Biocontrol of tobacco black shank disease (Phytophthora nicotianae) by Bacillus velezensis Ba168

Journal Pre-proof Biocontrol of tobacco black shank disease (Phytophthora nicotianae) by Bacillus velezensis Ba168

Dongsheng Guo, Chenhong Yuan, Yunyan Luo, YaHan Chen, Meihuan Lu, Guochan Chen, Guangwei Ren, Chuanbin Cui, Jiatao Zhang, Derong An PII:

S0048-3575(20)30004-3

DOI:

https://doi.org/10.1016/j.pestbp.2020.01.004

Reference:

YPEST 4523

To appear in:

Pesticide Biochemistry and Physiology

Received date:

16 October 2019

Revised date:

10 January 2020

Accepted date:

13 January 2020

Please cite this article as: D. Guo, C. Yuan, Y. Luo, et al., Biocontrol of tobacco black shank disease (Phytophthora nicotianae) by Bacillus velezensis Ba168, Pesticide Biochemistry and Physiology (2020), https://doi.org/10.1016/j.pestbp.2020.01.004

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© 2020 Published by Elsevier.

Journal Pre-proof

Biocontrol of tobacco black shank disease (Phytophthora nicotianae) by Bacillus velezensis Ba168 Dongsheng Guo a Chenhong Yuan a Yunyan Luo a YaHan Chen a Meihuan Lu a Guochan Chen b Guangwei Ren c a

*

Chuanbin Cuid Jiatao Zhang d Derong An a,

State Key Laboratory of Crop Stress Biology for Arid Areas, Northwest A&F University, Yangling,

Shaanxi 712100, China b Henan Academy of Sciences Institute of Biology, Limited Liability Company, Zhenzhou, Henan

d

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450000, China c China Tobacco Research Institute of CAAS, Qingzhou, Shandong 262500,China Shaanxi Tobacco Scientific Institution, Xian, Shaanxi 710000, China

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Abstract：

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Tobacco black shank (TBS) caused by Phytophthora nicotianae is destructive to almost all tobacco cultivars and is widespread in many tobacco- growing countries.

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Through lab study and field test, we isolated plant growth-promoting rhizobacteria

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(PGPR) strain Ba168 which is a promising biocontrol strain of TBS. Ba168 was isolated from 168 soil samples and identified as Bacillus velezensis by its genetic and phenotypic

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characteristics. A susceptibility test indicated that the P. nicotianae antagonistic materials of Ba168 in extracellular metabolites were composed of effective and stable

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proteins/peptides. P. nicotianae’s growth was suppressed by the ammonium sulfate

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precipitation of Ba168 culture filtrates (ASPBa) at a minimum inhibitory concentration of 5μg/mL. Extracellular conductivity, pH, and the wet/dry weights of P. nicotianae’s mycelia, along with scanning electron microscope analysis, suggested that Ba168-derived proteins/peptides could effectively inhibit P. nicotianae by causing irreversible damage to its cell walls and membranes. Protein identification of ASPBa supported these results and identified many key proteins responsible for various biocontrol-related pathways. Field assays of TBS control efficacy of many PGPRs and agrochemicals showed that all PGPR preparations reduced the disease index of tobacco, but Ba168 was the most effective. These results demonstrated the importance of Bacillus-derived proteins/peptides in the *

Corres ponding a uthor: a [email protected]

Journal Pre-proof inhibition of P. nicotianae through irreversible damage to its cell wall and membrane; and the effectiveness of PGPR strain B. velezensis Ba168 for biocontrol of the soil-borne disease caused by P. nicotianae. Keywords: Tobacco black shank, Phytophthora nicotianae, Bacillus velezensis Ba168, PGPR, proteins/peptides 1. Introduction Tobacco black shank (TBS) caused by Phytophthora nicotianae is a soil-borne

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disease which can cause serious stem and root rot, as well as folia blight from flash

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dispersal of propagules from soil in the entire growth period of the tobacco plant[1-3]. This pathogen can spread rapidly under conditions of high temperature (23℃-28℃) and

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high soil moisture, causing serious yield losses[4-7]. The traditional control strategies,

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such as crop rotation, resistant tobacco varieties, and fungicide application are not

lP

sufficient to control this soil-borne disease[8]. Fungicides, although being used in most cases, can exist long time in the field and lead to resistant pathogen strains [9, 10].

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Surprisingly, there were studies found that the crop rotation was even less efficient than continually culturing tobacco for 4-5 years to prevent the losses caused by soil-borne

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pathogens like Gaeumannomyces graminis var. tritici (take-all of wheat), Phytophthora cinnamon (root rot of eucalyptus) and Thielaviopsis basicola (black root rot of tobacco)

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et al[8, 11, 12]. The mechanism by which these phenomena occurred was later found to be that plant growth-promoting rhizobacteria (PGPR) recruited by the host plants in the soil suppressed soil-borne pathogens[8]. However, several years of monoculture is not practical for economic reasons and therefore not commonplace. Thus, it is urgent to explore more efficient and sustainable control methods for soil-borne diseases. Biocontrol, by using isolated PGPR, is a good option, because of its effectiveness and environmental- friendly side effects[13]. To date, many P. nicotianae control strains have been found, such as Bacillus subtilis Tpb55[14] and Bacillus atrophaeus HAB-5[15]; non-pathogenic binucleate Rhizoctonia fungi[16], Trichoderma[17, 18], Glomus mosseae,

Journal Pre-proof and Pseudomonas fluorescens[19]. Among these biocontrol agents, P. fluorescens is one of the most studied PGPRs[20], which has been used as a model to study of their disease suppression mechanisms. However, P. fluorescens’ less stable biocontrol efficacy, shorter storage period, and the toxic cyanide it produces[21] limit its application in the field in recent years. Bacillus products are tend to replace P. fluorescens due to their spores which are resistant to stresses and the size of spores are more suitable for product preparation [13, 22]. Additionally, Bacillus can release antimicrobial peptides and enzymes[23,

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24][21, 22] to suppress phytopathogens, and induce plant systemic resistant (ISR)

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response[25], as well as improve plant growth and resistance to abiotic stresses[26]. Biocontrol PGPR, sometimes unrelated to antibiotic production, elicits ISR, which allows

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plants to withstand pathogen attacks on leaves or roots [27, 28]. Bacillus-mediated ISR in

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plant disease suppression has been well studied [25, 29, 30], and it has been found to also

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be effective in P. nicotianae suppression. A recent study [31] showed that root colonization by B. amyloliquefaciens FZB42 (now considered as B. velezensis) would

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elicit ISR and restrict the reopening of stomata mediated by P. nicotianae in Nicotiana benthamiana, and thus block the pathogen’s ability to initiate penetration and infection.

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Nevertheless, rare studies have focused on whether Bacillus is effective for P. nicotianae suppression in the field. Additionally, molecules containing extracellular metabolites of

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PGPR have their own synthesis pathways corresponding to related genes, and in most cases[32] the protein- and peptide-related macromolecular compounds are dominant active fractions. However, small organic non-proteinaceous antimicrobial molecules (such as phenaminomethylacetic acid[33]), also have influence occasionally. Revealing the components that play a major role in P. nicotianae inhibition can help us determine the direction of further research. Protein identification of extracellular metabolites of PGPRs can denote their biocontrol potential [34], and lead to additional work to identify the biocontrol mechanism(s) using genetic approaches [35]. The objectives of this study were to isolate and identify antagonistic bacteria

Journal Pre-proof against P. nicotianae; analyze antagonistic material in cultured supernatant fluid of the biocontrol strain and its effects on the pathogen in vitro; and determine the bacteria’s biocontrol activity against TBS in the field. 2. Materials and methods 2.1 Materials Five previously isolated PGPRs (Bacillus licheniformis, B. subtilis, Brevibacillus laterosporus, B. methylotrophicus, and Bacillus pumilus) used in field trials and P.

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nicotianae used in lab studies were obtained from preserves in the Plant Virus and

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Microorganism Resource Laboratory in the College of Plant Protection, Northwest A&F University. Chemical fungicides, mixtures of Propamocarb hydrochloride(10%) and

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Azoxystrobin(20%) (MPA) and 80% Dimethomorph water dispersible granules, were

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purchased from the local market. B. velezensis FZB24 was provided by the State Key

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Laboratory of Crop Stress Biology for Arid Areas.

Tobacco varieties used in field trials were N. tabacum QinYan96 and NC89 (provided

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by Shaanxi Provincial Tobacco Research Institute). NC89: after topping, the plant height of NC89 was about 110 cm, the growth period in the field was 105-117 days, and 21–23

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leaves were available for harvest. NC89 is not drought-enduring, and it has resistance to tobacco black shank, tobacco root rot, tobacco brown spot, tobacco weather fleck, and

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tobacco root knot nematode, whereas it is susceptible to tobacco mosaic virus. Qinyan96: after topping, the plant height of Qinyan96 was about 143.5 cm, the growth period in the field was about 119 days, and 21–23 leaves were available for harvest. Qinyan96 is drought-enduring, and has resistance to tobacco black shank, tobacco bacterial wilt, and potato virus Y, whereas it is susceptible to tobacco mosaic virus, tobacco root knot nematode, and cucumber mosaic virus. 2.2 Identification of antagonistic bacteria 2.2.1 Soil sample collection, bacteria isolation and screening A total of 168 soil samples were collected in 3–15 cm columns by simple punches in

Journal Pre-proof an intact forest of the Qinling Mountains, using the multi-draw mixture method. Soil suspensions were identically prepared (1 g soil in 100 mL sterile water, 220 r/min shaking for 5 min) and serially diluted to 10-6 . A 0.25 mL supernatant from the final three dilutions of each sample was spread on Luria-Bertani (LB) medium plates and incubated at 28℃ for two days. A single bacterial colony from each sample was selected and sub-cultured on LB, and then stored at -80℃ in 20% sterile glycerol. Dual culture assay[36] was used to determine P. nicotianae inhibition activity of

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selected bacterial strains. After the mycelia of P. nicotianae fully covered a Potato

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Dextrose Agar (PDA) plate, a 5 mm diameter sterile cork borer was used to harvest fungal samples along the colony’s edge. Then, the fungus cake was placed at the center of

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a 90 mm wide petri dish containing 10 mL PDA. Using an aseptic 2.5 μL pipette, a 1μL

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overnight culture (OD600 = 0.8) of each bacterial strain was deposited at four

lP

perpendicular sites, each 25 mm from the fungus block. Each strain underwent three replications and the petri dishes were then incubated at 27℃ for 4 days. The antagonistic

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efficacy of each strain was observed by calculating the average value of two perpendicular diameters of the inhibitor zone, and the 4 best results were presented.

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2.2.2 Identification of biocontrol strains The strain Ba168 with the largest inhibitor diameter was identified as B. velezensis

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based on Gram staining, morphology, and physiological and biochemical tests according to Bergey’s Manual of Determinative Bacteriology[37]. Further identification of Ba168 was confirmed by multilocus phylogeny analysis combined with analysis of 16S rRNA and gyrB gene sequences[38]. The forward primer (5’-AGTTTGATCMTGGCTCAG-3’) and the reverse primer (5’-GGTTACCTTGTTACGACTT-3’) were used for 16S rRNA sequence

amplification[15].

(5’-GAAGTCATCATGACCGTTCTGCA-3’) (5’-AGCAGGGTACGGATGTGCGAGCC-3’)

The and were

forward the used

for

primer

reverse

primer

gyrB

sequence

amplification[39]. The phylogenetic analysis was conducted using representatives of

Journal Pre-proof Bacillus velezensis UCMB5033, Bacillus velezensis FZB42, Bacillus velezensis subsp. Plantarum NAU-B3, Bacillus velezensis CBMB205 Bacillus amyloliquefaciens DSM7, Bacillus subtilis subsp. Spizizenii TU-B-10, Bacillus subtilis subsp. Subtilis BCRC10255, Bacillus atrophaeus NRS213, Bacillus licheniformis ATCC 14580, Bacillus pumilus ATCC 7061, Bacillus cereus ATCC 14579, and Bacillus thuringiensis ATCC 10792. The reference strain Brevibacillus laterosporus LMG 15441 was used as an out-group. The sequences

of

these

strains

were

retrieved

from

the

NCBI

database

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(https://www.ncbi.nlm.nih.gov/). The amplifying procedure followed the method

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described by Jangir et al.[40], and the amplified fragments were then sequenced (Qingke, XiAn, China). The corresponding sequences of 16S rRNA and gyrB were aligned using

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MAFFT (https://mafft.cbrc.jp/alignment/server/index.html). The aligned sequence of 16S

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rRNA and gyrB were then spliced together. Phylogenetic trees were constructed with

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MEGA 7.0[41] software by using the Maximum Likelihood method with 1000 bootstrap replications.

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2.3 General properties of the antimicrobial substance 2.3.1 Crude extracts preparation

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2.3.1.1 Ammonium sulfate precipitation of Ba168(ASPBa) ASPBa was prepared via the following steps. To reduce error caused by foreign

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proteins, inorganic nitrogen source medium (INSM) was used. It was composed of 20 g/L glucose, 20 g/L (NH4 )2 SO4, 1 g/L K 2 HPO 4 , 0.5 g/L MgSO 4 , and 0.5 g/L NaCl, with the pH adjusted to 7.0. Initially, strain Ba168 was inoculated to the 250 mL conical flask containing 150 mL INSM media and cultured in a shaking table at 150 r/min, 28℃ for 60 h. The fermented liquid was subsequently separated in 50 mL centrifuge tubes and centrifuged at 10000 r/min for 20 min. Next, precipitation was removed and different quantities of ammonium sulfate were added to make saturations of 10%, 20%, 25%, 30%, 35%, 40%, 50%, 60%, 70%, 80%, 90%, and 100% ammonium sulfate mixtures. Those tubes were refrigerated at 4℃ for 12 h, centrifuged again, the supernatant was discarded,

Journal Pre-proof and sediment in each tube was dissolved in phosphate buffer (25 mmol/L PH = 7.4) of 0.5 mL and then passed across a bacteria filter (0.45 μm, MILLEX), finishing with dialysis desalting. The combined final product was lyophilized by using a vacuum freeze drying method, and then re-suspended in a proportionate amount of phosphate buffer solution. ASPBa was the resulting re-suspension across a bacteria filter (0.22 μm, MILLEX). 2.3.1.2 Butanol extraction of Ba168

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Fermented liquid (1000 mL) of Ba168 (prepared in 2.3.1) was centrifuged at10000

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r/min for 20 min, and then mixed with butanol at a ratio of 1:1. The mixtures were subsequently centrifuged (10000 r/min) in 50 mL centrifuge tubes to achieve phase

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separation. The organic phases were collected and evaporated, and the sediments were

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re-suspended with 10 mL of phosphate buffer (25 mmol/L pH = 7.4).

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2.3.2 Susceptibility to protease and heat

The treatment fluids of each group were prepared as follows: group A (198 μL

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ddH2 O and 2 μL protease K), group B (200 μL 4μg/mL ASPBa), group C (198 μL 4 μg/mL ASPBa and 2 μL protease), group D (198 μL culture filtrates and 2 μL protease),

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group E (200 μL culture filtrates), group F (200 μL 4 μg/mL ASPBa heated at 80℃ for 20 min), and group G (200 μL 4 μg/mL butanol extraction of Ba168).

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These experiments were performed using the same procedure as above. A 100 μL spore suspension of P. nicotianae was pipetted into a 90 mm wide petri dish containing 10 mL PDA. The spreading plate method was used, and then 4 Oxford cups were placed at 4 perpendicular sites. For each group, 100 μL of treatment fluid was aseptically pipetted into each cup. The antagonistic efficacy of each group was determined after culturing at 27℃ for 72 h by analyzing the average value of two perpendicular diameters of the inhibitor zone. 2.3.3 Minimum inhibitory concentration (MIC) of ASPBa and its effect on the mycelial weight of P. nicotianae

Journal Pre-proof Different volumes of ASPBa (<0.5 mL) were pipetted and dissolved into separate vessels, where sterile water was added to adjust the total volume to 0.5 mL to make a gradient concentration of ASPBa. PDA medium was melted and cooled to 40–45℃, then 9.5 mL was aseptically pipetted onto 90 mm wide petri dishes and mixed uniformly with the 0.5 mL ASPBa solution. Finally, the plates were mixed with different concentrations (1, 2, 3, 4, 5, 10, 20, and 50 ug/mL) of ASPBa. Control plates (without ASPBa) were prepared in the same method. A disk of mycelium harvested from the periphery of a

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four-day-old colony, using a sterilized puncher (diameter = 0.5 mm), was inoculated at

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the center of each petri dish. All tests were performed in triplicate. The plates were incubated at 27℃ for three days, at which time the antifungal efficacy of each treatment

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was evaluated and recorded. Results were then documented each day for the following 7

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days. Efficacy was evaluated by examining the mean of 2 perpendicular diameters of

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each colony. The lowest concentration of ASPBa that fully inhibited the growth of P. nicotianae (colony diameter was not changed) was considered the MIC100.

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The effect of ASPBa on mycelia weight of P. nicotianae was evaluated as described below. A 1% (v/v) spore suspension (4×106 /mL) of P. nicotianae was inoculated into five

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50 mL conical flasks containing 20 mL Potato Dextrose Broth (PDB) and ASPBa at 0, 1, 2, 3, and 4 μg/mL, respectively. After remaining in a shaking incubator at 28°C for 7 d,

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the mycelia produced in liquid cultures were filtered using Miracloth (Millipore) and washed 3 times with PBS. The wet weights of each group were measured when droplets barely dropped from the Miracloth, whereas the dry weight of each mycelium was determined after drying at 60°C for 24 h. 2.3.4 Cell wall and cell membrane permeability tests The 4×106 /mL spore suspension of P. nicotianae was inoculated in a conical flask (250 mL) containing 150 mL PDB at an inoculation volume of 1%, and incubated in a 150 r/min constant temperature shaker at 30℃ for 36 h. The fermented liquid was subsequently centrifuged in 50 mL centrifuge tubes at 3000×g/min for 10 min. The

Journal Pre-proof precipitated hyphae originating from each flask were collected in one 50 mL centrifuge tube and washed 3 times with sterilized H2 O. After exposure of the mycelium of P. nicotianae into 40 mL ASPBa solutions with concentrations of 0, 1, 2, 3, or 4×μg/mL, extracellular conductivity was immediately recorded by the electrical conductivity meter DDS-307 at 0, 30, 60, 90, and 120 min, and extracellular pH was measured by the pH meter FiveEasy Plus at 0, 15, 30, 45, 60, 75, 90, and 105 min. 2.3.5 Effect of ASPBa on the morphology of mycelia of P. nicotianae

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The preparation of the mycelia of P. nicotianae followed the method described in

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section 2.3.4. The collected mycelia was washed 3 times with PBS then equally inoculated into 50 mL sterilized centrifugal tubes containing 40 mL phosphate buffer

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saline (PBS) with 2% (w/v) glucose and ASPBa of different concentrations (0, 1, and 4

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×μg/mL). Next, the tubes were incubated in a 150 r/min constant temperature shaker at

lP

27℃ for 12 h. The mycelia were collected and then dried in liquid nitrogen and viewed under a scanning electron microscope (SEM).

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2.3.6 Protein identification of ASPBa

ASPBa at a volume of 100 μg was diluted to 100 μL with 100 mM NH4 HCO 3 , and a

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certain volume of 500 mM Dethiothreitol was added to bring the final concentration to 10 mM. The solution was then placed at 45℃ for 45 min (1M Dethiothreitol = 0.154 g/mL).

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After that, the solution was cooled down to room temperature and 500 mM Iodoacetamide was added to reach a final concentration of 20 mM. The obtained solution was placed in the dark for 30 min (500 mM Iodoacetamide = 0.0925 g/mL). A certain amount of 500 mM Dethiothreitol was added to the final concentration of 10 mM to terminate the alkylation. Enzymatic hydrolysis was achieved by adding trypsin with the ratio of 1:40 ~ 50 at 37 ℃ overnight under constant temperature for 12~16 h to start the enzymatic hydrolysis reaction. Finally, the protein samples were desalted using Pierce C18 Tips, 100 µL bed (Thermo). The proteins/peptides in the prepared samples were identified using a liquid chromatograph- mass spectrometer (LC-MS) (Orbitrap Fusion

Journal Pre-proof Lumos). 2.4 TBS suppression activity in plants 2.4.1 Experimental design The field test sites were locations where TBS occurs frequently. The test sites A and B were closely located, at about 1020 m above sea level with a longitude of 109°08′15″and latitude of 32°54′41″. From 2017 to 2018, tobacco variety Qinyan96 was planted in A, whereas NC89 was planted in B. The experiment (2 fungicides, 6 biocontrol strains, and

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a water control) in each test site was laid out in a randomized block design with 4

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replications and a plot of 667 m2 which contained about 1000 tobacco plants. 2.4.2 Formulations preparation

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Agrochemicals involved in field trials were diluted at 0.8 μg/mL. Six biocontrol

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solid formulations were identically prepared. Strains were inoculated on LB plates and

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then cultured at 27℃ for 24 h. Next, a single colony was selected and inoculated in conical flasks containing LB broth (150 mL conical flasks, 100 mL per each) and cultured

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in a shaking table at 150 r/min, at 30℃ for 48 h. Then, the fermentation broth was inoculated in a sterile solid fermentation medium in a volume ratio of 1/10. For instance,

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1 kg powder could be generated with solid fermentation material composed of 100 g sucrose, 100 g yeast extract, 0.2 g MnSO4 , 1 g MgSO4 , 600 g soybean meal, 600 g bran,

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and 1800 mL water. After preparation, the solid fermentation medium was packed in 250 mL conical flasks (80 g per flask) and incubated at 28℃ for 72 h. Finally, the semi-processed fermentation medium was dried in a 40℃ oven for 96 h, crushed by a grinder, and the powder was collected from across a 200- mesh sieve. Liquid formulations were solid ferments diluted into suspensions @ 2 g/L (1013 CFU/mL). 2.4.3 Field application and disease index analyzing In the experimental groups of the 6 biocontrol strains, tobacco plants were treated with a seed treatment (5 g/kg) + seedling dip (2 g/L) before transplant into experimental sites, whereas fungicides and control groups without treatment were transplanted

Journal Pre-proof simultaneously. This experiment was repeated for 2 consecutive years. The first application was conducted when the growth period of tobacco was at the rosette stage, and when mild symptoms of tobacco plants infected with P. nicotianae could be observed. Thereafter, preparations were applied as a combination of root inoculation (350 L/ha) + foliar spray (350 L/ha) every 10 days for a total of 3 times. Root inoculation of preparations was performed by pipetting 30 mL of the liquid formulations or fungicide suspension onto the roots of tobacco plants. Foliar spray was completed with

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a manual sprayer. Control groups were root-inoculated and sprayed with the same

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dosages of sterile distilled water.

The initial disease index was examined before the first treatment, and again 10

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days after each treatment. A five-point sampling method was used in each plot, the

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disease index of 50 tobacco plants were recorded at each point, and the total number of

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inspected tobacco plants and the number of diseased tobacco plants at each grade were recorded. The disease index was computed as previously described[14, 42, 43].

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(a×0)+(b×1)+(c×3)+(d×5）+(e×7)+（f×9） Disease index =

（a+b+c+d+e+f)

100 × 9

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Where 0 = no symptoms; 1, 3, 5, and 7 = 1/3, 1/3 to 1/2, 1/2 to 2/3, and 2/3 of the total leaves or the periphery of stems were wilted, respectively; 9 = tobacco plant was dead; a, b, c, d, e, and f note the

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numbers of tobacco plants in each disease grade.

（CDI-TDI）

Control efficacy =

×

100%

CDI Where CDI refers to the disease index increases of the control group, which means the disease index difference value between the first treatment and 10 days after the last treatment of the control group, TDI denotes the rise of disease index of treatment group.

2.5 Data analysis All data were analyzed using SPSS 22.0 with Duncan's new multiple range test (DMRT) (P-value of <0.05) and were expressed as the mean ± standard deviation (mean

Journal Pre-proof ± SD) from 3 parallel experiments. The graph was built by Excel 2013 and GraphPad prism8 after data processing. 3. Results 3.1 Identification of antagonistic bacteria Of the 12322 strains of bacteria isolated from soil samples, 3282 strains were antagonistic to P. nicotianae. The 4 strains with the best results are presented in Table 1, Figure 1. Strain Ba168 had the highest inhibitory activity against P. nicotianae. Table 1

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P. nicotianae

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Strain1326 Strain24

Inhibition diameter (mm) 28.42 ± 2.84a 24.70 ± 0.93b 26.38 ± 2.95b 22.01 ± 0.42c

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Biocontrol strains strain Ba168

Strain 24

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The inhibitory efficiency of top 4 strains

Strain Ba168

Strain284

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Note: Data were analyzed using SPSS 22.0 with Duncan's Strain 1326 Strain 284 new multiple range test (DMRT) (P-value of <0.05), Values Bacteria isolation 11111326B in the table are Mean ±SE. Fig. 1. Antagonistic bacteria isolation and screening. a168

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3.2 Identification of strain Ba168

Pure colonies of strain Ba168 on LB agar medium after 48 h incubation were canary

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yellow in appearance, had rough surfaces, were round as a whole with irregular but

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smooth margins, opaque, dry, and did not produce pigment. Cells of strain Ba168 were rod-shaped and peritrichous, and its spores were long and elliptical (Fig. 2A, 2B). Based on the morphology characteristics described above, the physiological and biochemical characteristics detailed in Table 2, and the results received from the NCBI database when using 16SrRNA or gyrB homology of partial sequences of strain Ba168 to conduct BLAST, we preliminarily determined Ba168 as B. velezensis. Other sequences were retrieved to conduct additional multilocus phylogenetic analysis. As shown in Figure 3, the genetic characteristics of Ba168 were inferred by using the Maximum Likelihood method based on the Tamura-Nei model[44]. The results clearly showed that strain Ba168 was B. velezensis.

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Fig. 2. (A): Scanning electron micrographs Bar=5μm. electron

of (B):

strain

Ba168.

Transmission

micrographs

of

strain

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lP

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-p

ro

of

Ba168. Bar=2μm.

Fig. 3. Phylogenetic tree of strain Ba168 based on Maximum Likelihood method analysis of combined

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16S rRNA and gyrB sequences data. Bootstrap values <51% are not shown. B. laterosporus strain IAM 12465 represents the out-group. Table 2

Comparison of biochemical and physiological characteristics of Ba168 strain and B. velezensis FZB24. Note: “+”Positive; “–”negative． Physicochemical indexes

Characteristics Ba168

FZB24

Gram staining

+

+

Methyl red test

-

-

Indole test

-

-

Growth in 10%NaCl

+

-

Anaerobic growth

+

+

Journal Pre-proof +

+

L-arabinose fermentation

+

+

D-xylose fermentation

+

+

D-mannitol fermentation

+

+

V-P detection

+

+

Catalase activity

+

+

Lipase activity

+

+

Oxidase activity Starch hydrolysis

+ +

+ +

Casamino acid hydrolysis

+

+

Gelatin hydrolysis

+

+

Citrate utilization

-

Nitrate reduction H2 S production

+ -

-

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+ -

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3.3 Susceptibility to proteases and heat

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D-glucose fermentation

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As shown in Table 3, small, organic non-proteinaceous antimicrobial molecules (such as phenaminomethylacetic acid[33]) were involved in P. nicotianae suppression (Group

lP

G). The suppression activity of ASPBa was much higher than Ba168 culture filtrates.

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Both ASPBa and Ba168 culture filtrates are resistant to proteaseK and heat. A previous study also revealed that the Bacillus-derived antagonistic proteins/peptides are resistant to

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proteaseK and heat[32]. These results suggested that antagonistic proteins/peptides in extracellular metabolism of Ba168 are rather effective and stable, which encourages

Table 3

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further study on ASPBa.

Protease and heat susceptibility of antagonistic materials. Group

Inhibition diameter (mm)

A(198μL ddH2O and 2μg protease K)

3.3 ± 0.03c

B(200μL 4μg/mL ASPBa) C(198μL 4μg/mL ASPBa and 2μg protease)

41.7 ± 2.36a 42.0 ± 2.13a

D (198μL culture filtrates and 2μg protease) E(200μL culture filtrates) F(200μL 4μg/mL ASPBa heated at 80℃ for 20 min)

10.5± 0.17b 8.3± 1.04b 41.0 ± 3.23a

G(200μL 4μg/mL Butanol extraction of Ba168)

15.3 ± 0.05c

Note: Data were analyzed using SPSS 22.0 with Duncan's new multiple range test (DMRT) (P-value of <0.05), Values in the table are Mean ±SE.

Journal Pre-proof 3.4 MIC of ASPBa and its effect on the mycelia weight of P. nicotianae The MIC of ASPBa that completely inhibited the growth of the mycelia of P. nicotianae was 5 μg/mL. Moreover, mycelia dry and wet weights decreased significantly as the concentration of ASPBa increased (Table 4). When the concentration of ASPBa exceeded 3~4 μg, mycelia essentially stopped growing. The results suggested that Ba168-derived antimicrobial proteins/peptides could strongly inhibit the mycelia growth of P. nicotianae. P. nicotianae Dry weight (mg)

3610.73 ± 31.94a

156.00 ± 0.62a

1366.65 ± 17.76b 510.25 ± 3.31c

84.45 ± 0.95b 28.4 ± 0.34c

134.325 ± 2.59d 99.00 ± 0.56d

10.48 ± 0.13d 1.73 ± 0.09e

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Wet weight (mg)

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Concentrations of crude extracts of Ba168（μg/mL) 0 1 2 3 4

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Table 4 Effect of ASPBa on the weight of mycelia of P. nicotianae.

Note: Data were analyzed using SPSS 22.0 with Duncan's new multiple range test (DMRT) (P -value

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of <0.05), Values in the table are Mean ±SE.

3.5 Effects of ASPBa on extracellular conductivity and pH of P. nicotianae

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Compared to that of the control group, the extracellular conductivity of mycelia

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exposed to ASPBa increased significantly with the extension of treatment time. This trend is positively correlated with the concentration of ASPBa (Fig. 4A). The extracellular conductivity of the 4 groups reached 94.12 μs/cm, 108.36 μs/cm, 116.46 μs/cm, and 123.13 μs/cm after 120 min, respectively (Fig. 4A). With the extension of time, the pH showed an overall downward trend. In addition, after the dosage increase, the pH of each treatment group was higher than that of the control group at any time, and the higher the dose, the higher the pH (Fig. 4B). After 105 min, the concentrations from 0 to 4 μg/mL in the five groups were 5.03, 5.16, 5.31, 5.42, and 5.72, respectively (Fig. 4B). These results indicated that the antifungal activity of ASPBa may be associated with irreversible damage to the cytoplasmic membranes and cell walls of P. nicotianae, which

Journal Pre-proof contributed to intracellular content leakage from the cells.

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Fig. 4. (A) Effect of ASPBa on extracellular conductivity of P. nicotianae. (B) Effect of ASPBa on extracellular pH of P. nicotianae. Note: Original data were analyzed by using SPSS 22.0 with Duncan's new multiple range test (DMRT) (P-value of <0.05).

3.6 Effect of ASPBa on the morphology of mycelia of P. nicotianae

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The mycelia of P. nicotianae not treated with ASPBa were plump under SEM analysis, and the mycelia’s surface was not damaged or punctured (Fig. 5A, 5C).

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However, the majority of mycelia presented shrinkage deformation after the treatment of 1 μg/mL ASPBa (Fig. 5B). When we treated the mycelia with an ASPBa concentration of

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4 μg/mL (Fig. 5D), the mycelia became severely deformed, wrinkled, and perforated on

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the surface. These findings supported the results presented in section 3.5.

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Fig.

5. (A)(6000×,

bar=20μm),

(C)

(16000×, bar=5μm): The untreated hyphae of P. nicotianae. (B) (6000×, bar=20μm): The hyphae treated with 1μg/mL ASPBa.

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(D) (16000×, bar=5

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μm):

hyphae

treated with 4μg/mL ASPBa.

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3.7 Protein identification of ASPBa

The

antifungal

polypeptides

(SH3b

domain-containing

proteins

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identified

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The identified proteins/peptides related to this study are listed in Table 5. The

(B8XCV1/I2C1I8))[45] and antimicrobial peptide LCI (C4P928/I2C153)[32] are

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effective against fungal and bacterial phytopathogens. Cellulose degradation enzymes (CDEs) A0A0K6KWN0, A0A0K6LUU2, Q8RPQ6,

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and A0A0D7XPS0 matched P. nicotianae suppression activity in SEM analysis. Flagellin (I2CAR1) is a classic ISR inducer[46], while phytase (A0A0D7XUH7) helps plants

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transform and utilize insoluble ions in bulk soil, and thus promotes plant growth[47]. Surfactin synthetases (Q70KJ6/ A0A0K6KNN0/ I2C187/ A0A142FB39/ I2C189/ E1UL91), linear gramicidin synthase (A0A0K6LRC2), iturin A synthetase (E1UV07), fengycin synthetase (H9TE69), tyrocidine synthase (A0A0K6M2E3), bacillaene synthase (A0A0A0TV34), bacteriocin

bacilysin

synthase

acetoine/butanediol

synthase (I2CBG4/ A0A0K6KA68/

(A0A0K6M2R3),

dehydrogenases

bacillomycin

(I2C0S7/

D

E1UMW4/

A0A0D7XR12),

synthetase

(H9TE64),

A0A0D7XSN3),

and

tryptophan synthase alpha/beta chain (A0A142F5W0) are responsible for surfactin synthesis, iturin A synthesis, gramicidin synthesis, tyrocidine synthesis, bacillaene

Journal Pre-proof synthesis, bacilysin synthesis, bacteriocin synthesis, bacillomycin D synthesis, 2,3-butanediol/acetoin (VOCs) synthesis, and auxin synthesis, respectively. These identified proteins/peptides imply that there are multiple biocontrol pathways related to antibiosis, and induced systemic resistance and specific pathogen–antagonist interactions are involved in the biocontrol activity of Ba168 against TBS. These results, combined with other studies presented above, indicated that Ba168 is a promising strain for the biocontrol of TBS.

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Table 5 Protein identification of ASPBa.

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Functional class

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Surfactin synthesis Iturin A synthesis Gramicidin synthesis Tyrocidine synthesis Bacillaene synthesis

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Accession number of key proteins Q70KJ6/ A0A0K6KNN0/ I2C187/ A0A142FB39/ I2C189/ E1UL91 E1UV07 A0A0K6LRC2 A0A0K6M2E3 A0A0A0TV34 I2CBG4/ A0A0K6KA68/ A0A0D7XR12 A0A0K6M2R3 H9TE64 A0A0K6KWN0, A0A0K6LUU2, Q8RPQ6, A0A0D7XPS0 A0A0D7XUH7/ A0A1J0C814/ A0A0K6M2L2 I2C0S7/ E1UMW4/ A0A0D7XSN3 A0A142F5W0/ A0A0K6M1G6 I2CAR1 B8XCV1/ I2C1I8 C4P928/ I2C153

Bacilysin synthesis Bacteriocin synthesis Bacillomycin D synthesis Cellulose degradation enzymes

3-Phytase 2,3-butanediol/acetoin (VOCs) synthesis Auxin synthesis Flagellin Antifungal polypeptides (SH3b domain containing protein ) Antimicrobial peptide, Lci

3.8 TBS inhibition activity in plants In Qinyan96, the 2 consecutive years of control efficacy of B. velezensis Ba168 (78.45%, 77.26% ) against TBS ranked behind B. methylotrophicus (82.83%, 83.18% ) and MPA (79.11%, 77.68% ), but higher than the 80% Dimethomorph water dispersible granules (73.75%, 73.42% ), B. licheniformis (73.05%, 74.82%), B. subtilis (73.24%, 74.78% ),

Journal Pre-proof B. laterosporus (71.18%, 69.82% ) and B. pumilus (69.48%, 71.41% ). The 2-year control efficacy of Ba168 (67.00%, 66.01% ) in NC89 was second only to MPA (71.46%, 67.21% ) and higher than the other tested subjects (Dimethomorph (58.22%, 60.99% ), B. licheniformis (53.83%, 53.29%), B. subtilis (54.18%, 58.19% ), B. laterosporus (53.03%, 55.62% ) and B. pumilus (51.16%, 55.04% )). After pooling analysis, it was found that the

disease control efficacy in NC89 was generally lower than in Qinyan 96, and B. velezensis Ba168 was the preferred biocontrol strain for TBS suppression in the two

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tobacco varieties.

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Fig. 6. TBS control efficacy of Qinyan96 and NC89 in two consecutive years. Note: Original data were analyzed by using SPSS 22.0 with Duncan's new multiple range test (DMRT) (P-value of <0.05). Fig.6 A, C: CDTI of Qinyan96 and NC89 in 2017-2018; CDTI=CDI-TDI: The difference value of disease index compared to control group after formulation treatment. Fig. 6 B, D:

TBS control efficacy of Qinyan96 and NC89 in 2017-2018. 4. Discussion In this study, an isolated strain, Ba168, was identified as B. velezensis by its

Journal Pre-proof morphology (Fig. 2), biochemical and physiological characteristics (Table 2), and multilocus phylogeny analysis (Fig. 3). Bacillus strains have been widely used in the fields of food industry[48], medicine[49], animal husbandry[50], aquaculture[51], and agriculture and forestry[52]. Although horizontal gene transfer exists in the bacteria[53, 54], nearly all the reported B. velezensis strains are capable of reducing disease incidence by inhibiting development and growth of fungal phytopathogens and inducing plant defense reactions.

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In in vitro studies, B. velezensis Ba168 has superior P. nicotianae inhibition activity

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than other tested biocontrol strains (Table 1). Extracellular metabolites (in this study, especially proteins/peptides (Table 3)) of a biocontrol strain can reflect the biocontrol

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potential of PGPRs after rhizosphere colonization in the field[34]. Therefore, we further

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examined the characteristics of ASPBa. Mycelia growth can be severely inhibited when

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exposed to ASPBa (Table 4), which is more effective than eugenol[43], Trichoderma EtOAc extract[17], and mixtures of plant extracts (such as clove oil, neem oil, mustard

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oil, pepper extract, and cassia extract)[3]. Intriguingly, the data of extracellular conductivity and extracellular pH (Fig. 4A, 4B) shows that ASPBa causes the outflow of

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mycelium contents in a dose-dependent way. It is not unexpected that ASPBa can exert irreversible damage on the cell membranes of P. nicotianae and ion channels on the

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membrane, as it is reminiscent of the function of the surfactin, iturin, and fengycins families. Surfactin families can easily combine with the lipid layer of the cell membrane to damage its integrity [60]; the iturin class does not destroy the membrane structure, but it can increase membrane permeability and form ionic conduction holes which interfere with the transport of transmembrane substances[61]; and while the mechanism of fengycins is unclear, they also tend to interact with lipid layers and retain the potential for dose-dependent changes in cell membrane structure and permeability[62]. This suggests the recorded SEM observation in Figure 5B, in which the mycelia wrinkled severely. Although these lipopeptide synthases were identified in ASPBa, few lipopeptides will

Journal Pre-proof exist with the current sample preparation method. As expected, they were not directly identified in the protein identification test. Combined with the butanol analysis, it is inferred that other antagonistic materials may play more important roles than lipopeptide in P. nicotianae inhibition. When the concentration of ASPBa was brought to 4 μg/mL (Fig. 5D), mycelia cell walls were dissolved to a certain extent. Interestingly, because the cell walls of P. nicotianae are composed of cellulose[63], we can infer that CDEs are also involved in

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ASPBa. In protein identification assays (Table 5), CDEs including beta-glucanase,

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endoglucanase, pectin lyase, and pectate lyase were identified. CDEs, either from plants or microbial organisms, may also play roles in anti-oomycete activity as reported in

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recent studies[64]. Beta-1,3-glucanase derived from pepper and tomato can inhibit

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hyphae growth of Phytophthora capsici[65] and cellulases from Bacillus are effective in

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degrading the cell walls of Phytophthora pathogens[64]. Nevertheless, extensive cell wall digestion of P. nicotianae may not bring death to oospores or a mycelial piece, since new

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hyphae grow even if others are being actively digested[66, 67]. Based on the experimental results, it is worth clarifying the role of CDEs by using a genetic approach

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of constructing CDE-deficient mutants and genetic complementation of CDEs. Effective biocontrol strains are capable of helping host plants withstand pathogens by

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producing various antimicrobial substances and inducing resistant responses. In protein identification analysis (Table 5), the identified phytase[47], flagellin[46] and other proteins related to the synthesis pathway of antimicrobial substances (such as surfactin[68], tyrocidine[69], gramicidin[70], bacillomycin D[71], bacillaene, bacilysin, bacteriocin[45], auxin[72], and 2-3-butanediol (VOCs)[73]) imply the comprehensive and promising biological control capability of strain Ba168. The presence of various identified antimicrobial peptides[32] and antifungal polypeptides[45], which are effective in controlling bacterial and fungal phytopathogens, also implies the broad inhibition spectrum of Ba168. Overall, lab studies show the promising P. nicotianae suppression

Journal Pre-proof activity of Ba168, and possible mechanisms involved in this process. However, whether it would work under field conditions remains unknown. In our field studies (Fig. 6), we adhered to set of techniques for the application of PGPRs in the field control of TBS, following a combination application method similar to Elanchezhiyan et al.[74], which was adopted to evaluate the TBS suppression activity of strain Ba168 and its rivals. Results showed that the disease control efficacy of strain Ba168 was lower than MPA in 2 tobacco varieties, but higher than Dimethomorph

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preparations. Dimethomorph, Propamocarb, and Azoxystrobin all have been applied to

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control TBS for many years, and consequently, drug-resistant oomycetes have emerged[75]. Of the three, Dimethomorph is more effective than Propamocarb or

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Azoxystrobin when used separately[75]. However, in this study, we demonstrated that

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MPA was more effective than Dimethomorph used alone. Despite the effectiveness of

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MPA, B. velezensis Ba168 has the benefit of being more environmentally friendly. It is also favorable in biocontrol stability and suppression activity in TBS control compared to

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B. licheniformis, B. subtilis, B. laterosporus, B. methylotrophicus, and B. pumilus. Under the same field conditions, the disease control efficacy for tobacco variety Qinyan96 was

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generally higher than variety NC89. Therefore, Qingyan96 may be more resistant to TBS than NC89. Additionally, the relative disease control efficacy of B. methylotrophicus

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against TBS had the greatest drop in the two tested varieties, from 83.22% to 51.48%. This may be because B. methylotrophicus is a methylotroph[76], which are more restricted in application conditions. In conclusion, results from our study indicate that the isolated PGPR strain B. velezensis Ba168 has the capability to be utilized in the biocontrol of TBS under field conditions, and additionally, the possible mechanisms involved were illustrated. Although differences exist in TBS control efficacy, the overall strategy of PGPR application is promising for biocontrol of soil-borne fungal diseases. Although the disease control efficacy of strain Ba168 is slightly lower than that of the conventional treatment MPA, it

Journal Pre-proof is environmentally friendly and would generate no residue, even after many years of repeated application. Further studies are warranted to focus on target gene identification and genetic engineering[35], as well as to research potential mixtures of microbial agents[77] or mixtures of biocontrol agents and harmless chemicals[30, 78] containing strain Ba168. Acknowledgements We would like to thank Life Science Research Core Services (LSRCS) and The State Key

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Laboratory of Crop Stress Biology for Arid Areas for providing us with the scanning electron microscope, transmission electron microscopy, and protein identification

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analysis. For revision advice, we would like to thank Professor Sun Guang-yu and

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Professor Wang yang. We also thank Tobacco Research Institution of C hinese Academy of Agricultural Sciences (CAAS) for providing funds (grant number: 110201601023,

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20161128000001). References

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Graphical abstract

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Highlights This work highlights the effectiveness of PGPR strain B. velezensis Ba168 for biocontrol of the soil-borne disease P. nicotianae in lab study and field trials; and the importance of Bacillus-derived proteins/peptides in the suppression of P. nicotianae through irreversible damage to its cell walls and membranes.