Identification of endophytic Bacillus velezensis ZSY-1 strain and antifungal activity of its volatile compounds against Alternaria solani and Botrytis cinerea

Accepted Manuscript Identification of endophytic Bacillus velezensis ZSY-1 strain and antifungal activity of its volatile compounds against Alternaria solani and Botrytis cinerea Zhenfeng Gao, Baojun Zhang, Huiping Liu, Jucai Han, Yongjie Zhang PII: DOI: Reference:

S1049-9644(16)30224-9 http://dx.doi.org/10.1016/j.biocontrol.2016.11.007 YBCON 3511

To appear in:

Biological Control

Received Date: Revised Date: Accepted Date:

30 May 2016 11 November 2016 17 November 2016

Please cite this article as: Gao, Z., Zhang, B., Liu, H., Han, J., Zhang, Y., Identification of endophytic Bacillus velezensis ZSY-1 strain and antifungal activity of its volatile compounds against Alternaria solani and Botrytis cinerea, Biological Control (2016), doi: http://dx.doi.org/10.1016/j.biocontrol.2016.11.007

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Identification of endophytic Bacillus velezensis ZSY-1 strain and antifungal activity of its volatile compounds against Alternaria solani and Botrytis cinerea Zhenfeng Gaoa, Baojun Zhanga, Huiping Liua, Jucai Hana*, Yongjie Zhangb a Plant Chemical Protection Laboratory, Shanxi Agriculture University, Taigu 030801, Shanxi, China b College of Life Sciences, Shanxi University, Taiyuan 030006, Shanxi, China

*Corresponding author: Jucai Han, phone: + 86 03546286399, e-mail: [email protected]

Abstract Soil microbes can release volatile organic compounds (VOCs) that influence plant growth and pathogen resistance. Screening these microbial VOCs for their antifungal activity is a new strategy for developing biopesticides. However, the ability of endophytic bacteria to synthesize VOCs is understudied. Here, we characterized VOCs produced by Bacillus velezensis ZSY-1, and tested their antifungal activity against plant pathogenic fungi. Strain ZSY-1, isolated from Chinese catalpa, was identified as B. velezensis, based on sequences of its 16S rRNA, gyrA, and gyrB genes. Bioassay of VOCs from ZSY-1 was conducted using the two-sealed base-plates method. Volatile organic compounds from ZSY-1 exhibited significant antifungal activity against Alternaria solani, Botrytis cinerea, Valsa mali, Monilinia fructicola, Fusarium oxysporum f. sp. capsicum, and Colletotrichum lindemuthianum; the inhibition rates were 81.1%, 93.8%, 83.2%, 80.9%, 76.7%, and 70.6%, respectively. Gas chromatography-mass spectrometry of solid phase microextraction samples (SPME-GC-MS) revealed 29 unique VOCs released by strain ZSY-1. Among these compounds, 2-tridecanone, pyrazine (2,5-dimethyl), benzothiazole, and phenol (4-chloro-3-methyl) had much higher peak areas than the main compound types. However, the most common VOC types were ketones (seven unique compounds), alcohols (six), and alkanes (six). Pyrazine (2,5-dimethyl), benzothiazole, 4-chloro-3-methyl, and phenol-2,4-bis (1,1-dimethylethyl) had significant antifungal activity against A. solani and B. cinerea. In fact, we report for the first time that phenol (4-chloro-3-methyl) synthesized by B. velezensis can inhibit the growth of plant pathogenic fungi. Based on our study of antifungal activity, pyrazine (2,5-dimethyl), benzothiazole, phenol (4-chloro-3-methyl), and phenol-2,4-bis (1,1-dimethylethyl) are promising bioagents for controlling tomato fungal diseases such as early blight and grey mold. Key words Strain ZSY-1, volatile organic compounds, A. solani, B. cinerea, Antifungal activity

1. Introduction Tomato is one of the most cultivated culinary vegetables in the world, and it is particularly popular in north China. However, fungal diseases such as early blight and grey mold seriously affect the yield and quality of the tomato crop (Bahraminejad et al., 2014; Kumar et al., 2013; Liu et al., 2016; Ye et al., 2015). To control these two diseases, strategies such as synthetic pesticide application, resistant-variety cultivation, and crop rotation have been applied; pesticide application remains the most common control strategy. However, intensive use of synthetic pesticides can cause problems such as pathogen resistance, pesticide residues, and environmental pollution (De Curtis et al., 2010; Harish et al., 2008; Ma et al., 2015; Yang et al., 2015). Dependence on agrochemicals is also unfavorable, because effective chemical treatments are lacking for several plant diseases. Moreover, consumers are increasingly demanding pesticide-free food (Wang et al., 2012; Wang et al., 2015). Therefore, there is an urgent need for new agents to be developed against plant diseases. Biopesticides may be desirable substitutes for traditional pesticides. Although non-volatile antifungal compounds have received much attention, research on microbial volatile organic compounds (VOCs) has been limited. Microbial VOCs easily evaporate at room temperature and atmospheric pressure (Wang et al., 2013). They also inhibit the growth of pathogenic fungi (Li et al., 2012; Minerdi et al., 2009; Raza et al., 2015; Yuan et al., 2012), improve plant growth (Asari, 2015; Hofmann, 2013; Park et al., 2015), mediate relationships, interactions, and communications between organisms (Cernava et al., 2015; Effmert et al., 2012; Fischer et al., 2015), identify bacterial species (Spinelli et al., 2011), and induce systemic resistance in plants (Kwon et al., 2010; Ryu et al., 2004; Zamioudis et al., 2015). In addition, biogenic VOCs can coexist in the environment and decompose easily under natural conditions. Diverse VOCs occur in plants, fungi, actinomycetes, and plant growth-promoting rhizobacteria (PGPR). Volatile organic compounds occur as alcohols, nitrogen compounds, aldehydes, ketones, alkanes, olefins, phenols, acids, esters, and ethers (Fernando et al., 2005; Li et al., 2014; Perestrelo et al., 2016). Among these VOCs, 13-tetradecadien-1-ol, 2-butanone, 2-methyl-n-1-tridecene, 2,3-butanediol, and acetoin can promote plant growth (Park et al., 2015; Piechulla and Degenhardt, 2014; Ryu et al., 2003). Benzothiazole, cyclohexanol, n-decanal, dimethyl trisulfide, 2-ethyl-1-hexanol, nonanal, and pyrazine (2,5-dimethyl) possess antifungal activity (Fernando et al., 2005; Munjal et al., 2016). In addition, 2,3-butanediol, acetoin, and tridecane can induce systemic resistance in plants (Farag et al., 2013). Therefore, VOCs may have numerous biotechnological applications. Endophytic bacteria can control plant disease (Nandhini et al., 2012; Zhang et al., 2015), promote crop growth (Chen et al., 2014; Faria et al., 2013), improve crop resistance (Kang et al., 2007; Pavlo et al., 2011), and promote degradation of organic pollutants (Kukla et al., 2014; Zhang et al., 2014). Among all biological effects, the antifungal activity of endophytic bacteria has been widely researched in recent years. Soluble non-volatile bioactive compounds such as lipopeptides (Gond et al., 2015; Zachow et al., 2015), proteins (Wang et al., 2016), pyoluteorin, phenazine (Kilani-Feki et al., 2010), and 2,4-diacetylphloroglucinol (DAPG) (Ramesh et al., 2009) are secondary metabolites of endophytic bacteria. Owing to the unique endophyte habitat, the VOCs of endophytic bacteria have been overlooked, with a few recent exceptions. This oversight seems particularly significant in light of the large number of identified species of endophytic bacteria. To investigate endophyte VOC metabolism and potential alternatives to chemical

pesticides, the present study screened VOCs from endophytic bacteria for antifungal activity. Strain ZSY-1, isolated from the leaf of Chinese catalpa, has significant antifungal activity against 12 plant diseases (unpublished results). In this study, we found that ZSY-1 could produce a strong odor. Therefore, we investigated whether VOCs with good antifungal activity could be produced by strain ZSY-1. Toward this objective, we characterized the VOCs of strain ZSY-1, and sequenced its 16S rRNA, gyrA, and gyrB genes. 2. Materials and Methods 2.1. Endophytic Bacteria and Plant Pathogenic Fungi Endophytic bacteria ZSY-1 was isolated from a Chinese catalpa leaf according to the method described by El-Deeb, with some modifications (El-Deeb et al., 2013). Specifically, after being washed thoroughly with tap water and sterile double distilled water, plant tissues were surface sterilized by immersion in 70% ethanol for 3 min, 3% sodium hypochlorite for 5 min, and 70% ethanol for 1 min. Sterilized plant tissues were rinsed three times with sterile distilled water, and remaining water (100 µ L) from the final wash was spread onto nutrient agar (NA; consisting of 3 g/L beef extract, 10 g/L peptone, 5 g/L NaCl, and 15 g/L agar; pH 7.0) to test the efficiency of our surface sterilization strategy. After drying on a piece of sterile filter paper, plant tissues were cut aseptically into 1 cm long segments using sterile blades or scissors. These plant segments were placed onto NA plates, and incubated for 5 d at 26 °C. Colonies with differing morphology and pigmentation were selected from each plate and streaked on fresh NA plates. Isolates were checked for purity by restreaking on the same NA plates; pure isolates were preserved in 25% glycerol at -80 °C, for further study. We isolated the fungal species Alternaria solani and Botrytis cinerea from tomatoes (Shi et al., 2015). The other fungi — Sclerotinia sclerotiorum, Fusarium oxysporum f. sp. niveum, Colletotrichum lindemuthianum, Rhizoctonia solani, Valsa mali, Monilinia fructicola, Fusarium graminearum, and Fusarium oxysporum f. sp. capsicum — were provided by the Department of Plant Pathology, Shanxi Agricultural University, Taigu, China, and stored in our laboratory. Fusarium oxysporum was identified using ISSR markers, and by comparing the morphological, cultural, spore, and mycelia characteristics with those of a standard culture. The other fungi were identified using ITS sequencing, and by comparing the morphological, cultural, spore, and mycelia characteristics with those of a standard culture. Pure culture isolates were grown on potato dextrose agar (PDA) at 26 °C for up to 7 d prior to use. 2.2. Antifungal activity of VOCs of strain ZSY-1 The two-sealed-base-plates method was used to test the antifungal activity of VOCs from strain ZSY-1. One base plate contained 15 mL NA, and another base plate contained 15 mL PDA. Strain ZSY-1 was streaked onto the NA plate, and a 5-mm-diameter spot of plant pathogenic fungi was placed on the PDA plate. Next, these two base plates were sealed with PE stretch-wrap, and cultured in a mold cultivation cabinet for 10 d at 26 °C. Every experiment was repeated three times. The inhibition rate against plant pathogenic fungi was calculated using the following formula：

Inhibition rate（%）=

Cd-Td Cd-5 mm

× 100%,

where Cd = fungal colony diameter on the control PDA base plate, and Td = fungal colony diameter on the treatment PDA base plate. 2.3. Analysis of ZSY-1 VOCs by Gas Chromatography-Mass Spectrometry (GC-MS) 2.3.1 Collection of VOCs We poured 3 mL NA into a 25-mL headspace-vial and inoculated the surface of the NA medium with 20 µ L of a ZSY-1 suspension (1.0×106 CFU/mL). As a control, we performed an inoculation with 20 µ L sterile water. Next, all treatments were incubated at 26 °C. After 7 d, the VOCs of each sample were characterized using solid-phase microextraction (SPME) and GC-MS. 2.3.2 Identification of volatile compounds from the strain ZSY-1 Samples were pretreated as the above section 2.3.1 describes, and subsequently characterized using SPME and GC-MS. SPME procedures: the extraction conditions were optimized according to the method of Zheng (Zheng et al., 2013). When optimizing the extraction temperature and time, we sought to maximize three metrics. We considered the number of phenols, heterocyclic compounds, and compounds containing N-, P-, Cl-, Br-, or another functional group; antifungal activity is overrepresented in these compounds. We favored extraction temperatures and times yielding relatively high peak area percentages, because temperatures and times that are too low or too high can lead to incomplete adsorption onto the extraction fiber. Furthermore, we favored conditions that led to confident compound identifications by our mass spectra reference database search software. Specifically, we considered SI and RSI indices of 800 and above to be reliable. To optimize the extraction temperature, the VOCs were extracted for 40 min at 60 °C, 70 °C, and 80 °C. Next, the extraction time was optimized at the optimal temperature for 20 min, 40 min, and 60 min. Finally, the VOCs were extracted using DVB/CAR/PDMS fiber (50/30 µm) at the optimized conditions. Before every extraction, the fiber was conditioned at 250 °C for 30 min. After extraction, the analytes were thermally desorbed for 5 min at 250 °C in the injector of the gas chromatograph. Chromatographic conditions: We used a TG-5 MS (30 m × 0.25 mm i.d. × 0.25 µm, Thermo) capillary gas chromatographic column to separate the volatiles. Helium was used as a carrier gas, and the flow rate was 1 mL/min. The temperature of the injection port was 250 °C. We used the following temperature program：begin at 50 °C for 2 min; increase to 150 °C at 4 °C min-1; hold at 150 °C for 1 min; increase to 250 °C at 5 °C min-1; and hold at 250 °C for 5 min. Conditions of MS: We used a Thermo Trace 1300-ISQ MS, with electrospray ionization (ESI). The quality range was 1.2~1095, and the quality precision was 1. All mass spectra were acquired in the electron impact of 70 eV. The m/z range was 45–500, with a scan rate of 0.2 scan/s. The temperatures of the ionization source and transfer lines were 280 °C, with an ionization mode of EI. The analysis was performed in full-scan mode. Compounds were identified by comparing their retention times, retention indices, and mass spectra with those of standard solutions injected at the same conditions; we also used the National Institute of Standards and Technology (NIST) mass spectral search program to search observed MS fragmentation patterns against the

NIST/EPA/NIH mass spectral library (version 2.0 g build May 19 2011). 2.4. Verification of screened VOCs against A. solani and B. cinerea All compounds were purchased from the Chengdu AIKE REGENT company. Each standard substance was dissolved with an appropriate solvent. We also used the two-sealed-base-plates method to evaluate the antifungal activity of the VOC standards. One base plate contained 15 mL Potato Dextrose Agar (PDA), and the other base plate was empty. A 5-mm-diameter spot of plant pathogenic fungi was placed on the PDA plate, and 80 µ L of VOC standard was added to the other base plate. Next, these two base plates were sealed with PE stretch-wrap, and cultured for 10 d in a mold cultivation cabinet at 26 °C. Every experiment was repeated three times. We calculated the inhibition rate as described above, in section 2.2. 2.5. Identification of strain ZSY-1 To identify strain ZSY-1, we extracted genomic DNA according to the method described by Chen and Hasan (Chen et al., 2010; Hasan et al., 2008), with some modifications. (1) 1.5 mL of bacterial suspension was collected in logarithmic phase, and centrifuged at 5000 rpm for 5 min at 4 °C. (2) Bacteria were collected, and 500 µ L of TE buffer was used to make a bacterial suspension. (3) To dissolve the cell walls, 30 µ L of lysozyme (50 mg/mL) and 25 µ L of RNase A (20 mg/mL) were added to this bacterial suspension. The mixture was vortexed for 50 s, and incubated at 37 °C for 30 min. (4) 55 µ L of 10% SDS and 3 µ L of proteinase K (20 mg/mL) were added to the suspension. The mixture was vortexed for 30 s, and incubated at 55 °C for 2 h. (5) An equal volume of phenol: chloroform: isoamyl alcohol (25:24:1) was added to the mixture. The sample was vortexed for a few seconds to form an emulsion. The emulsion was incubated at room temperature for 10 min. (6) We centrifuged the emulsion at 4 °C for 10 min at 12000 rpm. The top aqueous phase was pipetted off, without removing any precipitated material from the interphase, or any phenol. (7) We added an equal volume of isopropyl alcohol, and 1/10 volume of 3 M sodium acetate (pH 5.5). Next, the sample was mixed slowly by inversion, and incubated for 30 min at -20 °C. (8) We centrifuged the sample at 4 °C for 15 min at 12000 rpm. The precipitate was washed twice with 70% ethanol. (9) The precipitate was air-dried briefly, but not over-dried. The precipitate was dissolved in TE buffer with RNase A (final concentration 1 µ g/µ L), and incubated for 30 min at 37 °C. Finally, DNA was stored at -20 °C for further study. The 16S rRNA, gyrA, and gyrB genes were amplified using primers 27F/1492R (Li et al., 2013), gyrAF/gyrAR (Cao et al., 2008), and BS-F/R (Wang et al., 2007), respectively. PCR amplification was performed as follows: one cycle of 5 min at 94 °C, followed by 30 cycles of 45 s at 94 °C, 45 s at 55 °C, and 1.5 min at 72 °C, followed by one cycle of 10 min at 72 °C. PCR products were sequenced at BGI Tech in Beijing. 16S rRNA sequences were BLAST-queried against a reference database using the Ezbiocloud website. Type strains of the Bacillus subtilis group were chosen to construct an NJ phylogenetic trees, and used to analyze the taxonomic status of strain ZSY-1. All 16S rRNA, gyrA, and gyrB sequences of the type strains that we analyzed are derived from the GenBank/EMBL/DDBJ database. MEGA 5.0 was used for multiple sequence alignment and phylogenetic tree construction.

2.6. Statistical analysis Statistical significance was determined using the statistical program SPSS 17.0, by one-way analysis of variance (ANOVA) followed by Duncan’s multiple range test. 3. Results 3.1. The antifungal effect of VOCs against A. solani and B. cinerea The antifungal activity of VOCs was tested using the two-sealed-base-plates method. Volatile organic compounds of strain ZSY-1 had significant antifungal activity against A. solani (83.0% inhibition rate) and B. cinerea (92.1% inhibition rate) (Table 1). The VOCs of strain ZSY-1 can also significantly inhibit the sporulation of A. solani (Fig. 1). However, A. solani recovered the ability to sporulate when ZSY-1 was removed (Fig. 2). Therefore, the VOCs of strain ZSY-1 inhibited the spore production rate, rather than caused A. solani to die. 3.2. The antifungal spectrum of ZSY-1 VOCs The two-sealed-base-plates method was also used to determine the antifungal spectrum of ZSY-1 VOCs. Strain ZSY-1 VOCs exhibited significant antifungal activity against six plant pathogenic fungi. The inhibition rates were 83.2% against V. mali, 80.9% against M. fructicola, 76.7% against F. oxysporum f. sp. capsicum, 68.1% against S. sclerotiorum, 57.0% against F. oxysporum f. sp. niveum, and 70.6% against C. lindemuthianum (Figs. 3–4). The inhibition rates of ZSY-1 VOCs against R. solani and F. graminearum were only 43.6% and 37.3%, respectively. The VOCs of strain ZSY-1 have potential applications in biological control. 3.3. The effects of extraction time and temperature on GC-MS analyses of VOCs Extracting time and temperature are the main factors that affect SPME efficiency. In order to determine the SPME conditions for optimal GC results, we varied the extraction temperature and extraction duration. The results indicated that extracting temperature and time significantly impacted the GC-MS characterization of VOCs (Fig. 5 (A), Table 2). When the extracting temperature was 70 °C, the variety of VOCs was larger than that for extracts at other temperatures, as were the areas of the four main compounds. When the extracting temperatures were 60 °C and 80 °C, GC-MS resolved 34 and 33 distinct VOCs, respectively. Thus, when the extracting time was 40 min, the best extracting temperature was 70 °C. When the extracting temperature was 70 °C, extracting time also had a significant impact on VOC GC-MS outcomes (Fig. 5 (B)). When the extracting times were 20, 40, and 60 min, GC-MS resolved 31, 42, and 42 distinct VOCs, respectively. Eighteen distinct VOCs were detected consistently under the three different extraction temperatures, and 16 distinct VOCs were detected under the three different extraction times. Excluding compounds detected in control samples, we consistently detected 7 compounds under every extraction condition. Although 40- and 60-min extractions yielded the same number VOCs, the peak areas of the four main compounds were different. The peak areas of the four main compounds were maximal at 40 min, and significantly

diminished at 60 min. Extraction time is the time that the adsorption capacity of the extraction column is saturated. If the extraction time is too long, then the adsorbed compounds will degrade. The adsorption capacity of our extraction fiber was saturated between at 40–60 min. We selected 40 min as the best extraction time by considering the energy savings, the SPME fiber life, and the VOC stability. In addition, eight compounds were obtained only at 60 °C. Compared with the compounds only obtained at 80 °C, these compounds may have much higher molecular motion and volatility at room temperature. Such compounds have advantages as gas generating agents, gas regulators, or gaseous signal molecules at room temperature over the compounds extracted only at 80 °C. 3.4. Volatile organic compound detection by GC-MS The VOCs for ZSY-1 samples and control samples were tested using GC-MS under optimized extraction conditions. We detected 14 distinct VOCs in control samples and 42 distinct VOCs in ZSY-1 samples (Table 3). Twelve of the 42 distinct ZSY-1 VOCs also occurred in the control samples. In the control – but not treatment – samples, GC-MS detected acetophenone and nonanal. Acetophenone has the same structural formula as benzeneacetaldehyde, and nonanal is structurally similar to 2-nonanone. Therefore, we could not be sure that 2-nonanone was detected in the treatment samples. After excluding compounds potentially originating from contamination, we classified 29 distinct VOCs as authentically sourced from ZSY-1. Of these 29 compounds, seven were identified as ketones, one was an aldehyde, six were alcohols, six were alkanes, three were phenols, three were nitrogenous compounds, one was a quinone, one was an ether, and one was a naphthalene. Ketones, alcohols, and alkanes were the main compound types, and these three types accounted for 65.5% of the 29 detected compounds. Although phenols and nitrogenous compounds comprised only 10.3% of the 29 detected, their areas were much bigger than others. Based on the size of the areas, we concluded that pyrazine (2,5-dimethyl), benzothiazole, phenol (4-chloro-3-methyl), and 2-tridecanone were the main VOCs produced by strain ZSY-1. 3.5. Antifungal activity of the screened VOCs against A. solani and B. cinerea In order to determine which VOCs exhibited antifungal activity against A. solani and B. cinerea, 28 compounds were purchased and tested for antifungal activity. We were unable to obtain the 29th VOC — oxime (methoxy-phenyl) (C8H9 NO2) — because we could not find its Chemical Abstracts Service identifier. These tests determined that four compounds showed markedly stronger antifungal activity than did their peers against A. solani and B. cinerea (Table 4, Fig. 6). These compounds were pyrazine (2,5-dimethyl), benzothiazole, phenol (4-chloro-3-methyl), and phenol-2,4-bis (1,1-dimethylethyl). Their inhibition rates against B. cinerea, respectively, were 100%, 100%, 100%, and 91.19% (Table 4). The inhibition rates against A. solani, respectively, were 87.5%, 100%, 100%, and 89.14% (Table 4). Pyrazine (2,5-dimethyl), was more effective against B. cinerea than A. solani. Benzothiazole, phenol (4-chloro-3-methyl), and phenol-2,4-bis (1,1-dimethylethyl) had similar antifungal activity against B. cinerea and A. solani. In addition, Phenol-2,4-bis (1,1-dimethylethyl) could also inhibit sporulation in A. solani. Although other compounds such as phenol (2,4,6-trichloro) and some alcohols exhibited antifungal activity, their potencies were significantly lower than those of the above four

compounds (Table 4). However, the main compound 2-tetradecanone did not exhibit antifungal activity against these two plant pathogenic fungi. The antifungal mechanism of these four compounds may be different for different plant pathogenic fungi. These compounds are promising agents for controlling tomato grey mold and early blight, and one of them can affect the spore synthesis system of A. solani. 3.6. Identification of bacterial strain ZSY-1 Endophytic bacterium ZSY-1 was identified using sequencing of 16S rRNA, gyrA, and gyrB genes. The genomic DNA was of high quality and usable for PCR amplification, based on the results of 1.5%-agarose gel electrophoresis. PCR products of ~1400 bp, ~700 bp, and ~700 bp were amplified, respectively, from the 16S rRNA, gyrA, and gyrB genes of ZSY-1 genomic DNA. The 16S rRNA, gyrA, and gyrB NCBI sequence accession numbers are KT381096, KT381097, and KX855985, respectively. We conducted homology analysis of the 16S rDNA using Ezbiocloud. The results indicated that strain ZSY-1 matched with Bacillus amyloliquefaciens subsp. plantarum FZB42 T and Bacillus methylotrophicus KACC 13105T. The similarity of 16S rRNA gene sequences between ZSY-1, B. amyloliquefaciens subsp. plantarum FZB42T, and B. methylotrophicus KACC 13105T was 99.93%. The NJ phylogenetic tree also showed that strain ZSY-1 formed a cluster closely related to two type strains. This result showed that this strain was phylogenetically related to the members of genus Bacillus subtilis (Fig. 7). By itself, the 16S rRNA could not determine which of the above B. subtilis members were ZSY-1. To further classify strain ZSY-1, we amplified and sequenced its gyrA and gyrB gene. We used BLAST to query this sequence against an NCBI database. The sequence of ZSY-1 gyrA was 98% similar to B. amyloliquefaciens subsp. plantarum FZB42T, B. methylotrophicus KACC 13105T, and Bacillus velezensis NRRL B-41580T gyrA. Besides, the gyrA NJ phylogenetic tree also showed the same result as the 16S tree (Fig. 8). However, the NJ phylogenetic tree of 16S rRNA and gyrA sequences showed that strain ZSY-1 formed a cluster closely related to type strains FZB42T, KACC 13105T, and FZB42 T, which have also have been reclassified as later heterotypic synonyms of KACC 13105T (=Bacillus velezensis CBMB205T) (Dunlap et al., 2015). What is different is that gyrB sequence similarities between ZSY-1 and the type strains of NRRL B-41580T, FZB42T, and KACC 13105T were, respectively, 100%, 99%, and 99%. The gyrB NJ phylogeny showed that strain ZSY-1 formed a cluster closely related to type strain NRRL B-41580T, with a bootstrap value of 100 (Fig. 9). Although the gyrB NJ phylogeny showed a different result, B. amyloliquefaciens subsp. plantarum FZB42 T and B. methylotrophicus KACC 13105T have been reclassified as later heterotypic synonyms of B. velezensis NRRL B-41580T by genome analysis (Dunlap et al., 2016). Sequence analyses of the 16S rRNA, gyrA, and gyrB genes indicated that endophytic bacterium ZSY-1 is B. velezensis. 4. Discussion Endophytic bacteria living in plant tissue can harmoniously coexist with their hosts. The VOCs of endophytic bacteria exhibit no adverse effects toward their plant hosts. Naturally occurring VOCs are a promising source of new antifungal agents that are safe for humans and the

environment. In the present study, we characterized the VOCs of endophytic bacterial strain ZSY-1. To improve the utilization of ZSY-1 in developing biological pesticides, we screened novel ZSY-1 VOC agents for antifungal activity against A. solani and B. cinerea. By following the two-sealed-base-plates method, we found that the VOCs of strain ZSY-1 exhibit significant antifungal activity against A. solani and B. cinerea. To the best of our knowledge, we are the first to report that the VOCs of an endophytic bacterium can inhibit the growth of A. solani and B. cinerea. The VOCs also exhibit antifungal activity against four other plant pathogenic fungi (V. mali, M. fructicola, F. oxysporum f. sp. capsicum, and C. lindemuthianum), with inhibition rates above 70%. We identify strain ZSY-1 as B. velezensis based on its 16S rRNA, gyrA, and gyrB gene sequences. This result indicates that the gyrB gene can be used to identify Bacillus group strains. Until now, B. velezensis has been reported on in terms of strain functions, such as promoting plant growth (Meng et al., 2016), controlling plant diseases (Nam et al., 2009), and degrading pollutants (Bafana et al., 2008). Bacillus velezensis is an environmentally friendly bacterium with multiple biological functions. This study further elucidates its potential role as a biological agent for controlling the tomato diseases of gray mold and early blight. In order to know which compounds were the active ingredients, we performed SPME and GC-MS to analyze the composition of VOCs, and assayed the antifungal activity of each VOC in vitro. Extraction temperature and time influence experimental results (Diaz-Maroto et al., 2002; Hamm et al., 2003; Moreira et al., 2016; Perestrelo et al., 2016; Sousa et al., 2006), so the extracting temperature and time were optimized in our study. We found that 70 °C and 40 min were the optimal conditions for extracting VOCs from B. velezensis. The optimal extracting temperature and time are different for other organisms. For example, the VOCs of Pseudomonas sp. were extracted at 50 °C for 30 min (Elkahoui et al., 2015); the optimal extracting conditions of Gram-positive bacteria VOCs were 37 °C and 24 h (Dolch et al., 2012); bacterial VOCs were extracted at 30 °C for 30 min by SPME (Cernava et al., 2015); and volatiles produced by soil-borne endophytic bacteria were adsorbed for 20 min at 40 °C by SPME (D'Alessandro et al., 2014). The VOC types from strain ZSY-1 differ from those of other species. The main VOC compounds of ZSY-1 are ketones, alcohols, and alkanes. However, the main compounds in Pseudomonas sp. are dimethyl disulfide and dimethyl trisulfide (Elkahoui et al., 2015); in eight tested bacteria, ketones and alcohols (Li et al., 2014); and in Bacillus megaterium BP17, hydrocarbons (extracted by solvent) or pyrazine (extracted by dynamic head space) (Munjal et al., 2016). The high extraction temperature and VOC characteristics that we report for B. velezensis may be related to the good heat resistance of this organism, and the special habitat of endophytic bacteria. By screening 28 compounds, we found that pyrazine (2,5-dimethyl), benzothiazole, phenol (4-chloro-3-methyl), and phenol-2,4-bis (1,1-dimethylethyl) have antifungal activity. Although ketones, alcohols, and alkanes are the main types of VOC produced by strain ZSY-1, the compounds with antifungal activity are nitrogenous and phenolic compounds. According to the GC-MS peak areas, nitrogenous and phenolic compounds were much more abundant than the other compounds. Among the four active compounds, phenol (4-chloro-3-methyl) is recognized for the first time as a potential inhibitor of plant pathogenic fungi. Pyrazine (2,5-dimethyl) and benzothiazole occur in other bacteria and exhibit antifungal activity. For example, pyrazine (2,5-dimethyl) partially inhibits Magnaporthe oryzae and Phytophthora capsici (Munjal et al.,

2016). Benzothiazole exhibits a 100% inhibition rate against Fusarium oxysporum (Raza et al., 2015), and a comparably strong antifungal activity against Sclerotinia sclerotiorum (Fernando et al., 2005). Phenol-2,4-bis (1,1-dimethylethyl) also has been determined to inhibit plant pathogenic fungi (María Teresa et al., 2014). However, the antifungal activity of pyrazine (2,5-dimethyl) and benzothiazole is seldom reported, especially among inhibitors of A. solani and B. cinerea. Phenol-2,4-bis (1,1-dimethylethyl) also was first reported to inhibit the growth of A. solani and B. cinerea, and is the main substance that delayed A. solani spore production (Fig. 10). We did not observe sporulation changes resulting from the three other compounds. We further determined that pyrazine (2,5-dimethyl), benzothiazole, and phenol-2,4-bis (1,1-dimethylethyl) have good potential in plant disease control. Although many plant and microbe VOCs have been shown to benefit humans, plants, and animals (Kai et al., 2009; Mendes et al., 2013), VOC effects on animal health need further elucidation. Some VOCs may be harmless, some may be harmful, and some may benefit animal health only at relatively low concentrations. For example, 4-chloro-3-methyl phenol was often used as an antiseptic and disinfectant in healthcare facilities and animal houses (Krishnakumar et al., 2012), plant VOCs have nutritional and health benefits (Goff and Klee, 2006); marker-VOCs of MAP (Mycobacterium avium ssp. paratuberculosis) can be used to diagnose this chronic enteric disease in ruminants (Bergmann et al., 2015); mandala, marijuana, and ragweed can produce plant source contamination or cause neuropsychiatric disorders (Yamada, 2015; Yang, 2008); butyric acid, propionic acid, valeric acid, and isovaleric acid could inhibit the production and proliferation of lymphocyte cytokines (Kai et al., 2009; Kurita-Ochiai et al., 1995); and nonanoic acid, octadecanoic acid, and carboxylic acid methyl esters could stimulate oviposition of Aedes aegypti, and aid its search for a suitable egg-laying habitat (Kai et al., 2009; Ponnusamy et al., 2008). Moreover, benzothiazole can be used to produce riluzole and pramipexole (Faridbod, 2016; Powell, 2015); 2,5-dimethyl pyrazine can be used as a food additive at low concentration (Muller and Rappert, 2010); and 2,4-bis(1,1-dimethylethyl)-phenol can be used to produce antioxidants, stabilizers, and ultraviolet absorbers (Choi et al., 2013; Varsha et al., 2015). Nevertheless, the productive and safe deployment of VOCs and microbial VOCs necessitates further study of the effects that these compounds have on human and animal health. In summary, the four compounds reported presently are promising candidates for bio-pesticide development. Bacillus velezensis (ZSY-1) produces VOCs with antifungal activity against A. solani and B. cinerea. Although we found four compounds with significant in vitro antifungal activity against A. solani and B. cinerea, the practical field efficacy of these compounds merits future investigation. To critique an upstream element of our experimental design, the extraction fiber and GC conditions may affect detection efficiencies; although we detected 29 compounds by SPME-GC-MS at an optimized extraction time and temperature, variations to other experimental conditions could be studied further. Finally, we only tested antifungal activity against 10 plant pathogenic fungi; future work should examine additional pathogens. Phenol (4-chloro-3-methyl), pyrazine (2,5-dimethyl), benzothiazole, and phenol-2,4-bis (1,1-dimethylethyl) are promising biological agents with the potential to control the tomato diseases of grey mold and early blight. Acknowledgements

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Figure captions

Fig. 1 The antifungal effect of strain ZSY-1 VOCs against A. solani and B. cinerea. Fig. 2 The effect of strain ZSY-1 VOCs on spore production of A. solani. Fig. 3 The antifungal spectrum of strain ZSY-1 VOCs. Fig. 4 Inhibition rate of strain ZSY-1 VOCs against 10 plant pathogenic fungi. Fig. 5 Effect of different extracting temperatures (A) and times (B) on the GC-MS determination of VOC types. Fig. 6 Antifungal activity of four standard substances against A. solani and B. cinerea. Fig. 7 NJ tree showing the phylogenetic positions of strain ZSY-1 and other related taxa based on 16S rRNA gene sequences. Bootstrap values (expressed as percentages of 1000 replicates) are shown at branch points. Bar, 0.005 nt substitution rate (Knuc). Fig. 8 NJ tree showing the phylogenetic positions of strain ZSY-1 and other related taxa based on gyrA gene sequences. Bootstrap values (expressed as percentages of 1000 replicates) are shown at branch points. Bar, 0.05 nt substitution rate (Knuc). Fig. 9 NJ tree showing the phylogenetic positions of strain ZSY-1 and other related taxa based on gyrB gene sequences. Bootstrap values (expressed as percentages of 1000 replicates) are shown at branch points. Bar, 0.05 nt substitution rate (Knuc). Fig. 10 The effect of phenol-2,4-bis (1,1-dimethylethyl) on spore production of A. solani.

Table captions Table 1 Inhibition rate of strain ZSY-1 VOCs against A. solani and B. cinerea. Table 2 The area changes for four major VOCs under different incubation temperatures and extraction times. Table 3 Strain ZSY-1 VOCs were detected using SPME and GC-MS. Table 4 Antifungal activity of 28 compounds against A. solani and B. cinerea.

Tables Table 1 Inhibition rate of strain ZSY-1 VOCs against A. solani and B. cinerea. Fungal colony diameter (mm)

Plant pathogenic fungus

Control

Inhibition rate (%)

Treatment

80.11±1.45 17.78±1.79 A. solani 83.33±2.40 11.22±1.48 B. cinerea Note: Data are presented as means ± standard deviation.

83.0±2.2 92.1±1.9

Table 2 The area changes for four major VOCs under different incubation temperatures and extraction times. Area under different incubation temperatures (%)

Compound 2-Tridecanone Pyrazine， 2,5-dimethyl Benzothiazole 4-Chloro-3-methyl phenol

Area under different extraction times (%)

60 ℃

70 ℃

80 ℃

20 min

40 min

60 min

0.42±0.04 c

2.17±0.45 a

0.92±0.15 b

——

1.99±0.10 a

0.50±0.04 c

0.75±0.06 c

14.57±0.49 a

12.15±0.48 b

0.74±0.04 c

14.25±0.40 a

12.10±0.09 b

3.76±0.07 c

7.36±0.34 a

6.19±0.31 c

3.73±0.06 c

7.22±0.15 a

6.30±0.15 c

0.46±0.04 e

1.62±0.09 a

1.05±0.08 c

0.46±0.02 e

1.46±0.05 b

0.93±0.04 d

Note: “——” indicates that the compound was not detected. Different letters represents significant differences (P<0.05) per Duncan’s multiple range test. Data are presented as means ± standard deviation.

Table 3 Strain ZSY-1 VOCs were detected using SPME and GC-MS. Treatment

Compound

Retention

formula

time (min)

C6H14

Area (%)

SI

RSI

CAS#

2.30

0.28±0.03

809±3.51

827±7.64

110-54-3

C8H10

5.97

0.75±0.09

933±7.51

933±11.24

108-38-3

Benzaldehyde

C 7H6O

8.33

9.18±0.33

923±13.45

926±7.00

100-52-7

Phenol

C 6H6O

8.91

1.69±0.26

863±16.62

923±9.07

108-95-2

C 8H8O

10.71

0.83±0.05

863±11.93

895±6.03

122-78-1

Acetophenone

C 8H8O

11.41

0.14±0.01

865±12.01

919±2.89

98-86-2

Nonanal

C9H18O

12.48

1.49±0.15

932±4.04

933±1.73

124-19-6

Naphthalene

C10H8

14.82

0.49±0.10

914±7.02

932±3.46

91-20-3

C 10H 12O 2

15.12

1.60±0.25

919±8.02

920±4.04

93-92-5

Decanal

C10H20O

15.41

0.61±0.15

916±7.51

924±2.65

112-31-2

Undecanal

C11H22O

18.23

0.39±0.04

964±9.50

974±5.86

112-44-7

Dodecanal

C12H24O

20.92

0.52±0.05

843±21.50

905±8.62

112-54-9

n-Hexane Benzene, 1,3-dimethyl

Benzeneacetaldehyde Control

Chemical

Benzenemethanol, àmethyl, acetate

Hexadecanoic

C 17H 34O 2

32.55

0.57±0.04

902±3.21

906±2.89

112-39-0

C 19H 36O 2

35.99

12.07±0.38

913±4.58

911±2.52

112-62-9

C6H14

2.32

1.15±0.08

907±12.34

912±2.52

110-54-3

C8H10

5.98

1.07±0.08

936±4.58

948±4.16

108-38-3

C 8H9NO2

6.73

1.40±0.16

808±1.53

843±8.19

NA

C6H8N2

6.99

14.24±0.03

844±6.11

897±7.51

123-32-0

Benzaldehyde

C 7H6O

8.34

1.04±0.1

864±1.53

950±2.52

100-52-7

Phenol

C 6H6O

8.94

0.77±0.06

914±3.51

929±2.65

108-95-2

C 8H8O

10.74

0.87±0.04

954±6.11

976±9.71

122-78-1

C 13H28

11.13

0.48±0.08

885±4.51

894±16.62

17301-32-5

2-Nonanone

C9H18O

12.15

0.41±0.10

865±7.09

907±1.53

821-55-6

2-Decanol

C10H22O

14.27

0.15±0.02

805±3.21

828±6.51

1120-06-5

1-Nonanol

C9H20O

14.46

0.18±0.04

857±5.51

914±6.66

143-08-8

Naphthalene

C10H8

14.85

0.17±0.01

929±2.00

942±8.50

91-20-3

C 10H 12O 2

15.15

0.53±0.04

926±12.74

923±7.09

93-92-5

Decanal

C10H20O

15.43

0.29±0.06

912±2.65

919±6.51

112-31-2

2-Hexyl-1-octanol

C14H30O

15.67

0.16±0.02

833±2.08

865±6.00

19780-79-1

Benzothiazole

C7H5NS

16.02

7.17±0.09

937±11.15

946±7.55

95-16-9

C9H12O

16.93

1.45±0.07

803±2.00

834±2.64

2944-49-2

C8H18O

17.30

0.17±0.04

810±2.31

855±4.58

1653-40-3

C 16H34

17.52

0.28±0.07

838±1.53

880±4.51

544-76-3

C 7H7ClO

17.81

1.49±0.02

927±4.00

925±3.21

59-50-7

2-Undecanone

C11H22O

17.89

0.63±0.04

838±5.20

892±60.08

112-12-9

2-Undecanol

C11H24O

18.09

0.48±0.06

913±2.52

960±1.53

1653-30-1

Undecanal

C11H22O

18.25

0.80±0.03

961±1.53

973±1.73

112-44-7

C6H3Cl3O

19.51

0.42±0.09

915±4.36

938±4.04

88-06-2

2-Dodecanone

C12H24O

19.61

0.25±0.02

816±4.58

883±3.00

6175-49-1

Tetradecane

C 14H30

20.70

0.20±0.03

904±4.58

916±8.54

629-59-4

Dodecanal

C12H24O

20.94

7.89±0.09

922±2.65

922±6.25

112-54-9

acid, methyl ester 9-Octadecenoic acid (Z), methyl ester n-Hexane Benzene, 1,3-dimethyl Oxime, Methoxy-phenyl Pyrazine, 2,5-dimethyl

Benzeneacetaldehyde Undecane, 4,7-dimethyl

Benzenemethanol, àmethyl, acetate ZSY-1

2,3-Dimethylanisole 1-Heptanol, 6-methyl Hexadecane Phenol, 4-chloro-3-methyl

Phenol, 2,4,6-trichloro

2,5-Cyclohexadie ne-1,4-dione,2,6-

C 14H 20O 2

22.49

0.20±0.04

807±1.00

817±2.00

719-22-2

C13H26O

23.13

1.99±0.13

913±4.04

915±4.93

593-08-8

C14H22O

23.55

0.22±0.01

884±7.51

902±2.65

96-76-4

2-Tetradecanone

C14H28O

24.67

0.42±0.16

844±7.02

854±6.11

2345-27-9

2-Hexadecanol

C16H34O

25.00

0.33±0.02

836±5.03

865±4.36

14852-31-4

Tetradecanal

C14H28O

25.90

0.08±0.01

864±6.11

892±8.08

124-25-4

C 18H38

26.71

0.10±0.02

836±1.53

866±6.43

3892-00-0

C 16H18

27.52

0.10±0.02

832±2.52

856±7.02

26137-53-1

2-Pentadecanone

C15H30O

27.84

2.45±0.02

807±1.52

813±5.57

2345-28-0

2-Hexadecanone

C16H32O

29.23

0.20±0.02

833±8.14

858±10.02

18787-63-8

Octadecane

C 18H38

29.99

0.17±0.02

882±3.61

900±5.86

593-45-3

C 21H44

30.20

0.22±0.03

855±6.00

884±7.57

18344-37-1

C18H36O

30.96

0.23±0.03

907±6.00

917±3.21

502-69-2

C 17H 34O 2

32.56

3.53±0.02

924±8.02

924±3.06

112-39-0

C 19H 36O 2

35.93

5.89±0.13

930±2.65

932±2.52

112-62-9

Bis (1,1-dimethylethyl) 2-Tridecanone Phenol,2,4-bis(1,1 -dimethylethyl)

Pentadecane, 2,6,10-trimethyl Naphthalene,1,2,3 -trimethyl-4propenyl, (E)

Heptadecane, 2,6, 10,14-tetramethyl 2-Pentadecanone, 6,10,14-trimethyl Hexadecanoic acid, methyl ester 9-Octadecadienoc acid (Z), methyl ester

Note：The number of SI and RSI of VOCs more than 800 are listed in this table. NA indicates that the compound was not matched to the database. Data are presented as means ± standard deviation.

Table 4 Antifungal activity of 28 compounds against A. solani and B. cinerea. Colony diameter of Colony diameter of control (mm) treatment (mm) Compound

Inhibition rate (%)

A. solani

B. cinerea

A. solani

B. cinerea

A. solani

B. cinerea

84.67±0.33

85.0±0.58

13.67±0.81

5.0±0

87.5±1.86

100±0

84.67±0.33

85.0±0.58

80.0±1.15

80.0±0.58

5.84±1.81

6.23±1.22

2-Decanol

84.67±0.33

85.0±0.58

81.0±0.58

81.0±0.58

4.60±0.83

5.0±0.04

1-Nonanol 2-Hexyl-1-octanol

84.67±0.33 84.67±0.33

85.0±0.58 85.0±0.58

82.67±0.88 85.0±0.58

79.33±0.67 82.0±0.58

2.52±0.74 ——

7.07±1.06 3.75±0.71

Pyrazine, 2,5-dimethl Undecane, 4,7-dimethyl

Benzothiazole 2,3-Dimethylanisole 1-Heptanol, 6-methyl Hexadecane Phenol, 4-chloro3-methyl 2-Undecanone 2-Undecanol Phenol,2,4,6trichloro 2-Dodecanone Tetradecane 2,5-Cyclohexadiene -1,4-dione,2,6-bis(1, 1-dimethylethyl) 2-Tridecanone Phenol, 2,4-bis(1,1dimethylethyl) 2-Tetradecanoe 2-Hexadecanol Tetradecanal Pentadecane, 2,6,10-trimethl Naphthalene,1,2,3trimethyl-4propenyl,(E) 2-Pentadecanoe 2-Hexadecanoe Octadecane Heptadecane,2,6, 10,14-tetramethyl 2-Pentadecanoe,6, 10,14-trimethyl

84.67±0.33

85.0±0.58

5.0±0

5.0±0

100±0

100±0

84.67±0.33

85.0±0.58

81.33±0.88

85.33±0.33

4.19±0.84

——

84.67±0.33

85.0±0.58

80.67±0.88

79.33±0.33

5.01±1.43

7.07±1.06

84.67±0.33

85.0±0.58

83.33±1.20

81.33±0.67

1.68±1.12

4.56±1.47

84.67±0.33

85.0±0.58

5.0±0

5.0±0

100±0

100±0

84.67±0.33 84.67±0.33

85.0±0.58 85.0±0.58

80.33±0.33 84.67±0.33

82.33±0.88 74.67±0.67

5.43±0.82 ——

3.33±0.83 12.91±1.04

84.67±0.33

85.0±0.58

71.67±1.20

69.33±0.88

16.32±1.45

19.56±1.57

84.67±0.33 84.67±0.33

85.0±0.58 85.0±0.58

79.0±1.15 82.67±0.88

76.67±0.67 81.67±0.67

7.12±1.13 2.52±0.74

10.40±1.01 4.15±1.39

84.67±0.33

85.0±0.58

79.0±1.45

78.33±0.58

7.11±1.53

8.32±0.68

84.67±0.33

85.0±0.58

85.0±0.58

82.33±0.68

——

3.32±1.09

84.67±0.33

85.0±0.58

14.94±0.76

12.06±0.26

89.14±1.96

91.19±0.44

84.67±0.33 84.67±0.33 84.67±0.33

85.0±0.58 85.0±0.58 85.0±0.58

81.33±0.67 82.0±0.58 82.67±0.88

84.67±0.68 76.67±0.33 77.0±1.00

4.19±0.44 3.35±0.42 2.52±0.74

0.42±0.42 9.15±0.69 9.99±1.42

84.67±0.33

85.0±0.58

76.33±1.45

81.33±0.33

10.46±1.81

4.58±0.39

84.67±0.33

85.0±0.58

77.0±1.15

82.67±0.33

9.62±1.16

2.90±1.08

84.67±0.33 84.67±0.33 84.67±0.33

85.0±0.58 85.0±0.58 85.0±0.58

77.0±0.58 80.0±0.58 77.67±1.45

81.0±1.15 80.67±0.68 81.0±0.58

9.61±0.46 5.86±0.43 8.78±1.89

4.98±1.90 5.41±1.07 5.00±0.04

84.67±0.33

85.0±0.58

76.67±0.88

77.33±0.88

10.04±1.23

9.56±1.62

84.67±0.33

85.0±0.58

83.33±0.29

82.67±0.39

1.68±0.74

2.91±1.61

Note: “——” indicates no antifungal activity. Data are presented as means ± standard deviation.

Highlights ∙ Bacteria ZSY-1 synthesizes volatile organic compounds with antifungal activity. ∙ Phenol (4-chloro-3-methyl) could inhibit the growth of plant pathogenic fungi. ∙ Pyrazine (2,5-dimethyl) and benzothiazole could inhibit A. solani and B. cinerea. ∙ Phenol-2,4-bis (1,1-dimethylethyl) is main substance to delay pigment production.