently stored at −20 °C for downstream molecular analyses. Quantitative real time polymerase chain reaction (qPCR) amplifications were performed in 8-well tubes on the CFX96TM Real-Time System (Bio-Rad Laboratories Inc., Hercules, CA, USA) to enumerate the populations of bacteria, fungi, and F. oxysporum. The reaction mixtures and thermal conditions of qPCR were according to Liu et al. (2016). The respective primer sets of bacteria (Eub338/Eub518), fungi (ITS1-f/ 5.8s), and F. oxysporum (ITS1 F/AFR 308) were listed in Table S1. Melt curves were detected to ensure the amplification specificity after each amplification. The standard curves were generated according to Huang et al. (2015b) and the slopes of bacteria, fungi, and F. oxysporum were −3.003, −3.359, and −3.368, respectively.

2.3. Measurement of soil characteristics and total microbial activity

The soil used in this experiment was collected from the Luhe Animal Science Base, Jiangsu Academy of Agriculture Sciences, Jiangsu Province, China (32°2′N, 118°8′E). The watermelon was continuously cultivated for several years in this soil and ultimately suffered from severe Fusarium wilt disease. The soil was a Tye-Fel-Stagnic Antrosols clay (IUSS Working Group WRB, 2015), with the following initial properties: pH 7.03; total organic carbon (TOC) 19.46 g kg−1; total nitrogen (TN) 3.74 g kg−1, and F. oxysporum 1.02 × 106 ITS copies·g−1 soil. The organic material sugar fermentation liquor (SF), a coproduct of ethanol fermentation using molasses, used in the RSD treatment was obtained from a sugar factory in Guangxi, China (22°48′, 108°22′). The TOC, TN, and easily oxidized organic carbon (EOC) contents of SF were 59.27 g kg−1, 5.89 g kg−1, and 58.20 g kg−1, respectively. 2.2. Experimental design and watermelon cultivation

2.5. MiSeq sequencing and raw data processing

The F. oxysporum infected soil described above was treated by RSD before watermelon planting. Briefly, the experiment had a completely randomized design with three replications and each replicate contained 10 pots that had the following treatments, 1) CK, 4 kg soil was equipped into a pot (22 × 15 × 20 cm, Top × bottom diameter × high) without the organic matter amendment, irrigating and sealing; 2) RSD, 4 kg soil incorporated with 2% (v/w) SF was equipped into a pot, irrigated to soil saturation and sealed with plastic film. The soils in the pots were incubated for 20 days at 35 °C. During the incubation period, the soil water content in the CK treatment was maintained at 15%–18% to

In order to characterize the microbial communities in response to RSD treatment and watermelon cultivation, DNA extracted from all soil samples were selected for Miseq sequencing. The bacterial V4 region and fungal ITS1 region were respectively amplified using the primer sets 515F/806R and ITS1F/ITS2 (Table S1). The PCR procedures containing the reaction mixture and thermal profile were according to Zhao et al. (2014). After PCR amplification, the PCR products were purified using the SequalPrep Normalization Plate (96) Kit, pooled in equimolar 2


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total microbial activity in the RSD treatment significantly (P < 0.05) increased by 7.84 μg g−1 h−1 in comparison to CK treatment, whereas the microbial activity in the RSD treatment significantly decreased after watermelon planting and had no obviously difference compared to CKC soil (Table 1).

concentrations, and then subjected to sequencing at Genesky Biotechnologies, Inc. (Shanghai, China) on an Illumina MiSeq instrument (USA). The sequencing data has been uploaded in the NCBI Sequence Read Archive (SRA) database (accession number SRP117934). Quantitative Insights into Microbial Ecology (QIIME) software package (version 1.9.1) (Caporaso et al., 2010) coupled with a previously established procedure (Zhao et al., 2016) were used to process the sequencing data. Briefly, the paired-end sequences were merged and quality-controlled using the default arguments in the multiple_joined_paired_ends.py and multiple_split_libraries_fastq.py, respectively. The quality-filtered sequences of bacteria and fungi were then clustered into operational taxonomic units (OTUS) at 97% similarity against the bacterial Greengenes 13_8 database (McDonald et al., 2012) and fungal UNITE database (Kõljalg et al., 2013), respectively. UCHIME (Edgar et al., 2011) was used to remove the chimera sequences and the singletons were excluded from downstream analysis. Finally, the obtained representative sequences were taxonomically classified using RDP naïve Bayesian rRNA Classifier (Wang et al., 2007) with confidence threshold of 80% and 50% for bacteria and fungi according the aforementioned databases, respectively. The bacterial and fungal sequences in all samples were respectively rarefied to 26,000 and 12,000.

3.2. The fresh biomass, length, and disease index of watermelon plant The disease index was consistently (P < 0.05) higher in the CK (7.78%–73.33%) treatment than that of RSD (4.44%–42.22%) treatment during the cultivation from 40 d to 90 d (Fig. S1a), and the shoot length in the RSD treatment was 37.0%–23.7% higher than that in CK (Fig. S1b). After 90-day cultivation, the fresh biomass and plant length of both shoot and root were respectively and significantly (P < 0.05) increased by 47.67%, 83.33%, 23.67% and 62.88% in the RSD treatment than those in the CK treatment (Table 2). In addition, the disease index significantly (P < 0.05) decreased by 42.42% in the RSD treatment compared with that in the CK (Table 2). 3.3. The soil bacteria, fungi, and F. oxysporum populations Overall, RSD treatment had no significant influence on the population sizes of bacteria and fungi when compared to the CK treatment. However, the population of F. oxysporum in the RSD soil (4.34 × 103 gene copies g−1 dry soil) significantly decreased by 99.22% of that in CK (5.58 × 105 gene copies g−1 dry soil) (Fig. 1). After watermelon cultivation, the populations of bacteria had no significant increases in both treatments, while the populations of fungi respectively considerably (P < 0.05) increased by 2.49 and 2.27 times in the CK-C and RSD-C treatments (Fig. 1a, b). The number of soil F. oxysporum in the RSD-C treatment was considerably (P < 0.05) resurged and still significantly (P < 0.05) lower 42.74% than that in the CK-C treatment (Fig. 1c). RSD treatment had no significant impact on the ratio of bacteria/fungi, while it notably (P < 0.05) declined by 64.48% and 59.29% after watermelon cultivation in the CK and RSD treatments, respectively. (Fig. 2a). Conversely, the ratio of F. oxysporum/fungi was considerably (P < 0.05) reduced by 99.22% in the RSD treatment and the trend was steady even it was rapidly restored in both treatments after watermelon cultivation (Fig. 2b).

2.6. Community data analysis The changes of microbial alpha-diversity after RSD treatment and watermelon cultivation were both calculated based on the rarefied OTU tables. To visualize the pairwise community dissimilarity between samples, principle coordinates analysis (PCoA) was performed based on the Bray-Curtis distances. Principal Components Analysis (PCA) was performed to analyze the relationship between the dominant phyla and different soil samples using the CANOCO for Windows (version 4.5). Redundancy analysis (RDA) was performed to examine the relationship between the dominant genera and environmental factors. 2.7. Statistical analysis Microbial count data were log10-transformed prior statistical analysis. The significant differences among treatments were tested using one-way ANOVA by the LSD test (p ≤ 0.05) in SPSS 19.0 (SPSS Inc., Chicago, USA).

3.4. Soil microbial community alpha-diversity 3. Results In total, 179,995 bacterial 16S rDNA and 338,047 fungal ITS highquality sequences were generated from the 12 soil samples (2 treatments × 2 time points × 3 biological replicates), respectively. These sequences were respectively clustered into 24,937 OTUs for bacteria and 2768 OTUs for fungi at 97% sequence similarity. Compared with the CK treatment, although RSD treatment had no significant effect on richness, diversity, and evenness for both bacteria and fungi, the bacteria Chao richness, fungal Shannon diversity, and equitability showed a slight increase after RSD treatment (Table S2). After watermelon cultivation, the fungal richness and the diversity, evenness for both bacteria and fungi significantly (P < 0.05) decreased in both CK-C and RSD-C soils, whereas the bacterial Chao richness had no significant decrease in both treatments (Table S2). With the exception of fungal evenness considerably higher in RSD-C treatment than the CK-C treatment, no significant differences were detected for the rest of bacterial and fungal alpha-diversity between CK-C and RSD-C soils (Table S2).

3.1. The soil pH and total microbial activity After 20-day incubation, the soil pH in the RSD treatment significantly (P < 0.05) increased by 0.81 compared that in the CK treatment, and the similar trend was found after the watermelon cultivation although the soil pH respectively considerably (P < 0.05) decreased by 2.03 and 2.65 in the CK and RSD treatments coinciding with watermelon growth in both treatments (Table 1). Likewise, the Table 1 The changes of soil pH and microbial activity after RSD treatment and watermelon cultivation. Treatment†

pH

CK RSD CK-C RSD-C

7.01 7.92 4.89 5.27

Fluorescein (μg g−1 h−1) ± ± ± ±

0.01b‡ 0.01a 0.00d 0.05c

3.89 ± 0.16b 11.73 ± 0.48a 8.77 ± 0.76c 7.78 ± 0.11c

3.5. Soil microbial community structures and compositions

† CK indicates the untreated soil; RSD indicates the soil applied with 2% (v/ w) sugar fermentation liquor and irrigated to saturate followed by sealing, “-C” represents after the watermelon cultivation. ‡ Values (means ± SE, n = 3) within the same column followed by different letters are significantly different at P < 0.05 according to LSD’s tests.

PCoA plot revealed that RSD treatment considerably (P < 0.05) influenced both soil bacterial and fungal community structures when compared to the CK treatment (Fig. 3a, b). However, both soil bacterial and fungal communities changed again and tended to appear similar after watermelon cultivation (CK-C vs. RSD-C), especially for the 3


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Table 2 The watermelon plant biomass, length, and disease index in the treatments of CK and RSD. Treatment†

Plant fresh biomass Shoot (g)

CK RSD † ‡

31.88 ± 1.69b 47.08 ± 1.56a

‡

Plant length

Disease Index (%)

Root (g)

Shoot (cm)

Root (cm)

1.98 ± 0.17b 3.63 ± 0.22a

108.70 ± 4.83b 134.43 ± 4.83a

23.33 ± 2.62b 38.00 ± 2.59a

73.33 ± 0.12a 42.22 ± 0.16b

CK indicates the untreated soil; RSD indicates the soil applied with 2% (v/w) sugar fermentation liquor and irrigated to saturate followed by sealing. Values (means ± SE, n = 3) within the same column followed by different letters are significantly different at P < 0.05 according to LSD’s tests.

(Table 3).

bacterial community (Fig. 3a, b). Moreover, both RSD treatment and watermelon cultivation significantly shifted the microbial communities from phylum (Table 3) to genus level (Fig. S2a, b).

3.5.2. For the bacterial and fungal genus level Compared with the CK treatment, the relative abundances of bacterial dominant genera Uc-Bacteroidales, Flavisolibacter, UcIgnavibacteriaceae and Gracilibacter as well as fungal dominant genera Peziza, Uc-Basidiomycota, and Ciliophora significantly (P < 0.05) increased in the RSD treatment. Moreover, all of the significant increase of bacterial dominant genera changed by tens of percent in abundances in comparison to CK. The relative abundances of bacterial genera UcTM7-1, Uc-Cytophagaceae, Uc-A4b, and Uc-CFB-26 as well as fungal genera Davidiella and Uc-Pezizomycetes dramatically (P < 0.05) decreased in the RSD treatment (Fig. 4). Similar to the changes of phylum level, the differences for bacterial and fungal genera compositions between CK-C and RSD-C were also shrunken and tended to be appear similar coupling with the watermelon cultivation. Moreover, the relative abundances of Uc-Chitinophagaceae, Arthrobotrys and Ascobolus were significantly (P < 0.05) higher in the RSD-C soil than those of CK-C soil, whereas the Uc-TM7-1, Uc-Xanthomonadaceae, and Conocybe revealed an opposite pattern (Fig. 4). In addition, the significantly increased genera that described above in the RSD treatment were considerably (P < 0.05) decreased with watermelon growth, whereas the bacterial genera Uc-Chitinophagaceae, Uc-TM7-1, Uc-Xanthomonadaceae and fungal genera Conocybe, Arthrobotrys, Davidiella, as well as

3.5.1. For the bacterial and fungal phylum level Compared with the CK treatment, the relative abundances of bacterial dominant phyla Firmicutes, OD1, Acidobacteria, and Chlorobi as well as fungal dominant phylum Ciliophora dramatically (P < 0.05) increased in the RSD treatment, whereas the relative abundances of bacterial phyla Actinobacteria, TM7, Chloroflexi, and Gemmatimonadetes in the RSD treatment significantly decreased (P < 0.05) (Table 3). Both bacterial and fungal community compositions changed after watermelon planting and the differences between CK-C and RSD-C soils were shrunken which were revealed by the relative abundances of most dominant phyla tended to be similar (Fig. 3c, d). Moreover, the relative abundances of Bacteroidetes (bacteria) and Ascomycota (fungi) were significantly (P < 0.05) higher in the RSD-C soil than that in the CK-C soil, whereas the TM7, Acidobacteria, and Basidiomycota (fungi) exerted an opposite pattern (Table 3). In addition, the phyla Firmicutes, OD1, Acidobacteria, Chlorobi, and Ciliophora significantly (P < 0.05) increased after RSD treatment and considerably (P < 0.05) decreased with watermelon growth. However, the Actinobacteria, TM7, Gemmatimonadetes, and Ascomycota showed an opposite pattern, being reduced after RSD treatment and increased after watermelon cultivation

Fig. 1. The populations of soil bacteria (a), fungi (b), and F. oxysporum (c) after RSD treatment and watermelon cultivation. Error bars represent the standard error of the means of three replicates. Bars with different letters represent significant differences according to LSD’s tests (P < 0.05). CK indicates the untreated soil; RSD indicates the soil applied with 2% (v/w) sugar fermentation liquor and irrigated to saturate followed by sealing, “-C” represents after the watermelon cultivation. 4


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Fig. 2. The ratios of soil bacteria to fungi (a) and F. oxysporum to fungi (b) after RSD treatment and watermelon cultivation. Error bars represent the standard error of the means of three replicates. Bars with different letters represent significant differences according to LSD’s tests (P < 0.05). CK indicates the untreated soil; RSD indicates the soil applied with 2% (v/w) sugar fermentation liquor and irrigated to saturate followed by sealing, “-C” represents after the watermelon cultivation.

Fig. 3. Principal coordinates analysis (PCoA), principal components analysis (PCA), and redundancy analysis (RDA) for the dissimilarity of the microbial communities in the different soils after RSD treatment and watermelon cultivation. PCoA for the visualization of pairwise community dissimilarity was calculated based on Bary-Curits distances (a, bacterial; b, fungal). PCA based on bacterial (c) and fungal (d) dominant phyla distribution. RDA ordination plots showed the relationships between dominant general (e, bacterial; f, fungal) and environmental factors, respectively. CK indicates the untreated soil; RSD indicates the soil applied with 2% (v/w) sugar fermentation liquor and irrigated to saturate followed by sealing, “-C” represents after the watermelon cultivation.

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Table 3 The dominant phyla with the relative abundance larger than 2% were found either in the CK or RSD treatment after pre-planting treatment and watermelon cultivation. Dominant phyla

Relative abundance CK† (%)

RSD (%)

CK-C (%)

RSD-C (%)

Bacteria

Proteobacteria Bacteroidetes Firmicutes Actinobacteria TM7 Chloroflexi OD1 Gemmatimonadetes Acidobacteria Chlorobi

18.91 ± 0.60bc 15.16 ± 0.67c 7.26 ± 0.71b 15.76 ± 1.25a 3.81 ± 0.21c 14.30 ± 0.53a 1.03 ± 0.02b 2.94 ± 0.21a 1.67 ± 0.09b 0.31 ± 0.02b

18.06 ± 0.38c‡ 16.59 ± 0.66bc 16.93 ± 0.52a 3.17 ± 0.24c 0.86 ± 0.07d 6.07 ± 0.15b 6.10 ± 0.42a 1.23 ± 0.02c 2.13 ± 0.09a 3.94 ± 0.26a

21.23 ± 1.05ab 19.59 ± 0.59b 6.48 ± 0.35b 6.98 ± 0.24b 14.32 ± 0.20a 2.24 ± 0.08c 1.73 ± 0.27b 1.76 ± 0.11b 1.27 ± 0.15c 0.11 ± 0.02b

21.59 ± 0.77a 24.18 ± 1.78a 6.06 ± 0.44b 7.40 ± 0.52b 11.78 ± 0.24b 2.13 ± 0.04c 1.72 ± 0.10b 2.14 ± 0.19b 0.88 ± 0.04d 0.07 ± 0.01b

Fungi

Ascomycota Basidiomycota Ciliophora Chytridiomycota

40.56 ± 8.04b 14.17 ± 3.71b 10.11 ± 1.46b 1.11 ± 0.41a

27.81 ± 4.08b 14.50 ± 4.38b 19.73 ± 4.88a 2.89 ± 2.73a

30.51 ± 5.99b 61.09 ± 6.04a 4.96 ± 1.86b 0.24 ± 0.18a

60.08 ± 3.96a 24.04 ± 2.92b 8.47 ± 1.54b 0.13 ± 0.05a

† CK indicates the untreated soil; RSD indicates the soil applied with 2% (v/w) sugar fermentation liquor and irrigated to saturate followed by sealing, “-C” represents after the watermelon cultivation. ‡ Values (means ± SE, n = 3) within the same column followed by different letters are significantly different at P < 0.05 according to LSD’s tests.

watermelon growth significantly supported the F. oxysporum proliferation. The population of F. oxysporum was lower in the RSD-C treatment than that of CK-C treatment, suggesting that the RSD treatment still had the impact on pathogen inhibition after one crop season. Furthermore, the ratios of bacteria/fungi and F. oxysporum/fungi are two important indicators of soil health (Janvier et al., 2007). In this study, RSD increased the bacteria/fungi ratio and significantly declined the F. oxysporum/fungi ratio, and consequently, both ratios showed an opposite pattern after watermelon cultivation, yet the F. oxysporum/ fungi ratio was still significantly lower in the RSD-C soil compared to the CK-C soil, respectively. These results indicated that the soil health might be improved after RSD treatment and then declined after watermelon cultivation. It is well recognized that soil community structure plays an important role on plant growth and disease suppression (Mendes et al., 2011; Weller et al., 2002). Thus, many researchers devoted to improve soil health and promote plant growth by improving soil community structure, such as applying bioorganic fertilizer and replacing continuous cultivation by intercropping (Ling et al., 2010; Ren et al., 2008; Wu et al., 2009). In this study, Miseq sequencing showed that the soil microbial communities significantly improved after RSD treatment compared with CK, which was coincide with our previous studies (Huang et al., 2016; Liu et al., 2016). For example, the relative abundances of bacterial genera Uc-Bacteroidales, Flavisolibacter, Uc-lgnavibacteriaceae, and Gracilibacter as well as fungal genus Peziza significantly increased after RSD treatment, especially these increases of bacterial dominant genera changed by tens of percent in relative abundances. Previous studies have demonstrated that Flavisolibacter (Yoon and Im, 2007) and Peziza (Bojsen et al., 1999) are positive in producing some β-glucosidase, N-acetyl-β-glucosaminidase, and α-1, 4glucan lyase. Coincidentally, the facultative anaerobic genera Uc-Bacteroidales and Uc-lgnavibacteriaceae can decompose some polysaccharides, such as amylose, dextran, D-mannose, D-maltose, etc. (Bäckhed et al., 2004; Iino, 2014; Salyers et al., 1977). In addition, although the Gracilibacter was the only dominant genus belonging to Firmicutes that was in consistent with the RSD treatment with the incorporation of solid organic matters (Huang et al., 2015b, 2016; Liu et al., 2016), the functions of producing organic acids and ethonal to suppress pathogens were still conformed to the characteristics of Firmicutes (Lee et al., 2006). Unsurprising, these three types of genera significantly increased mainly due to the stimulation of SF and probably cooperated to suppress F. oxysporum. Furthermore, Davidiella (Bensch

Monosporascus showed an opposite pattern, being reduced after RSD treatment and increased more than tens of percent after the watermelon cultivation (Fig. 4). Moreover, the fungal genera Arthrobotrys, Davidiella, and Monosporascus re-assembled after watermelon cultivation were accounting for more than 47.61% of the Ascomycota phylum. 3.6. The relationship between soil dominant genera and environmental factors The redundancy analysis (RDA, Fig. 3e, f) showed that the significantly increased bacterial and fungal genera that described above in the RSD treatment had a significant (P < 0.05) positive correlation with soil pH and negative correlations with disease index (DI) as well as the populations of F. oxysporum. However, the relative abundances of bacterial genera Uc-TM7-1 and Uc-Xanthomonadaceae as well as fungal genera Uc-Stephanosporaceae, Conocybe, Arthrobotrys, and Monosporascus showed an opposite pattern, which had a significant (P < 0.05) negative correlation with soil pH and positive correlations with DI and the populations of F. oxysporum. 4. Discussion Pathogens suppression is likely a key factor in controlling soil-borne disease (Klein et al., 2013; Zhao et al., 2017). In this study, RSD significantly inhibited the population size of F. oxysporum, which was consistent with several studies on potato (Messiha et al., 2007), carnation (Yossen et al., 2008), spinach (Mowlick et al., 2013a), and strawberry (Ebihara and Uematsu, 2014). Our previous study identified that the organic acids produced during RSD was one of the determinants on soil-borne pathogens suppression, and its production was mainly driven by the microbial decomposition of EOC in organic materials (Huang et al., 2015b; Liu et al., 2016). The good pathogen reduction in this study was likely due to the high EOC content in SF. However, the population of F. oxysporum rapidly resurged during watermelon growth in the RSD-C soil, which was possibly due to the positive stimulation by specific root exudates such as salicylic acids and cinnamic acids (Ren et al., 2016; Ye et al., 2006). This result was coincided with Zhao et al. (2017), observing that the F. oxysporum was considerably suppressed after ammonia gas fumigation and significantly restored with the cucumber growth. In addition, the population of F. oxysporum also remarkably increased in the CK treatment (CK vs. CK-C), indicating that the root exudates released during 6


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Fig. 4. The relative abundance of the top ten bacterial and the top ten fungal general found in all of the treatments after RSD treatment and watermelon cultivation. Error bars represent the standard error of the means of three replicates. Bars with different letters represent significant differences according to LSD’s tests (P < 0.05). The letters following “Uc-” represent the most detailed classifications of the unclassified genus. CK indicates the untreated soil; RSD indicates the soil applied with 2% (v/w) sugar fermentation liquor and irrigated to saturate followed by sealing, “-C” represents after the watermelon cultivation.

soil microbial community responds to plant cultivation and re-assembles during its growth are still unclear. In the present study, the RSDregulated soil microbial community shaped again with the watermelon cultivation, and the difference between CK-C and RSD-C treatments was shrunken, especially the bacterial community. It was inconsistent with the findings of Mowlick et al. (2013a) which using clone library analyses based on 16S rRNA gene sequences revealed that the soil microbial community in the RSD treated soil did not recover fully to the levels of untreated soil with the cultivation of spinach. It was likely due to that the root exudates released by watermelon had enough powerful driving force than those released by spinach or other crops and thus selected similar taxa by filtering the microbial seed bank. Interestingly, Mowlick et al. (2013b) also found that the recover potential of soil microbial community in the Chloropicrin treated soil is weaker than that in the RSD treated soil when after the cultivation of spinach, similar to the observations in the RSD treatment with watermelon cultivation. It was probably because of that the soil microbial community in the fumigated soil changed more intensely than that in the RSD

et al., 2012) and Uc-Pezizomycetes (Hansen et al., 2013) reported as pathogenic fungi significantly decreased after the RSD treatment. Thus, the decreases in these potential pathogenic fungi and the increases in the potential beneficial microorganisms after RSD treatment could improve soil health and provide a safe environment for the watermelon growth. In addition to the RSD treatment, plants also have the capacity to shape the soil microbial community in turn mainly by root exudates (Haichar et al., 2008). For example, Chaparro et al. (2013) revealed that Arabidopsis could secrete high levels of sugars and sugar alcohols in the early developmental stage, thus randomly recruit certain soil microbial taxa from bulk soil to colonize the rhizosphere. However, in the late developmental stage, Arabidopsis root will release higher levels of phenolic acids to further exert a selection on other specialized microbial taxa presumably for specific functions such as disease suppression, nutrient cycling’s improvement, etc. (Chaparro et al., 2014). These results indicate that plant is capable to select a distinct microbial community from microbial seed banks. Nonetheless, how RSD-regulated 7


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Bacteroidales, Flavisolibacter, Uc-lgnavibacteriaceae, Gracilibacter and Peziza significantly increased and negatively correlated with watermelon DI. However, this non-pathogenic consortium was quickly reshaped to a non-plant-preferred microbiome and similar with CK when after the watermelon cultivation, such as the potential pathogenic microorganisms Uc-Xanthomonadaceae, Conocybe, Davidiella, and Monosporascus significantly increased and positively correlated with watermelon DI. These results illustrated the hypothesis 2 that watermelon cultivation is capable to neutralize the dissimilarity of microbial community created by RSD treatment.

treatment, and thus resulted in the root exudates released by plant could not recruit all of the microorganisms. In details, these beneficial dominant genera in the RSD soil dramatically decreased with the watermelon cultivation, meanwhile, the Uc-Chitinophagaceae, Uc-TM7-1, Uc-Xanthomonadaceae, Conocybe, Arthrobotrys, Davidiella, and Monosporascus changed by tens of percent due to the pathogenic process and became the common dominant genera in both CK-C and RSD-C soils. The genus of Uc-Chitinophagaceae can degrade chitin and cellulose (Rosenberg, 2014), and its increase might be due to the stimulation of substrates from watermelon. Previous studies have reported that the genera of Uc-TM7-1 (Winsley et al., 2014) and Arthrobotrys (Liou and Tzean, 1997; Stirling et al., 1998) favor colonizing in the acidic soil, which is in consistent with our study that there was a significant negative correlation between soil pH and the relative abundances of these two genera. Terribly, the Uc-Xanthomonadaceae as a xylem-limited plant pathogen could colonize in plant vascular and possess a cellulose-binding domain by releasing endoglucanase EngXCA and 1, 4-beta cellobiosidase CbhA (Pieretti et al., 2009). In addition, previous study has demonstrated that the Conocybe could produce some toxins, such as ama toxins and phallo toxins (Hallen et al., 2003), were harmful to plants or fruits growth. Moreover, the genera of Davidiella (as a synonym of Cladosporium) and Monosporascus respectively induce the most common leaf spot and root rot diseases during the watermelon plant growth (Bensch et al., 2012; Cohen et al., 2000; Kwon et al., 1999). Thus, the resurgence of F. oxysporum combined with the increases in these potential plant pathogens severely restricted the watermelon growth in the late developmental stage. Collectively, RSD treatment could considerably reduce soil conduciveness to Fusarium wilt and these conduciveness was quickly resurged after watermelon planting via the influence of root exudation by watermelon. These results were accorded with the previous findings that the beneficial microbiome reshaped to a pathogenic microbiome via the influence of root exudation by peanut is the main mechanism leading to the occurrence of soil-borne disease (Li et al., 2014). Besides, the micro-environmental conditions employed in this study cannot perfectly reflect the open field conditions, further field experiment is needed to verify these results. After the 90 days cultivation, the disease index was significantly reduced to 42.22% in the RSD treatment, with a control efficacy of 42.42%. This efficacy was lower than the control strategies such as intercropping with aerobic rice (Ren et al., 2008) or applying bioorganic fertilizer application (Wu et al., 2009). It was likely due to the relative short planting time in these control strategies, such as 40 days cultivation of Ren et al. and 63 days cultivation of Wu et al., whereas the high-incidence of watermelon Fusarium wilt often occurs in the late developmental stage of watermelon planting (Martyn, 2014). Recent research also revealed that biogas slurry amendment had no significant effect on watermelon disease suppression after transplanting for 70 days but it considerably delayed the incidence time and alleviated the disease symptom compared to control (Cao et al., 2016), similar to the observations in this study. Thus, additional strategies such as bioorganic fertilizer application might still be required to develop the sustainable disease suppression of watermelon following RSD treatment.

Acknowledgements This study was financially supported by the National Key Research and Development Program of China (2017YFD0200600), the National Natural Science Foundation of China (Grant No. 41771281, 41701277), the Postgraduate Research and Practice Innovation Program of Jiangsu Province, China (KYCX17_1062), the Priority Academic Program Development (PAPD) of Jiangsu Higher Education Institutions and the Key Subjects of Jiangsu Province, China (Ecology). Declaration of interest. The authors declare that they have no conflicts of interest. This research did not involve human participants or any animal experimentation. Appendix A. Supplementary data Supplementary data associated with this article can be found, in the online version, at http://dx.doi.org/10.1016/j.apsoil.2018.05.004. References Adam, G., Duncan, H., 2001. Development of a sensitive and rapid method for the measurement of total microbial activity using fluorescein diacetate (FDA) in a range of soils. Soil Biol. Biochem. 33, 943–951. Bäckhed, F., Ding, H., Wang, T., Hooper, L.V., Koh, G.Y., Nagy, A., Semenkovich, C.F., Gordon, J.I., 2004. The gut microbiota as an environmental factor that regulates fat storage. PNAS 101, 15718–15723. Bensch, K., Braun, U., Groenewald, J.Z., Crous, P.W., 2012. The genus Cladosporium. Stud. Mycol. 72, 1–401. Blok, W.J., Lamers, J.G., Termorshuizen, A.J., Bollen, G.J., 2000. Control of soilborne plant pathogens by incorporating fresh organic amendments followed by tarping. Phytopathology 90, 253–259. Bojsen, K., Yu, S., Marcussen, J., 1999. A group of α-1,4-glucan lyase genes from the fungi Morchella costata, M.vulgaris and Peziza ostracoderma. Cloning, complete sequencing and heterologous expression. Plant Mol. Biol. 40, 445–454. Bremner, J.M., Jenkinson, D.S., 1960. Determination of organic carbon in soil. Eur. J. Soil Sci. 11, 394–402. Bremner, J.M., 1960. Determination of nitrogen in soil by the Kjeldahl method. J. Agric. Sci. 55, 11–33. Cao, Y., Wang, J.D., Wu, H.S., Yan, S.H., Guo, D.J., Wang, G.F., 2016. Soil chemical and microbial responses to biogas slurry amendment and its effect on Fusarium wilt suppression. Appl. Soil Ecol. 107, 116–123. Caporaso, J.G., Kuczynski, J., Stombaugh, J., Bittinger, K., Bushman, F.D., Costello, E.K., Fierer, N., Peña, A.G., Goodrich, J.K., Gordon, J.I., 2010. QIIME allows analysis of high-throughput community sequencing data. Nat. Methods 7, 335–336. Cebolla, V., Busto, J., Ferrer, A., Miguel, A., Maroto, J.V., 2000. Methyl bromide alternatives on horticultural crops. Acta Hortic. 532, 237–242. Chaparro, J.M., Badri, D.V., Bakker, M.G., Sugiyama, A., Manter, D.K., Vivanco, J.M., 2013. Root exudation of phytochemicals in Arabidopsis follows specific patterns that are developmentally programmed and correlate with soil microbial functions. PLoS One 8, 525–534. Chaparro, J.M., Badri, D.V., Vivanco, J.M., 2014. Rhizosphere microbiome assemblage is affected by plant development. ISME J. 8, 790–803. Cohen, R., Pivonia, S., Burger, Y., Edelstein, M., Gamliel, A., Katan, J., 2000. Toward integrated management of monosporascus wilt of melons in Israel. Plant Dis. 84, 496–505. Ebihara, Y., Uematsu, S., 2014. Survival of strawberry-pathogenic fungi Fusarium oxysporum f. sp. fragariae, Phytophthora cactorum and Verticillium dahliae under anaerobic conditions. J. Gen. Plant Pathol. 80, 50–58. Edgar, R.C., Haas, B.J., Clemente, J.C., Quince, C., Knight, R., 2011. UCHIME improves sensitivity and speed of chimera detection. Bioinformatics 27, 2194–2200. Gamliel, A., Austerweil, M., Kritzman, G., 2000. Non-chemical approach to soil-borne pest management–organic amendments. Crop Prot. 19, 847–853. Goud, J.C., Termorshuizen, A.J., Blok, W.J., van Bruggen, A.H.C., 2004. Long-term effect of biological soil disinfestation on Verticillium wilt. Plant Dis. 88, 688–694.

5. Conclusion To the best of our knowledge, this is the first study to evaluate the effects of crop cultivation on the abundance of soil-borne pathogen and community assembly after RSD treatment. This research revealed that RSD with SF could significantly inhibit the F. oxysporum and suppress the Fusarium wilt of watermelon. Nevertheless, the population of F. oxysporum would be considerably recovered after planting watermelon for one season. Moreover, RSD treatment could control the Fusarium wilt of watermelon by enhancing soil conduciveness to a non-pathogenic consortium, such as the potential beneficial microorganisms Uc8


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Mowlick, S., Yasukawa, H., Inoue, T., Takehara, T., Kaku, N., Ueki, K., Ueki, A., 2013a. Suppression of spinach wilt disease by biological soil disinfestation incorporated with Brassica juncea plants in association with changes in soil bacterial communities. Crop Prot. 54, 185–193. Mowlick, S., Inoue, T., Takehara, T., Kaku, N., Ueki, K., Ueki, A., 2013b. Changes and recovery of soil bacterial communities influenced by biological soil disinfestation as compared with chloropicrin-treatment. AMB Express. 3, 46–57. Pieretti, I., Royer, M., Barbe, V., Carrere, S., Koebnik, R., Cociancich, S., Couloux, A., Darrasse, A., Gouzy, J., Jacques, M.A., Lauber, E., Manceau, C., Mangenot, S., Poussier, S., Segurens, B., Szurek, B., Verdier, V., Arlat, M., Rott, P., 2009. The complete genome sequence of Xanthomonas albilineans provides new insights into the reductive genome evolution of the xylem- limited Xanthomonadaceae. BMC Genomics 10, 616–630. Qiu, M.H., Zhang, R.H., Xue, C., Zhang, S.S., Li, S.Q., Zhang, N., Shen, Q.R., 2012. Application of bio-organic fertilizer can control Fusarium wilt of cucumber plants by regulating microbial community of rhizosphere soil. Biol. Fert. Soils 48, 807–816. Ren, L.X., Su, S.M., Yang, X.M., Xu, Y.C., Huang, Q.W., Shen, Q.R., 2008. Intercropping with aerobic rice suppressed Fusarium wilt in watermelon. Soil Biol. Biochem. 40, 834–844. Ren, L.X., Huo, H.W., Zhang, F., Hao, W.Y., Xiao, L., Dong, C.X., Xu, G.H., 2016. The components of rice and watermelon root exudates and their effects on pathogenic fungus and watermelon defense. Plant Signal. Behav. 11, e1187357. Rosenberg, E., 2014. The Family Chitinophagaceae. In: Rosenberg, E., Delong, E.F., Lory, S., Stackebrandt, E., Thompson, F. (Eds.), The prokaryotes: Other Major Lineages of Bacteria and The Archaea. Springer-Verlag Berlin Heidelberg Publishing, Germany, pp. 493–495. Salyers, A.A., Vercellotti, J.R., West, S.E.H., Wilkins, T.D., 1977. Fermentation of mucin and plant polysaccharides by strains of Bacteroides from the human colon. Appl. Environ. Microb. 33, 319–322. Shennan, C., Muramoto, J., Lamers, J., Mazzola, M., Rosskopf, E.N., Kokalis-Burelle, N., Momma, N., Butler, D.M., Kobara, Y., 2014. Anaerobic soil disinfestation for soil borne disease control in strawberry and vegetable systems: current knowledge and future directions. Acta Hortic. 1044, 165–175. Shinmura, A., 2000. Causal agent and control of root rot of welsh onion. PSJ soil-borne disease workshop report. 20, pp. 133–143 (in Japanese with English Summary). Shinmura, A., 2004. Principle and effect of soil sterilization method by reducing redox potential of soil. PSJ soil-borne disease workshop report. 22, pp. 2–12 (in Japanese with English Summary). Stirling, G.R., Smith, L.J., Licastro, K.A., Eden, L.M., 1998. Control of root-knot nematode with formulations of the nematode-trapping fungus Arthrobotrys dactyloides. Biol. Control. 11, 224–230. Wang, Q., Garrity, G.M., Tiedje, J.M., Cole, J.R., 2007. Naïve bayesian classifier for rapid assignment of rRNA sequences into the new bacterial taxonomy. Appl. Environ. Microb. 73, 5261–5267. Wei, Z., Yang, X.M., Yin, S.X., Shen, Q.R., Ran, W., Xu, Y.C., 2011. Efficacy of Bacillusfortified organic fertiliser in controlling bacterial wilt of tomato in the field. Appl. Soil Ecol. 48, 152–159. Weller, D.M., Raaijmakers, J.M., Gardener, B.B.M., Thomashow, L.S., 2002. Microbial populations responsible for specific soil suppressiveness to plant pathogens. Annu. Rev. Phytopathol. 40, 309–348. Winsley, T.J., Snape, I., McKinlay, J., Stark, J., van Dorst, J.M., Ji, M., Ferrari, B.C., Siciliano, S.D., 2014. The ecological controls on the prevalence of candidate division TM7 in polar regions. Front. Microbiol. 5, 345–354. Wu, H.S., Yang, X.N., Fan, J.Q., Miao, W.G., Ling, N., Xu, Y.C., Huang, Q.W., Shen, Q.R., 2009. Suppression of Fusarium wilt of watermelon by a bio-organic fertilizer containing combinations of antagonistic microorganisms. BioControl 54, 287–300. Ye, S.F., Zhou, Y.H., Sun, Y., Zou, L.Y., Yu, J.Q., 2006. Cinnamic acid causes oxidative stress in cucumber roots, and promotes incidence of Fusarium wilt. Environ. Exp. Bot. 56, 255–262. Yoon, M.H., Im, W.T., 2007. Flavisolibacter ginsengiterrae gen. nov., sp. nov. and Flavisolibacte ginsengisoli sp. nov., isolated from ginseng cultivating soil. Int. J. Syst. Evol. Micr. 57, 1834–1839. Yossen, V., Zumelzu, G., Gasoni, L., Kobayashi, K., 2008. Effect of soil reductive sterilisation on Fusarium wilt in greenhouse carnation in Córdoba, Argentina. Australas. Plant Path. 37, 520–522. Zhao, J., Ni, T., Li, Y., Xiong, W., Ran, W., Shen, B., Shen, Q.R., Zhang, R.H., 2014. Responses of bacterial communities in arable soils in a rice-wheat cropping system to different fertilizer regimes and sampling times. PLoS One 9, e85301. Zhao, J., Ni, T., Li, J., Lu, Q., Fang, Z.Y., Huang, Q.W., Zhang, R.H., Li, R., Shen, B., Shen, Q.R., 2016. Effects of organic–inorganic compound fertilizer with reduced chemical fertilizer application on crop yields, soil biological activity and bacterial community structure in a rice-wheat cropping system. Appl. Soil Ecol. 99, 1–12. Zhao, J., Mei, Z., Zhang, X., Xue, C., Zhang, C.Z., Ma, T.F., Zhang, S.S., 2017. Suppression of Fusarium wilt of cucumber by ammonia gas fumigation via reduction of Fusarium population in the field. Sci. Rep. 7, 43103.

Haichar, F.E., Marol, C., Berge, O., Rangel-Castro, J.I., Prosser, J.I., Balesdent, J., Heulin, T., Achouak, W., 2008. Plant host habitat and root exudates shape soil bacterial community structure. ISME J. 2, 1221–1230. Hallen, H.E., Watling, R., Adams, G.C., 2003. Taxonomy and toxicity of Conocybe lactea and related species. Mycol. Res. 107, 969–979. Hansen, K., Perry, B.A., Dranginis, A.W., Pfister, D.H., 2013. A phylogeny of the highly diverse cup-fungus family Pyronemataceae (Pezizomycetes, Ascomycota) clarifies relationships and evolution of selected life history traits. Mol. Phylogenet. Evol. 67, 311–335. Hopkins, D.L., Elmstrom, G.W., 1984. Fusarium wilt in watermelon cultivars grown in a 4-year monoculture. Plant Dis. 68, 129–131. Huang, X.Q., Wen, T., Zhang, J.B., Meng, L., Zhu, T.B., Liu, L.L., Cai, Z.C., 2015a. Control of soil- borne pathogen Fusarium oxysporum by biological soil disinfestation with incorporation of various organic matters. Eur. J. Plant Pathol. 143, 223–235. Huang, X.Q., Wen, T., Zhang, J.B., Meng, L., Zhu, T.B., Cai, Z.C., 2015b. Toxic organic acids produced in biological soil disinfestation mainly caused the suppression of Fusarium oxysporum f. sp. cubense. BioControl 60, 113–124. Huang, X.Q., Liu, L.L., Wen, T., Zhang, J.B., Wang, F.H., Cai, Z.C., 2016. Changes in the soil microbial community after reductive soil disinfestation and cucumber seedling cultivation. Appl. Microbiol. Biotechnol. 100, 5581–5593. Iino, T., 2014. The Family Ignavibacteriaceae. In: Rosenberg, E., Delong, E.F., Lory, S., Stackebrandt, E., Thompson, F. (Eds.), The prokaryotes: Other Major Lineages of Bacteria and The Archaea. Springer-Verlag Berlin Heidelberg Publishing, Germany, pp. 701–703. IUSS, 2015. IUSS Working Group WRB. World Reference Base for Soil Resources 2014, update 2015 International soil classification system for naming soils and creating legends for soil maps. World Soil Resources Reports No. 106. FAO, Rome. Janvier, C., Villeneuve, F., Alabouvette, C., Edel-Hermann, V., Mateille, T., Steinberg, C., 2007. Soil health through soil disease suppression: which strategy from descriptors to indicators? Soil Biol. Biochem. 39, 1–23. Klein, E., Ofek, M., Katan, J., Minz, D., Gamliel, A., 2013. Soil suppressiveness to Fusarium disease: shifts in root microbiome associated with reduction of pathogen root colonization. Phytopathology 103, 23–33. Kõljalg, U., Nilsson, R.H., Abarenkov, K., Tedersoo, L., Taylor, A.F.S., Bahram, M., Bates, S.T., Brus, T.D., Bengtsson-Palme, J., Callaghan, T.M., 2013. Towards a unified paradigm for sequence-based identification of fungi. Mol. Ecol. 22, 5271–5277. Kwon, M.K., Hong, J.R., Cho, B.H., Ki, U.K., Kim, K.C., 1999. A scab disease caused by Cladosporium cucumberinum on watermelon seedlings. Plant Pathol. J. 15, 72–75. Lee, Y.J., Romanek, C.S., Mills, G.L., Davis, R.C., Whitman, W.B., Wiegel, J., 2006. Gracilibacter thermotolerans gen. nov. sp. nov. an anaerobic, thermotolerant bacterium from a constructed wetland receiving acid sulfate water. Int. J. Syst. Evol. Micr. 56, 2089–2093. Li, X.G., Ding, C.F., Hua, K., Zhang, T.L., Zhang, Y.N., Zhao, L., Yang, Y.R., Liu, J.G., Wang, X.X., 2014. Soil sickness of peanuts is attributable to modifications in soil microbes induced by peanut root exudates rather than to direct allelopathy. Soil Biol. Biochem. 78, 149–159. Ling, N., Xue, C., Huang, Q.W., Yang, X.M., Xu, Y.C., Shen, Q.R., 2010. Development of a mode of application of bioorganic fertilizer for improving the biocontrol efficacy to Fusarium wilt. BioControl 55, 673–683. Liou, G.Y., Tzean, S.S., 1997. Phylogeny of the genus Arthrobotrys and allied nematodetrapping fungi based on rDNA sequences. Mycologia 89, 876–884. Liu, L.L., Kong, J.J., Cui, H.L., Zhang, J.B., Wang, F.H., Cai, Z.C., Huang, X.Q., 2016. Relationships of decomposability and C/N ratio in different types of organic matter with suppression of Fusarium oxysporum and microbial communities during reductive soil disinfestation. Biol. Control. 101, 103–113. Martyn, R.D., 2014. Fusarium wilt of watermelon: 120 years of research. Hortic. Rev. 42, 349–442. McDonald, D., Price, M.N., Goodrich, J., Nawrocki, E.P., DeSantis, T.Z., Probst, A., Andersen, G.L., Knight, R., Huagenholtz, P., 2012. An improved Greengenes taxonomy with explicit ranks for ecological and evolutionary analyses of bacteria and archaea. ISME J. 6, 610–618. Mendes, R., Kruijt, M., de Bruijn, I., Dekkers, E., van der Voort, M., Schneider, J.H.M., Piceno, Y.M., DeSantis, T.Z., Andersen, G.L., Bakker, P.A.H.M., Raaijmakers, J.M., 2011. Deciphering the rhizosphere microbiome for disease-suppressive bacteria. Science 332, 1097–1100. Messiha, N.A.S., van Diepeningen, A.D., Wenneker, M., van Beuningen, A.R., Janse, J.D., Coenen, T.G.C., Termorshuizen, A.J., van Bruggen, A.H.C., Blok, W.J., 2007. Biological Soil Disinfestation (BSD), a new control method for potato brown rot, caused by Ralstonia solanacearum race 3 biovar 2. Eur. J. Plant Pathol. 117, 403–415. Miguel, A., Maroto, J.V., San Bautista, A., Baixauli, C., Cebolla, V., Pascual, B., López, S., Guardiola, J.L., 2004. The grafting of triploid watermelon is an advantageous alternative to soil fumigation by methyl bromide for control of Fusarium wilt. Sci. Hortic. 103, 9–17. Momma, N., Kobara, Y., Uematsu, S., Kita, N., Shinmura, A., 2013. Development of biological soil disinfestations in Japan. Appl. Microbiol. Biotechnol. 97, 3801–3809.

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