IJAMBR 8 (2020) 21-32 ISSN 2053-1818

Plant-derived allyl isothiocyanate enrich biocontrol doi.org/10.33500/ microbes of tomato plant rhizosphere in vegetable ijambr.2020.08.003 greenhouse

Xiangmei Yao1,3*, Zhijun Lu2,3, Cece Yin1, Qiong Fu2 and Jian Ye1*

1State Key Laboratory of Plant Genomics, Institute of Microbiology, Chinese Academy of Sciences, Beijing, 100101, China. 2Laboratory of Pests Detection, Beijing Plant Protection Station, Beijing, 100029, China. 3These authors contributed equally to this work.

Article History ABSTRACT Received 20 May, 2020 Natural plant-derived metabolites have been widely used for human nutrients, Received in revised form 17 antitumor, antimicrobial agents. Glucosinolate is the most abundant secondary June, 2020 Accepted 18 June, 2020 metabolites in the botanical order Brassicales. Recently allyl isothiocyanate (AITC) has been applied as a soil fumigant in greenhouse vegetable production to Keywords: control soil-borne diseases. Although AITC is known to directly inhibit bacterial Allyl isothiocyanate, and fungal pathogens and nematodes, it is not clear if it could also enrich the Rhizosphere beneficial microbes associated with soil. In this study, bacterial and fungal microbiome, communities from AITC-treated soil, with- or without- tomato plants, were studied Bacterial community, in a greenhouse vegetable production facility. Microbiome structures differed Fungal community, between the AITC-treated soil, with- or without- tomato plants. Fungal community Soil-borne diseases. diversity was significantly decreased in phylum and species level in two types of AITC-treated soils. The fungal phyla Basidiomycota, Glomeromycota, Zygomycota, and Chytridiomycota were decreased in AITC-treatment soils when the plants were presented. Populations of the fungal pathogens in the genus Alternaria, Aspergillus sesamicola and Aspergillus westerdijkiae were decreased in the AITC-treated tomato rhizosphere. Three potential beneficial bacterial isolates were enriched from the genera Acinetobacter and Pseudomonas communities. These were confirmed to have antifungal and antibacterial properties in pot growth plants. Further bioassays confirmed their roles in the suppression of bacterial wilt pathogens. The data suggest that AITC was critical to the characteristic microorganism of the microbial community in bulk soil, but when tomato plants were present, the plant had a greater effect on soil characteristic microorganism than AITC. AITC not only diminished some Article Type: pathogenic fungi, but also enriched beneficial biocontrol microbes and reduced Full Length Research Article disease of tomato by suppressing bacterial and fungal pathogens in the soil. ©2020 Blue Pen Journals Ltd. All rights reserved

INTRODUCTION

Healthy soil is critical for food safety and human health. microbial community composition and structure (Berg and Unfortunately, serious deterioration of agricultural soil has Smalla, 2009; Lu et al., 2013; Li et al., 2014). An been exacerbated by over cropping, enrichment of incremental accumulation of soil-borne pathogens, allelopathic substances and degradation of soil microbial simplification of the beneficial microbial community and communities (Vargas et al., 2009; Napper and Thompson, decreased microbial diversity are important factors that 2019). Continuous cropping results in disruption of soil influence plant growth and yields caused by continuous

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cropping (Xiong et al., 2015; Li et al., 2016a). To ensure activities, including anti-, anti-fungi, anti-tumor and quantity and quality of plant products, modern agricultural anti-proliferation of cancer cells (Szymanska et al., 2018). practices largely rely on pesticides to protect crops against Treatment of soil with fumigants prior to planting has been various plant diseases and insect pests. Pesticide shown to reduce soil-borne diseases. Earlier studies also applications can dramatically deteriorate soil health, either show that ITCs treatment or glucosinolate-containing by inhibiting microbial biology processes (Puglisi et al., plants have a less impacts on soil bacterial composition, 2012; Feld et al., 2015), or by reducing the abundance but significant reduction in fungal populations (Hollister et and/or diversity of key microbial populations (Tsiamis et al., al., 2013; Rumberger and Marschner, 2003; Hu et al., 2014; Romdhane et al., 2016). Some pesticides can also 2014). AITC, one kind of ITCs, has been widely used as a impose a selection on certain microbial populations soil fumigant agent for controlling soil-borne diseases harboring specific catabolic genes that allow them to use (Romeo et al., 2018; Ren et al., 2018). AITC is rapidly pesticides as nutrient or/and energy source(s) (Devers et sorbed and degraded in soil and has a low risk of al., 2008; Gallego et al., 2019). The widely used pesticides persistence. Moreover, the flashpoint and acute toxicity of oxamyl, hexachlorocyclohexanes (HCHs) and AITC are safer and have a lower toxicity than methyl-ITC dichlorodiphenyltrichloroethanes (DDTs) induced changes (Vig et al., 2009; Ren et al., 2018). Recently, AITC has of function microorganisms and microbial community been further evaluated as a potential replacement for the structure (Sun et al., 2019; Gallego et al., 2019). Pesticide fumigant, methyl bromide, which can cause ozone overuse has also led to progressive contamination on depletion, central nervous system depression and damage natural resources and destruction of microbial to the olfactory epithelium (Ren et al., 2018). Several soil communities (Gilden et al., 2010). Therefore, there is an fumigants methyl iodide have been phased out or their urgent need for a safer and healthier alternative product registration canceled in some countries or worldwide used for plant protection. because of their potential or actual environmental risk or Over the past three decades, natural plant-derived their mammalian toxicity (Yin et al., 2014; Guo et al., 2004). metabolites, especially the usage of Brassicaceae plants However, it is not known if AITC is a microbiome-friend residues for control of plant diseases and pests provided which could promote enrichment of beneficial microbes alternative opportunities for plant protection for modern associated with soil. For this reason, our research agriculture. Plants possess an almost limitless ability to highlights the efforts needed to gain a better understanding synthesize aromatic substances, most of which are of the efficacy of plant extracts such as AITC effect soil terpenoids, alkaloids, ketones, polyphenols, phenols, microbial community structure with or without AITC flavonoids, ketones, and essential oils, and these aromatic treatment, and enrich beneficial biocontrol microbes by substances have an important role in self-defense suppressing bacterial and fungal pathogens in soil. mechanisms against microbes, herbivores, and insect In this study, we adopted a high-throughput sequencing predation (Benelli et al., 2016). These natural-derived method to compare the bacterial and fungal community compounds cause no or less damage to the environment structures with or without AITC treatment under and human health (Szymanska et al., 2018; Sut et al., greenhouse conditions. We found that bacterial 2017). To dampen the problem of continuous cropping community changes are only observed in AITC treatment obstacle, there will be an increasing demand to discover tomato plant rhizosphere group. Some enriched beneficial more reliable and eco-friendly alternative products with bacterial isolates from AITC-treated tomato plant lower risks for environmental damage to replace high toxic rhizosphere soil have antifungal and antibacterial chemical by plant-derived metabolites (Yang et al., 2014; activities. At the same time, some plant-pathogenic fungi Sun et al., 2016). such as Alternaria, Aspergillus sesamicola and Aspergillus Natural plant protection has been commonly used by westerdijkiae were significantly decreased in phylum and human being since the origins of agriculture. Brassicaceae species level in two types of AITC-treated tomato plant plants are rich in secondary metabolites and contain rhizosphere group. These data suggest that AITC will be a numerous antioxidants and phytoalexins substances such microbiome-friend plant derived metabolite used for plant as glucosinolate (GLs)-related contents. Isothiocyanates protection as an alternative to chemical pesticides. (ITCs), hydrolysis product of GLs, including allyl, butyl, phenyl, and benzyl ITC are the most abundant bioactive compounds of Brassicaceae plants. The characteristic MATERIALS AND METHODS flavors and odors of isothiocyanates are effective at repelling insects, and also possess several biological Experiment design, soil samples collection and DNA extraction

We set up two similar field greenhouses having identical *Corresponding authors. E-mail: [email protected], soil conditions. Each greenhouse was about 667 square [email protected]. meters. The two neighboring greenhouses were at a farm

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of Chang ping district, Beijing, China (N40° 12′ 12.99″, reference operational taxonomic units (OTUs) picking E116° 22′ 51.66″). In August, the outside average protocol in the UPARSE-pipeline was used to cluster at the temperature was about 31.4°C in Beijing, China. We ≥97% sequence similarity level of bacteria and fungil deeply plowed the land and then sprayed AITC at a (Edgar, 2010, 2013), and the most abundant sequences concentration of about 40 mg m-2. Subsequently, we from each OTU were taken as representative sequences covered the soil with a layer of plastic film to maintain a for the respective OTU. The taxonomic configuration of high temperature and humidity conditions for three days. bacterial OTUs was performed using the Silva database At that time we uncovered the plastic film. The same (Quast et al., 2013), and taxa were assigned to all OTUs treatment was done for the control greenhouse except using the RDP classifier within QIIME (Cole et al., 2009). without AITC treatment. At the end of September, cherry tomato plants (Lycopersicon esculentum var. cerasiforme) were transplanted into both soils. Data analyses Soil samples were collected at two time periods. Firstly, AITC-treated (AITC1) and non-treated bulk soil (CK1) Before downstream data analysis, contaminating were collected immediately after uncovering the plastic chloroplast sequences were firstly removed by BBSplit film. Five-point sampling method was used for sample from BBMap to bin the reads (chloro and non-chloro). collection about 15~20 cm depth, followed by soil mixture QIIME were used to calculate the α-Diversity, including the and then divided it into three equal parts. The non-treated observed species and Shannon diversity index based on a (CK2) and AITC-treated tomato plant group (AITC2) subsample of a minimum number of sequences (Caporaso rhizosphere soil were randomly collected from three et al., 2010). Principal component analysis (PCA) was individual plants in each of the two greenhouses in used to analyze the bacterial and fungal community November about two months after transplant. For the variation through the Canoco program for Windows 4.5 rhizosphere soil, the plant roots were gently removed from (Biometris, Wageningen, the Netherlands). A non-metric soil. The soil samples were mixed and placed in 50 mL multidimensional scaling (NMDS) ordination was used to Falcon tubes, frozen in dry ice and stored at -80°C until illustrate the clustering of bacterial and fungal community DNA extraction. DNA was extracted according to the composition variation using the Vegan software based on standard protocols using Power Soil DNA extraction kit the Bray-Curtis distance of OTUs. The significant (MoBio, Carlsbad, CA, USA). The extracted soil DNA was taxonomic differences analysis was used by linear eluted with 80 μL elusion buffer, and then the DNA was discriminant analysis (LDA) together with effect size stored at -80°C for further polymerase chain reaction measurements (LEfSe) (Segata et al., 2011). The (PCR) amplification. The DNA concentration was magnitude effect of differentially abundant taxa was quantified on a Nanodrop 2000 spectrophotometer analyzed by LDA (LDA>4), and the significantly different (Thermo Scientific, Wilmington, DE, USA). abundance of each taxa was calculated by the factorial Kruskal–Wallis sum-rank test (α=0.05). The differences in microbial community among all samples were shown by a Bacterial 16s rDNA and fungal ITS amplicon cladogram which was generated using the taxa. The sequencing analysis was performed in the ‘Galaxy’ module of LEfSe.

High-throughput sequencing was used to investigate the soil microbial diversity, the bacterial and fungal Bioassay of beneficial microbes from AITC-treated composition. Bacterial primers (515F: 5′- rhizosphere soil GTGCCAGCMGCCGCGG-3′, 806R: 5′- GGACTACHVGGGTWTCTAAT-3′) was used to amplify About 5 g AITC-treated rhizosphere soil was suspended the V4 region. For fungal the ITS1 region was amplified by into 95 ml sterilized distilled water, and the soil suspension PCR using primers (ITS5-1737F: 5′- was gradient dilution spread onto Nutrient Agar (NA) GGAAGTAAAAGTCGTAACAAGG-3′, ITS2-2043R: 5′- medium. There were three replicated culture plates for GCTGCGTTCTTCATCGATGC-3′). The paired-end each concentration gradient. The culture plates were sequencing of the bacterial and fungal amplicons was placed in a 28°C incubator and incubated in dark for 2 to 3 sequenced by Novogene (Beijing, China) on the Illumina days. Single colonies were separated from 10-5~10-6 MiSeqPE300 platform (Illumina Inc., San Diego, CA). dilution plates. The separated bacterial colonies were First, the sequence reads were quality filtered by identified by sequence determination through amplification Quantitative Insights Into Microbial Ecology (QIIME) toolkit of the full-length 16S rDNA gene by PCR (universal primer (Caporaso et al., 2011). Chimeras were removed using 27F and 1492R). The isolates were preserved in 50% USEARCH with the UCHIME algorithm (Edgar et al., glycerol and frozen at -80°C for bacterial storage. The 2011). The sequences were analyzed by UPARSE with the isolates were cultured separately in 5 mL NA liquid medium UPARSE-OTU and UPARSE-OTUref algorithms. An open- in glass tube for 200 rpm. The culture broth was

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centrifuged at 12,000 g, and the isolated pellet was total number of plants used in each treatment; V, is the washed three times with sterilized distilled water to remove highest disease index (4) (Li et al., 2017). any supernatant. The bacterial pellet was re-suspended in sterilized distilled water, and the final concentration was diluted to approx. OD600=0.60. Antibacterial activity of the Accession number bacterial isolates against Ralstonia solanacearum was evaluated by co-culturing with three selected isolates on The partial bacterial and fungal OTU data were deposited NA medium plate covered with R. solanacearum. NA in the National Center for Biotechnology Information plates were evenly streaked with 10 µl of 108 CFU/ml R. Sequence Reads Archive with accession number solanacearum suspension. A sterilized Oxford cup with a MK544526 - MK544792 for bacterial and MK564759 - diameter of 10 mm was placed in the central of the plate, MK566180 for fungal. and 100 µl of 108 CFU/ml bacterial isolates suspension was added to the Oxford cup. Inoculated plates were incubated at 28°C for 48 h. And the average diameter of RESULTS the inhibition zone was used as an indicator of antagonistic anti-R. solanacearum effect. The bacterial isolates AITC treatment soil microbiome with- or without- antifungal activity was investigated against five different tomato plants soil-borne fungal pathogens. First, five different soil-borne fungal pathogens growths were cultured in 60 mm Petri The sequencing of the V4 region produced high-quality dishes filled with PDA solid medium for 4 days. Next, the effective tags (942,336) from 12 soil samples (mean center of each Petri dish was inoculated with 5 mm 78,528 sequences per sample, max = 86,040, min = diameter disc of fungal, taken from pure culture (4 days 70,042, SD = 3,687). These reads corresponded to a total old). And 5 µl of 108 CFU/ml fresh bacterial isolates of 10575 OTUs in all four group samples, and there were suspension was added to the Petri dish with 5 cm diameter common 4149 OTUs for bacteria. Each sample had their away from the fungus. Then, all inoculated dishes were own unique OTUs: 515 for CK1 (bulk soil), 671 for AITC1 incubated at 28°C for 3 days. Finally, the antifungal activity (AITC treated bulk soil), 310 for CK2 (control with tomato of each bacterial was calculated in terms of inhibition plants), 570 for AITC2 (Figure 1A). There were substantial percentage of fungal growth. differences in the bacterial and fungal community structures of AITC-treated soil with or without plant. The top five bacterial phyla accounted for about 74.8% of the Tomato plant growth and inoculation assay bacteria microbiome (Figure 1C and Table 1). At the class level, Betaproteobacteria, Deltaproteobacteria and To evaluate whether the enriched bacterial isolates can unidentified Acidobacteria were enriched in AITC2 suppress disease development, we performed a pot compared with other three groups (Table 1). Remarkably, experiment. Tomato Zhongza No.9 seeds were in the presence of tomato plants, Actinobacteria surfacesterilized according to previously reported dramatically decreased by about 34.42 times (Figure 1D protocols (Kwak et al., 2018). The tomato surface- and Table 1). Meanwhile, Firmicutes and Actinobacteria sterilized seeds were germinated in sterilized distilled were enriched about by 20 times in AITC2, and the water and planted into pots filled with soils which contain bacterial species Acinetobacter johnsonii (57 times), 8 10 CFU/g bacterial isolates each gram of soil. For bacterial Acinetobacter calcoaceticus (27 times) and Pseudomonas wilt response, tomato plants were grown for 3~4 weeks at citronellolis (about 3 times) were also considerably 28°C in light for 14 h and in dark for 10 h. Individual plants enriched (Table 1). 8 were watered with 5 ml of 10 CFU/ml bacterial wilt The sequencing of the ITS1 region produced high quality suspension. Disease progress was scored at day 7, 10 and sequences (3,489,918) from 11 soil samples (mean 14 on a disease index scale (0, no wilt symptoms; 1, one 33,237 sequences per sample, max = 38,417; min = leaf on disease side wilted; 2, two or three leaves on 18,844; SD = 3,687; the librarby construct failed for one of disease side wilted; 3, more than two-thirds of the leaves AITC treated bulk soil samples). Similarly, the high-quality on disease side wilted; 4, all the leaves on disease side reads corresponded to a total of 1405 OTUs in all four wilted). The disease index (DI) was calculated using the group samples, and there were common 233 OTUs in all following formula: four groups for fungi. Each group had their own unique OTUs: 131 for CK1, 119 for AITC1, 151 for CK2, 107 for (ni × vi) DI = [∑ ] × 100 AITC2 (Figure 1B). The fungal phyla Ascomycota, (N × V) Basidiomycota, Glomeromycota and Zygomycota accounted for 82.4% of the total fungal microbiomes Where ni, is the number of plants with the respective (Figure 1D and Table 1). The fungal phyla Basidiomycota, disease index; vi, is disease index (0, 1, 2, 3, 4); N, is the Glomeromycota, Zygomycota, and Chytridiomycota were

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Figure 1. Venn diagram show the overlap and distinct among the enriched dynamic taxa OTUs. A, Venn diagram shows composition of the enriched bacterial taxa, the four sample groups showing as four different colors. Sample group names are bulk soil (CK1), AITC1 bulk soil, rhizosphere soil from tomato plant (CK2) and rhizosphere soil from AITC2. B, Venn diagram shows composition of the enriched fungal taxa. C-D, Relative abundances of bacterial (C) and fungal (D) community structures at the phylum level in 4 group samples. Top 10 abundant phyla showing as different colors are defined as those the relative abundances larger than 0.01 of total sequences across all samples, the remainder are combined into a category designated as ‘Others’.

Table 1. The relative abundance in phylum, class and species level for bacterial and fungal OTUs for each sample. The Relative abundance in phylum level for Bacterial and Fungal were about top 10.

CK2 AITC2 CK1 (Bulk AITC1 AITC1/CK1 AITC2/CK2 Kingdom level (rhizosphere (rhizosphere soil) (Bulk soil) (the ratio) (the ratio) soil) soil) Bacterial Phylum 0.457747 0.438918 0.417642 0.44228 0.958866 1.058993 Bacteroidetes 0.066571 0.072482 0.126856 0.133452 1.088792 1.051996 Acidobacteria 0.127592 0.124474 0.140064 0.091849 0.975563 0.655765 Gemmatimonadetes 0.089401 0.09319 0.084049 0.085232 1.042382 1.014075 Firmicutes 0.008764 0.00943 0.008673 0.04859 1.075993 5.602444 Actinobacteria 0.038115 0.04076 0.02736 0.054567 1.069395 1.994408 Verrucomicrobia 0.037617 0.042802 0.041512 0.022124 1.137837 0.532954 Nitrospirae 0.039633 0.029208 0.016666 0.018697 0.736962 1.121865

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Table 1. Contd.

Planctomycetes 0.028888 0.030315 0.043766 0.025491 1.049398 0.582438 Chloroflexi 0.028228 0.038333 0.019377 0.019443 1.357978 1.003406 Others 0.077443 0.080089 0.074036 0.058274 1.034167 0.787104 Class 0.095784 0.099979 0.136555 0.170135 1.043796 1.245908 unidentified_Acidobacteria 0.099669 0.097557 0.122139 0.073706 0.97881 0.60346 Sphingobacteriia 0.033768 0.037449 0.061737 0.074889 1.109009 1.213033 Betaproteobacteria 0.12549 0.123225 0.098643 0.079169 0.981951 0.802581 Deltaproteobacteria 0.120712 0.100898 0.079215 0.067831 0.835857 0.85629 Alphaproteobacteria 0.109611 0.107707 0.098963 0.122672 0.982629 1.239574 unidentified_Gemmatimonadetes 0.089401 0.09319 0.084049 0.085232 1.042382 1.014075 species A. johnsonii 0.000056 0.00003 0.000162 0.009201 0.535714 56.7963 Acinetobacter calcoaceticus 0.000132 0.000152 0.000152 0.004169 1.151515 27.42763 P. citronellolis 0.000295 0.000437 0.00032 0.000884 1.481356 2.7625 Fungal Phylum Ascomycota 0.409096 0.646335 0.761605 0.943696 1.57991 1.239089 Basidiomycota 0.156913 0.134857 0.129478 0.036593 0.859438 0.282619 Glomeromycota 0.051488 0.003648 0.001168 0.000078 0.070851 0.066781 Zygomycota 0.003931 0.002917 0.022083 0.006261 0.74205 0.283521 Chytridiomycota 0.003498 0.004019 0.003648 0.000825 1.148942 0.226151 Neocallimastigomycota 0 0.000009 0.000012 0 0 Others 0.375074 0.208214 0.082006 0.012547 0.555128 0.153001 Class Dothideomycetes 0.020115 0.014531 0.011632 0.56324 0.722396 48.4216 Saccharomycetes 0.234994 0.429424 0.006785 0.004341 1.827383 0.639794 Sordariomycetes 0.003426 0.000713 0.520909 0.003606 0.208114 0.006923 Pezizomycetes 0.104288 0.174529 0.199376 0.207931 1.673529 1.042909 Eurotiomycetes 0.034438 0.013817 0.008302 0.138021 0.401214 16.62503 Agaricomycetes 0.144535 0.120281 0.128111 0.021855 0.832193 0.170594 Glomeromycetes 0.051488 0.003648 0.000458 0.000078 0.070851 0.170306 Leotiomycetes 0.003046 0.000858 0.003197 0.010819 0.281681 3.38411 Incertae_sedis_Zygomycota 0.003931 0.002917 0.022083 0.006261 0.74205 0.283521 Tremellomycetes 0.003594 0.003955 0.000572 0.008826 1.100445 15.43007 species Alternaria dauci 0.002161 0.002014 0.001632 0.000765 0.931976 0.46875 A. sesamicola 0.005798 0.000226 0.000265 0.000084 0.038979 0.316981 A. westerdijkiae 0.000211 0.000018 0 0 0.085308 -- Periconia byssoides 0.000205 0 0 0 --- -- Curvularia verruculosa 0.000054 0 0 0 --- --

decreased in AITC-treatment soils when plant presented. enriched in AITC2 compared with other group samples At the same time, the fungal Ascomycota (class level) were (Table 1). Compared with bulk soil samples, the fungal phylum Saccharomycetes was significantly decreased by group samples (Figure 1C and D). More specifically, bulk about 100 times when tomato plants were present (Figure soil had the highest alpha-diversity both in bacteria and 1D and Table 1). In species level, especially for those fungi, while AITC-treatment tomato plant group had the fungal pathogens Alternaria, A. sesamicola and A. lowest alpha-diversity in bacteria and fungi (Figure 2A). westerdijkiae were significantly diminished (Table 1). There were no significant differences in fungal communities among four groups, but the patterns of fungal alpha-diversity had the similar trend with bacteria (Figure AITC treatment exhibit overlap and distinct microbial 2B). PCA results showed that bacterial communities were communities with- or without- tomato plant more sensitive to the AITC treatment with or without tomato plants as compared to the fungal community. In A common core microbial community containing the top bulk soil, the bacterial communities were further apart from five bacterial and four fungal phyla were found in all four AITC treated group in the PC2 ordination. Nevertheless,

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Figure 2. Estimated values of bacterial and fungal community relative abundances. Shannon Index represents alpha diversity of bacterial A and fungal B community. C-D, Principle coordinate analysis showing that the bacterial rhizosphere microbiota shifts with AITC treatment and tomato plant in the first axis (C) and the fungal rhizosphere microbiota separated by AITC treatment in the first axis (D). The values of axes 1 (PC1) and axes2 (PC2) are the percentages that can be explained by the corresponding axis. Analysis of variance (ANOVA) was used to analysis the difference between groups. The difference of values was represented by a, b and c, and the same letter indicate no significant difference (P>0.05).

when tomato plants appeared, the bacterial communities AITC played more important role in the formation of clustered more closely together with or without AITC bacterial than fungal community structure (Figure 2). treatment (Figure 2C). AITC and tomato plant microbiomes had less impact on fungal community distribution than bacterial community (Figure 2D). For Distinct community composition in bacterial and fungal community distribution, only bulk soil group without fungal in AITC treatment with or without tomato plant AITC treatment was separated from the other three groups in the PC1 ordination. Therefore, both tomato plants and Linear discriminant analysis (LDA) of effect size (LEfSe)

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Figure 3. Cladogram showing the phylogenetic distribution of the bacterial and fungal lineages associated with AITC-treated soil with- or without- plant. A, Indicator bacteria with LDA scores of 4 or greater in bacterial communities associated with rhizosphere soil from the four group samples. B, Different-colored regions represent different constituents (red, CK1; green, CK2; blue, AITC1; purple, AITC2). C, Indicator fungal with LDA scores of 4 or greater in fungal communities associated with soil from the four group samples. D, Different-colored regions represent different constituents (red, CK1; green, CK2). Circles indicate phylogenetic levels from domain to genus. The diameter of each circle is proportional to the abundance of the group.

was used to determine the taxa that most likely explain the was enriched at at the phylum level, and no significant differences between AITC-treatment with or without enrichment was detected at any other clade (Figure 3A tomato plants. The large number of OTUs identified in this and B). When tomato plants appeared, the characteristic study would be computationally too complex for statistical bacteria changed significantly. The bacteria Subgroup-6, analysis and thus we analyzed the domain only to the (orders) and Xylariaceae (family) were genus level. Groups were shown in cladograms, and LDA detected to be significantly enriched in no AITC treatment scores of 4 or greater were confirmed by LEfSe (Figure 3). group (Figure 3A and B). Three groups of bacteria were In bulk soil, three distinct types of bacteria namely detected to be significantly enriched in AITC treatment Nitrospirae, which itself contained one order group, inculding Acitinobacteria (Phylum), (Nitrospirales), Betaproteobacteria (the class to its order Sphingobacteriaceae (Family), Gammaproteobacteria and Nitrosomonadales), Deltaproteobacteria (the class), and Pseudomonadales (order) (Figure 3A and B). The fungal only one type of fungi were significantly enriched. Glomeromycota was enriched only at the phylum level, However, in AITC-treatment bulk soil, only one Chloroflexi and there was no significant enrichment at any other clade

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(Figure 3C and D). Therefore, AITC was critical to the and an increase in plant pathogens (Avis et al., 2008), characteristic microorganism of the microbial community in which ultimately result in the occurrence of several soil- bulk soil, but when tomato plants were present, the plant borne diseases. Soil-borne diseases, which are capable of had a greater effect on soil characteristic microorganism surviving for long periods on soil hosts or plant debris, are than AITC. relatively more difficult to control than stem or leaf diseases, particularly those caused by Fusarium spp., Phytophthora spp., Pythium spp. and Meloidogyne spp. AITC treated plant rhizosphere-associated enriched Fumigation is the most direct approach for altering the soil isolates cultivation and bacteria wilt resistance microbiota and is a common practice for controlling soil- borne disease (Li et al., 2016b). The bioactive hydrolysis We analyzed the relative abundance in phylum and products of glucosinolates obtained from damaged species level for bacteria and fungi. Compared with the Brassica tissues, such as ITCs, applied to the soil are untreated tomato plant rhizosphere soil (CK2), the relative relatively rapid sorbed and degraded. abundance for some bacteria and fungi in AITC2 were ITCs and host plants had an interaction with the significantly enriched or diminished. Hence, we structure of microbiomes which were thought to have an investigated whether the enriched rhizosphere important role in shaping soil composition of microbiomes communities were associated with resistance or (Hollister et al., 2013; Hu et al., 2014; Rumberger and susceptibility to plant pathogens. We hypothesized that the Marschner, 2003; Troncoso-Rojas et al., 2009). AITC as enriched bacteria in AITC-treatment tomato plant one of ITCs hydrolyzed from a specific GLs have received rhizosphere soil may be associated with resistance to plant particular attention since they strongly inhibit a variety of pathogens such as bacterial wilt. About one thousand soil-borne plant pathogens including Rhizoctonia spp. cultured bacterial isolates from AITC-treatment tomato (Cole, 1976), Aphanomyces euteiches f. sp. pisi plant rhizosphere soil were isolated and identified. But only (Smolinska et al., 1997), Phymatotrichopsis omnivora three enriched bacterial isolates were obtained (Table 1). (Duggar) Hennebert (Hu et al., 2011), and changed the soil They were tentatively identified through 16S rRNA gene composition of microbiomes (Hu et al., 2014; Ren et al., sequencing as P. citronellolis SY9 (GenBank accession 2018; Yu et al., 2019; Deng et al., 2020). Nowadays more No. MK361107), Acinetobacter schindleri SY10 (GenBank increasing demand to discover reliable and eco-friendly accession No. MK361105) and A. johnsonii SY11 products with low risks for environmental. Plant extracted (GenBank accession No. MK361106). Antibacterial activity AITC was widely used as a soil and warehouse fumigant of SY9, SY10 and SY11 were tested against R. agent for controlling soil-borne diseases, plant parasitic solanacearum (RS2). SY10 and SY11 (in the center) nematodes and weeds (Tracz et al., 2017; Ren et al., showed obviously effective antibacterial activity with an 2018; Wang and Mazzola, 2019). Our study provided the inhibition zone of 43 mm after 12~14 h (Figure 4A). SY10 most comprehensive characterization of the microbial and SY11 showed the highest antifungal activity against community structure composition and dynamic changes five different fungal pathogens included FoPVo1 under greenhouse condition for tomato (L. esculentum (Fusarium oxysporum), V991 (Verticillium dahliae Kleb), Mill.) plant. In this study, we examined the role of tomato FJ-6 (Aspergillus ruber), PH-1 (Fusarium graminearum plant in the establishment of soil composition of Schw) and XXE-F (Alternaria solani) (Figure 4A). SY9 microbiomes, especially beneficial microbes following showed very slight inhibition against these bacterial and AITC treatment. According to our findings AITC and other fungal pathogens. The plate bioassay results indicated ITCs hydrolyzed products influence the bacterial and that SY10 and SY11 have a broad spectrum of antifungal fungal communities of AITC-treated soil with or without activity against plant pathogenic fungi. SY10 showed host plant. Some beneficial bacterial populations ability to suppress bacterial wilt disease caused by RS2 associated with plant fungal disease suppression were (Figure 4B), and the disease index showed dramatically prominently enriched in AITC treated soil harboring the decrease from day 7 to day 14 (Figure 4C). Therefore, we host plant. Beneficial soil microbes improve plant growth concluded that AITC could not only diminish some by exerting profound impacts that increase mineral pathogenic fungi, but also induce tomato plants to enrich solubilization (Bever et al., 1997), modulate plant hormone some antibacterial microorganisms and help plant improve production (Hayat et al., 2010) and directly inhibit plant resistance to soil-borne disease. pathogens by producing antimicrobial compounds (Mendes et al., 2011). AITC used as a pre-plant soil fumigant can effectively DISCUSSION AND CONCLUSION control certain soil-borne bacteria and fungi, nematodes and weeds (Ren et al., 2018). Hu et al. (2014) also Continuous intensive cropping often causes drastic reported that the application of allyl ITC in the presence of changes to the soil microbiome, leading to an unbalanced flax temporarily decreased soil fungal populations. AITC is community represented by a loss of beneficial microbes reported to control R. solani (Sarwar et al., 1998; Chung et

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Figure 4. Three AITC recruited bacterial strains suppressed the growth of soil-borne pathogens. A, Antifungal and antibacterial activities of three selected recruited strains suppressed five soil-borne fungal pathogens FoPVo1 (F. oxysporum), V991 (V. dahliae Kleb), FJ-6 (A. ruber), PH-1 (F. graminearum Schw) and XXE-F (A. solani), and one soil- borne bacterial wilt disease RS2 (R. solanacearum). Three AITC recruited bacterial strains were SY9 (Pseudomonas citronellolis), SY10 (A. schindleri), SY11 (A. johnsonii). Bacterial DH5α (Escherichia coli) was control. The antifungal and antibacterial activities have been repeated at least three times. Scale bars, 2 mm. B, Phenotype of tomato plant Zhongzha No.9 infested with RS2 after 11 days with or without strain SY10 treated. Scale bars, 5 mm. C, The disease index of SY10 suppressed bacterial wilt in tomato plant Zhongzha No.9. Each Point represent means ±SEM (n=6). Student t-test was used to analyzed significant difference (*p<0.05, ***p<0.001, Student t-test).

al., 2003), V. dahlia (Olivier et al., 1999), F. oxysporum which are associated with suppression of fungal (Smolinska and Horbowicz, 1999), P. aphanidermatum pathogens and R. solanacearum, were dramatically (Chung et al., 2003) and P. capsica (Chung et al., 2003). enriched by tomato plantation. We hypothesize that AITC Our results show that after AITC-treatment both fungal and could influence Solanaceae plants shaping their core bacterial populations were considerably influenced in bulk rhizosphere microbiome and may induce plants to enrich soil and in the tomato plant rhizosphere soil. The fungal some beneficial bacteria to suppress soil-borne fungal communities, especially some soil-borne fungal pathogens. Therefore, we isolated three enriched bacterial pathogens, were significantly decreased. Thus, when species and detected whether these bacterial communities plants are present, there is a shift in response to AITC that have the function of inhibiting pathogens. is in a different direction then in bulk soil, indicating that the Two enriched cultured bacterial isolates from AITC- plant rhizosphere effect is synergistic with the amendment treated plant rhizosphere soil have a broad spectrum of of AITC. Interestingly, beneficial biocontrol microbes, antifungal activity against soil-borne pathogens. One including P. citronellolis, A. schindleri and A. johnsonii, Acinetobacter bacterial isolate with a high identity to the

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reported bacteria, which could degrade malathion with 336. cometabolism and exhibiting nematicidal activity against Caporaso J. G., Lauber C. L., Walters W. A., Berg-Lyons D., Lozupone C. A., Turnbaugh P. J., Fierer N. & Knight R. (2011). Global patterns of Caenorhabditis elegans (Xie et al., 2009; Tian et al., 2016), 16S rRNA diversity at a depth of millions of sequences per sample. has a beneficial role in against bacterial diseases. We Proc. Natl. Acad. Sci. USA. 1:4516-4522. assume that AITC induced plant to shape their unique Chellemi D. O., Ajwa H. A., Sullivan D. A., Alessandro R., Gilreath J. P. & rhizosphere microbiome, and some core benificial Yates S. R. (2011). Soil fate of agricultural fumigants in raised-bed, plasticulture systems in the Southeastern United States. J. Environ. microbiota may have a biological interactions and Qual. 40:1204. competition with the altered microbial community to induce Chung W., Huang J., Huang H. C. & Jen J. (2003). 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