Bacillus amyloliquefaciens ALB65 Inhibits the Growth of Listeria monocytogenes on Cantaloupe Melons

Listeria monocytogenes is estimated by the Centers for Disease Control and Prevention and the U.S. Food and Drug Administration to cause disease in approximately 1,600 to 2,500 people in the United States every year. The largest known outbreak of listeriosis in the United States was associated with intact cantaloupe melons in 2011, resulting in 147 hospitalizations and 33 deaths. In this study, we demonstrated that Bacillus amyloliquefaciens ALB65 is an effective biological control agent for the reduction of L. monocytogenes growth on intact cantaloupe melons under both pre- and postharvest conditions. Furthermore, we demonstrated that B. amyloliquefaciens ALB65 can completely inhibit the growth of L. monocytogenes during cold storage (<8°C). ABSTRACT Listeria monocytogenes is a foodborne pathogen that causes high rates of hospitalization and mortality in people infected. Contamination of fresh, ready to eat produce by this pathogen is especially troubling because of the ability of this bacterium to grow on produce under refrigeration temperatures. In this study, we created a library of over 8,000 plant phyllosphere-associated bacteria and screened them for the ability to inhibit the growth of L. monocytogenes in an in vitro fluorescence-based assay. One isolate, later identified as Bacillus amyloliquefaciens ALB65, was able to inhibit the fluorescence of L. monocytogenes by >30-fold in vitro. B. amyloliquefaciens ALB65 was also able to grow, persist, and reduce the growth of L. monocytogenes by >1.5 log CFU on cantaloupe melon rinds inoculated with 5 × 103 CFU at 30°C and was able to completely inhibit its growth at temperatures below 8°C. DNA sequence analysis of the B. amyloliquefaciens ALB65 genome revealed six gene clusters that are predicted to encode genes for antibiotic production; however, no plant or human virulence factors were identified. These data suggest that B. amyloliquefaciens ALB65 is an effective and safe biological control agent for the reduction of L. monocytogenes growth on intact cantaloupe melons and possibly other types of produce. IMPORTANCE Listeria monocytogenes is estimated by the Centers for Disease Control and Prevention and the U.S. Food and Drug Administration to cause disease in approximately 1,600 to 2,500 people in the United States every year. The largest known outbreak of listeriosis in the United States was associated with intact cantaloupe melons in 2011, resulting in 147 hospitalizations and 33 deaths. In this study, we demonstrated that Bacillus amyloliquefaciens ALB65 is an effective biological control agent for the reduction of L. monocytogenes growth on intact cantaloupe melons under both pre- and postharvest conditions. Furthermore, we demonstrated that B. amyloliquefaciens ALB65 can completely inhibit the growth of L. monocytogenes during cold storage (<8°C).

economic burden of approximately $2.6 billion, making it the third most costly foodborne illness in the United States (7).
Since 2011, several multistate foodborne listeriosis outbreaks have been associated with milk, soft cheeses, deli meats, and fresh produce (8). Especially concerning is the increased rate of contamination of fresh, ready-to-eat produce, including cantaloupe melons (2,8,9). In 2011, the largest outbreak of listeriosis in the United States was associated with uncut cantaloupe melons and resulted in 147 illnesses and 33 deaths in 28 states (10). Fresh produce can become contaminated with L. monocytogenes during the pre-and postharvest stages via direct or indirect contact with infected animals, contaminated soil, water, equipment, and human processors (2,10). The prevention of L. monocytogenes contamination of fresh, ready-to-eat fruit and vegetables is a serious food safety challenge, and a method to prevent L. monocytogenes from colonizing produce at both the pre-and postharvest stages would be advantageous to both producers and consumers.
Biological control is a bioeffector method of pest control and has been used to control the growth of insects (11), invasive weeds (12,13), plant pathogens (14)(15)(16), and other unwanted organisms. Several studies have proposed the use of lytic bacteriophages for the biological control of L. monocytogenes because of their ability to infect and lyse this bacterium (1,(17)(18)(19)(20)(21). Virulent bacteriophages have been used as biological control agents in many ready-to-eat foods such as milk, cheese, hot dogs (20), and raw meat (17,19). Application of mixtures of the phages LM-103 and LMP-102 with the bacteriocin nisin significantly reduced L. monocytogenes populations on sliced honeydew melons and apples (21). Bacteria have also been used as biological control agents. For example, Leverentz et al. (22) reported the use of bacteria that naturally occur on the surfaces of apples to inhibit the growth of L. monocytogenes on fresh-cut apples. Others have shown that bacteriocinogenic lactic acid bacteria, such as Leuconostoc citreum MB1 and Enterococcus mundtii CRL35, were able to delay L. monocytogenes growth in milk at refrigeration temperatures (23) and during sausage fermentation (24), respectively. In addition, Sharma et al. (25) showed that Azotobacter chroococcum, Bacillus megaterium, and Pseudomonas fluorescens were effective as biopesticides for the reduction of L. monocytogenes in the rhizosphere of pigeon pea plants (Cajanus cajan). However, bacterial biocontrol agents have rarely been examined for the ability to prevent L. monocytogenes from colonizing and persisting on the surfaces of intact, unprocessed fruits such as cantaloupe melons. Here, we used a novel, high-throughput in vitro screening methodology to identify plant phyllosphere associated bacteria that can be used as biological control agents to prevent/reduce the growth of L. monocytogenes on cantaloupe melons.

RESULTS
Identification of PPAB capable of inhibiting the growth of L. monocytogenes in vitro. We created and screened a plant phyllosphere associated bacterial library for the ability to inhibit the growth of L. monocytogenes RM2387-pNF8 in an in vitro fluorescence assay. Twenty PPAB isolates, named ALB65 to ALB84 for anti-Listeria bacteria, were identified that could inhibit the fluorescence of L. monocytogenes RM2387-pNF8 by 13.6-to 31.3-fold after 48 h, compared to the control (data not shown). We also assayed 18-h cell-free culture supernatants from these bacteria for the ability to inhibit the growth of this bacterium and found that 7 of the 20 isolates were able to inhibit the fluorescence of L. monocytogenes RM2387-pNF8 by 10.4-to 22.2-fold after 48 h (data not shown). To identify these isolates, we sequenced their 16S rRNA genes and performed BLAST analysis against the NCBI nucleotide database that revealed all of them were ϳ99% identical to Bacillus spp. Isolate ALB65 produced the greatest fluorescence inhibition of L. monocytogenes RM2387-pNF8 in vitro (Table 1) and was selected for further analysis.
Genomic DNA sequence analysis of ALB65 revealed that it was 98.72% identical to the plant-associated bacterium Bacillus amyloliquefaciens FZB42 and 95.49% identical to the soil-associated bacterium B. amyloliquefaciens DSM7 (26,27). To confirm the ability of B. amyloliquefaciens ALB65 to inhibit the growth of multiple L. monocytogenes outbreak strains, we performed disk diffusion assays using cell-free culture supernatant from B. amyloliquefaciens ALB65 against L. monocytogenes RM2387-pNF8 and 10 additional L. monocytogenes outbreak strains, all of which produced zones of inhibition of 10 to 14 mm (Table 2).
Growth and persistence of B. amyloliquefaciens ALB65 on cantaloupe melon rinds. We examined the ability of B. amyloliquefaciens ALB65 to grow and persist on the rinds of cantaloupe melons. Growth assays showed that when B. amyloliquefaciens ALB65 was inoculated onto cantaloupe rinds at approximately 1 ϫ 10 4 CFU rind Ϫ1 , it was able to multiply to Ͼ1 ϫ 10 6 CFU rind Ϫ1 after 48 h at 30°C (Fig. 1A). We also examined the ability of B. amyloliquefaciens ALB65 to persist on cantaloupe rinds kept under refrigeration temperatures. When B. amyloliquefaciens ALB65 was inoculated at 1 ϫ 10 7 CFU rind Ϫ1 and incubated at 8°C, we observed a significant (P Ͻ 0.05) decrease of ϳ2 logs after 6 days. However, after that time point the number of CFU rind Ϫ1 did not decrease significantly throughout the 9-day experiment (Fig. 1B). To determine whether B. amyloliquefaciens ALB65 was able to persist on the surfaces of preharvested, immature cantaloupe melons still attached to the plant, we sprayed greenhouse-grown melons with B. amyloliquefaciens ALB65 and quantified the number of CFU on the melon rinds. After 1 day, the number of B. amyloliquefaciens ALB65 was approximately 1 ϫ 10 5 CFU rind Ϫ1 and did not change significantly (P Ͼ 0.05) throughout the 6-day experiment (Fig. 1C).
Inhibition of L. monocytogenes growth on cantaloupe melon rinds. We examined the ability of B. amyloliquefaciens ALB65 to inhibit the growth of L. monocytogenes RM15995, an isolate from the 2011 U.S. multistate cantaloupe outbreak (28), on cantaloupe rinds under conditions that simulated both pre-and postharvest contamination. We coated cantaloupes with B. amyloliquefaciens ALB65 or phosphate-buffered saline (PBS [control]), inoculated them with approximately 5 ϫ 10 3 CFU L. monocytogenes RM15995 rind Ϫ1 , and incubated them at 30°C. L. monocytogenes RM15995 grew rapidly on the control rinds, reaching Ͼ2.9 ϫ 10 6 CFU rind Ϫ1 in 24 h; however, on the  B. amyloliquefaciens ALB65-treated rinds, L. monocytogenes only reached 4.5 ϫ 10 4 CFU rind Ϫ1 , an ϳ1.5-log CFU reduction in growth ( Fig. 2A). We also examined B. amyloliquefaciens ALB65 for the ability to inhibit the growth of L. monocytogenes under simulated postharvest cold-storage conditions. We applied B. amyloliquefaciens ALB65 to cantaloupe rinds, inoculated them with L. monocytogenes as described above, and incubated them at 8°C. After 6 days L. monocytogenes grew to approximately 8.3 ϫ 10 4 CFU on the PBS control rinds; however, on the B. amyloliquefaciens ALB65 treated rinds, we did not observe any increase in the number of L. monocytogenes CFU rind Ϫ1 (Fig. 2B). Predicted secondary metabolite gene clusters in the B. amyloliquefaciens ALB65 genome. We sequenced the genome of B. amyloliquefaciens ALB65 using a combination of Pacific Biosciences (PacBio) RS II and Illumina MiSeq platforms (27). In silico analysis of the B. amyloliquefaciens ALB65 genome via antiSMASH 4.0 (29) revealed six gene clusters, representing ϳ5.7% of the genome, that were predicted to encode genes for the biosynthesis of the antibiotic compounds macrolactin, difficidin, bacillaene, bacilysin, bacillibactin, and amylocyclicin (Table 3). All the gene clusters shared 90 to 100% similarity with the corresponding gene clusters in B. amyloliquefaciens FZB42, except for the bacillibactin gene cluster that shared 100% similarity to that found in B. subtilis subsp. subtilis 168.

DISCUSSION
The contamination of fresh, ready to eat produce by human-pathogenic bacteria continues to be a challenge for the food industry, government agencies and ultimately the consumer (30). Several methods to reduce/eliminate pathogens from produce have been implemented, the most commonly used methods employ physical or chemical treatments (31,32). A third approach to preventing/reducing pathogen contamination of produce is biocontrol, a bioeffector method of controlling pests using other living organisms (33). In order to be an effective biocontrol agent, the organism must be able to grow and persist on the surface of the target produce (31). We demonstrated that B. amyloliquefaciens ALB65 was able to grow and persist on cantaloupe melons under both pre-and postharvest conditions, as well as during cold storage. In addition, biological control agents must not induce early rot of the produce, impart off colors or smells, or produce virulence factors against the host plant or humans (34). B. amyloliquefaciens ALB65 did not induce early rot, nor did it impart off colors or smells to the cantaloupes after treatment and genome sequence analysis did not detect any virulence associated genes (data not shown). If applied to plants preharvest, biological control agents must not inhibit plant growth or fruit production. When we tested B. amyloliquefaciens ALB65 for its effects on cantaloupe plant growth, we observed that treated cantaloupes grew twice as fast in length as untreated cantaloupes and produced fruit significantly earlier as well (data not shown). This finding is consistent with previously published studies that have shown that B. amyloliquefaciens FZB42 enhances plant growth (26,35). Lastly, effective biocontrol agents must be able to inhibit the target pathogen's growth on produce (34). We demonstrated that B. amyloliquefaciens ALB65 could significantly reduce the growth of L. monocytogenes on cantaloupe rinds  at 30°C (P Ͻ 0.05) and completely inhibit the growth of this pathogen on cantaloupe rinds at temperatures below 8°C. It is unclear how B. amyloliquefaciens ALB65 inhibits the growth of L. monocytogenes on produce. However, genome sequence analysis identified multiple gene clusters that are predicted to produce antibacterial compounds, including macrolactin, difficidin, bacillaene, bacilysin, bacillibactin, and amylocyclicin. Macrolactin, difficidin, and bacillaene are polyketide antibiotics that act by inhibiting protein synthesis and have been shown to be active against both Gram-positive and -negative bacteria, including Staphylococcus aureus, Clostridium perfringens, Escherichia coli, and Ralstonia solanacearum (36). Bacilysin is a dipeptide antibiotic composed of anticapsin and alanine moieties and has been shown to inhibit cell wall biosynthesis in both Gram-positive and -negative bacteria, including Erwinia amylovora (37) and Staphylococcus aureus (38). Bacillibactin is a lipopeptide siderophore that sequesters iron, making it unavailable for other microorganisms and thus inhibits their growth (39). Finally, amylocyclicin is a circular bacteriocin produce by several species of Bacillus that are most effective against Gram-positive bacteria by forming pores in their cell membranes (40,41). Recently, amylocyclicin was purified and shown to have strong inhibitory activity against L. monocytogenes and B. cereus (41).
Previously, strains of B. amyloliquefaciens have been show to exert biocontrol activities against plant-pathogenic fungi and bacteria (15,42), including brown rot of stone fruit caused by Monilinia fructigena (42), root rot of ginseng caused by Cylindrocarpon destructans (43), root and stem rot of soybeans caused by Phytophthora sojae (44), bacterial canker disease caused by Clavibacter michiganensis (45), tomato wilt caused by Ralstonia solanacearum (46), and crown gall caused by Agrobacterium tumefaciens (47). Strains of B. amyloliquefaciens are also commercially used as safe and effective biofertilizers for plant growth promotion in multiple crops (48,49). In the present study, we demonstrated that B. amyloliquefaciens ALB65 is also an effective, food-grade, biological control agent for the reduction of L. monocytogenes growth on cantaloupe melons during both the pre-and postharvest periods.

Inhibition of L. monocytogenes growth by PPAB in vitro.
To identify PPAB that could inhibit the growth of L. monocytogenes, we used a modification of the method described by McGarvey et al. (34). Briefly, frozen stock cultures of PPAB in 96-well plates were thawed at 25°C, inoculated into fresh TSB in 96-well plates, and incubated at 37°C for 18 h without shaking. L. monocytogenes RM2387 harboring the green fluorescent protein encoding plasmid pNF8 (50) was cultured in brain heart infusion broth (BHI; Difco) supplemented with 25 g ml Ϫ1 lincomycin and 1 g ml Ϫ1 erythromycin (BHIϩEryϩLin) at 37°C for 18 h with shaking at 200 rpm and diluted to approximately 5 ϫ 10 3 CFU ml Ϫ1 with BHIϩEryϩLin, and then 50 l was placed into each well of black 96-well plates (Thermo Fisher Scientific, Roskilde, Denmark). Portions (25 l) of the PPAB 18-h cultures were added to each L. monocytogenes-containing well, followed by incubation at 30°C for 48 h; the samples were then analyzed for fluorescence in a Victor3 multilabel counter (Perkin-Elmer, Waltham, MA). PPAB isolates from the wells with the least amount of fluorescence, and thus the least amount of L. monocytogenes pNF8 growth, were selected for further analysis.
The ability of the PPAB isolates to inhibit the growth of 10 different L. monocytogenes outbreak strains was assayed using a filter disk diffusion assay as previously described (51). Briefly, 10 ml of 18-h PPAB cultures were centrifuged at 5,000 ϫ g for 10 min, and the supernatants were filtered through a 0.2-m PES syringe filter (Corning, Corning, NY) and then concentrated 20ϫ in a Savant SpeedVac concentrator (Thermo Fisher Scientific, Waltham, MA) without heat. A 20-l aliquot was applied to a 6-mm filter paper disk and placed onto BHI agar plates previously overlaid with 10 ml of BHI soft agar (0.7% agar) seeded with 0.5 ml of an 18-h culture of each of the L. monocytogenes outbreak strains (RM2387-pNF8, RM2199,  RM3180, RM3177, RM3176, RM15994, RM15995, RM15996, RM15997, RM20667, and RM20669) (Table 2), followed by incubation at 30°C for 16 h.
Growth and persistence of B. amyloliquefaciens ALB65 on cantaloupe melon rinds. The growth of B. amyloliquefaciens ALB65 on the surfaces of cantaloupe melons was carried out as described previously (34). Briefly, B. amyloliquefaciens ALB65 was grown for 18 h in TSB at 37°C with shaking at 150 rpm and diluted to ϳ1 ϫ 10 6 CFU ml Ϫ1 with sterile PBS. Sections (5 by 5 cm) of cantaloupe rinds (ϳ0.5 cm deep) were removed from melons using a sterile scalpel and placed into sterile petri plates (100 ϫ 25 mm deep; Falcon, Corning, NY). Ten l-l drops of the diluted culture were deposited onto the surface of the melon rinds, followed by incubation at 30°C. The growth of B. amyloliquefaciens ALB65 on the melon rinds was quantified daily for 2 days by homogenizing the sections in 100 ml of PBS in a sterilized blender jar with an Osterizer Beehive blender (Oster, Neosho, MO) on low speed for 30 s, followed by 30 s on high speed. The resulting solutions were serially diluted with PBS and plated onto Bacillus cereus selective agar plates (BCA) composed of B. cereus agar base (Oxoid), egg yolk emulsion (Remel), and polymyxin B supplement (Oxoid). The plates were incubated at 37°C for 24 h and counted.
To investigate the ability of B. amyloliquefaciens ALB65 to persist on cantaloupe rinds after a simulated postharvest dunk tank inoculation and subsequent cold storage, we grew B. amyloliquefaciens ALB65 in 600 ml of TSB for 18 h at 37°C with shaking at 150 rpm. The culture, containing approximately 3.0 ϫ 10 8 CFU ml Ϫ1 , was placed into a sterile 3.8-liter plastic bag containing a cantaloupe melon to mimic a dump tank washing. The air from the plastic bag was carefully removed by hand so that the culture was in constant contact with the cantaloupe, followed by incubation at 25°C for 30 min (34). After 30 min. the melons were blotted dry with paper towels and air dried 18 h at 25°C. Next, 5-cm 2 sections of the melon rinds were removed using a sterile scalpel and placed into sterile petri plates (100 ϫ 25 mm deep; Falcon), followed by incubation at 8°C. The persistence of B. amyloliquefaciens ALB65 on the melon rinds was quantified every third day for 9 days as described above (34).
To evaluate the persistence of B. amyloliquefaciens ALB65 on the surfaces of preharvest melons, we grew cantaloupe plants (Cucumis melo var. reticulatus) in a greenhouse from seeds (Park Seed, Greenwood, SC) and pollinated them by hand. When the melons were fully formed but still unripe (ϳ30 days postemergence), they were sprayed with 100 ml of an 18-h culture of B. amyloliquefaciens ALB65, containing approximately 3 ϫ 10 8 CFU ml Ϫ1 , and the number of B. amyloliquefaciens ALB65 CFU on 5-cm 2 sections of the melon rinds was quantified at days 0, 1, 3, and 6 as described above.
Inhibition of L. monocytogenes growth on cantaloupe melon rinds by B. amyloliquefaciens ALB65. To evaluate the ability of B. amyloliquefaciens ALB65 to inhibit the growth of L. monocytogenes RM15995, an isolate from the 2011 US multistate cantaloupe outbreak (28), on cantaloupe rinds after a simulated postharvest dunk tank inoculation, we grew B. amyloliquefaciens ALB65 and mimicked a dunk tank inoculation as described above under growth and persistence. PBS was used in place of B. amyloliquefaciens ALB656 cultures for the control melons. Sections (5 cm 2 ) of the melon rinds were removed using a sterile scalpel and placed into sterile petri plates (100 ϫ 25 mm deep; Falcon). L. monocytogenes RM15995 was grown for 18 h in BHI at 37°C shaking at 200 rpm and diluted to approximately 5 ϫ 10 5 CFU ml Ϫ1 in PBS, and 10 1-l drops were placed onto the cantaloupe rinds. The rinds were incubated at 30°C for 24 h and quantified for L. monocytogenes by homogenizing the rinds in 100 ml of PBS in a sterilized blender jar at low speed for 30 s, followed by high speed for 30 s using an Osterizer Beehive blender (Oster). The resulting solution was serially diluted in PBS, plated onto modified Oxford agar plus 50 g/ml nalidixic acid (MOXϩNal) agar plates (Oxoid), incubated at 37°C in a 2.5-liter Oxoid AnaeroJar with a Remel Oxoid AnaeroGen 3.5-liter sachet (Remel) for 2 days, and counted.
To assay for the ability of ALB65 to inhibit the growth of L. monocytogenes strain RM15995 on cantaloupe rinds under simulated postharvest cold storage conditions, we coated cantaloupe melons as described above with B. amyloliquefaciens ALB65 (34), removed 5-cm 2 sections, and inoculated them with L. monocytogenes RM15995 as described above. The rinds were incubated for 6 days at 8°C, and the number of L. monocytogenes RM15995 on the rinds was quantified as described above.
Identification of B. amyloliquefaciens ALB65 by use of 16S rRNA gene and genomic DNA sequence analysis. For 16S rRNA gene sequence analysis, B. amyloliquefaciens ALB65 was inoculated into 3 ml of TSB, followed by incubation for 18 h at 37°C shaking at 150 rpm. The culture was centrifuged at 10,000 ϫ g for 10 min, and the pellet was suspended in PBS. DNA was extracted using the Wizard Genomic DNA purification kit (Promega, Madison, WI) according to the manufacturer's instructions. The 16S rRNA gene was PCR amplified in 50-l reaction mixtures containing 25 l of high-fidelity PCR master mix (Roche, Nutley, NJ), 10 ng of DNA, and 10 M concentrations of the primers 27F (AGAGTTTGATCM TGGCTCAG) and 1492R (TACGGYTACCTTGTTACGACTT) (52) in a C1000/S1000 Touch thermocycler (Bio-Rad, Hercules, CA) under the following conditions: 1 cycle of 95°C for 5 min; 30 cycles of 94°C for 30 s, 55°C for 30 s, 72°C for 1.5 min; and 1 cycle of 10 min at 72°C. The resulting DNA was cloned using the TOPO TA cloning kit (Invitrogen, Carlsbad, CA) according to the manufacturer's instructions and transformed into E. coli TOP10 competent cells (Invitrogen). Transformants were grown on LB agar plates containing 50 g ml Ϫ1 kanamycin (Km). Individual colonies were picked and streaked onto LB Km agar plates. Plasmid minipreps were performed using a QIAprep spin miniprep kit according to the manufacturer's instructions. Plasmid inserts were sequenced using the 27F and 1492R primers and a BigDye Terminator v3.1 cycle sequencing kit (Applied Biosystems, Foster City, CA). Sequencing reactions were purified using a BigDye XTerminator purification kit (Applied Biosystems); electrophoresis and readout were performed using an Applied Biosystems 3730XL genetic analyzer (Applied Biosystems). The forward and reverse sequences were aligned using SeqManII software (DNASTAR, Inc., Madison, WI). Sequences were analyzed using the BLAST software and the NCBI nr/nt database set to exclude models and uncultured/environmental sample sequences and to limit to sequences from type material (available at http://blast.ncbi.nlm.nih.gov/).
To resolve B. amyloliquefaciens ALB65 to the species level and identify possible antibiotic and secondary metabolite operons, we sequenced the genome using a combination of Pacific Biosciences (PacBio) RS II and MiSeq Illumina platforms (27). Genomic DNA was extracted by the phenol-chloroform method (53). Briefly B. amyloliquefaciens ALB65 was cultured for 18 h in 100 ml of TSB at 37°C with shaking at 150 rpm. Cells were harvested by centrifugation at 5,000 ϫ g for 5 min and resuspended in 50 mM Tris buffer (pH 8.0) to an optical density at 600 nm of 1.6 to 1.8. A portion (1.5 ml) of the suspension was transferred to a 15-ml Falcon tube, and 250 l of 10 mg ml Ϫ1 lysozyme solution and 600 l of 100 mM EDTA (pH 8.0) was added. The mixture was incubated on ice for 10 min; then, 300 l of 5% SDS was added, and the mixture was vortexed vigorously for 10 s. RNA was removed by the addition of 5 l of 100 mg ml Ϫ1 RNase A, followed by incubation at 37°C for 24 h with intermittent inversion. Next, 10 l of 15 mg ml Ϫ1 proteinase K was added, and the solution was incubated at 37°C for 4 h with inversions every 1 h. Genomic DNA was precipitated with 265 l of 3.0 M sodium acetate (pH 5.5) and 6 ml of ice-cold ethanol, dried in a Savant SpeedVac concentrator, and resuspended in 400 l of 10 mM Tris (pH 8.0). DNA was treated with phenol-chloroform-isoamyl alcohol (25:24:1), precipitated with 100% ethanol, and resuspended in 100 l of 10 mM Tris (pH 8.0).
PacBio template preparation was carried out as described for the procedures and checklist for the 20-kb template preparation using the BluePippin size-selection system (53). The genome sequence was assembled and analyzed as described previously (27).
Data availability. The sequence of the B. amyloliquefaciens ALB65 16S rRNA gene was deposited into GenBank (accession number MN538240). The genome sequence of B. amyloliquefaciens ALB65 was also deposited in GenBank (accession number CP029069).
Statistical analysis. All experiments were carried out using a complete randomized design. Experimental data were analyzed with one-way analysis of variance analysis using Sigma Plot (SSPS, version 12). The results are presented as mean values Ϯ the standard deviations of at least three independent experiments in which each rind was a replicate.