Biological Control 90 (2015) 141–147

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Biological Control

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Expression of Paenibacillus polymyxa b-1,3-1,4-glucanasein Streptomyces lydicus A01 improves its biocontrol effectagainst Botrytis cinerea

http://dx.doi.org/10.1016/j.biocontrol.2015.06.0081049-9644/� 2015 The Authors. Published by Elsevier Inc.This is an open access article under the CC BY-NC-ND license (http://creativecommons.org/licenses/by-nc-nd/4.0/).

⇑ Corresponding authors.E-mail addresses: liuwich@163.com (W. Liu), wuhuiling925@126.com (H. Wu).

Jinjin Li a, Weicheng Liu a,⇑, Lijin Luo b, Dan Dong a, Ting Liu a, Taotao Zhang a, Caige Lu a, Dewen Liu a,Dianpeng Zhang a, Huiling Wu a,⇑a Institute of Plant and Environment Protection, Beijing Academy of Agriculture and Forestry Sciences, Beijing 100097, Chinab Fujian Provincial Key Laboratory of Screening for Novel Microbial Products, Fujian Institute of Microbiology, Fuzhou 350007, China

h i g h l i g h t s

� Strain A21 was isolated from special eco-environment in Tibet, China.� First report on expression of P. polymyxa glucanase in industrial strain.� Enhanced antifungal activity is due to synergy of three factors.

a r t i c l e i n f o

Article history:Received 8 May 2015Revised 20 June 2015Accepted 24 June 2015Available online 25 June 2015

Keywords:b-1,3-1,4-GlucanaseAntifungal activityPaenibacillus polymyxaStreptomyces lydicusNatamycin

a b s t r a c t

Streptomyces lydicus strain A01, which can produce natamycin and chitinase, has a significant inhibitioneffect on gray mold disease caused by Botrytis cinerea. However, it has no detectable glucanase activity.Strain A21 isolated from the snow covered high altitude area in Tibet, China, also has a high antagonisticactivity against B. cinerea. It displayed an obvious halo on lichen polysaccharides plates by congo redstaining, indicating a strong glucanase activity. A21 was identified as Paenibacillus polymyxa using 16SrDNA gene analysis and biochemical and physiological analysis. To obtain the synergistic antifungaleffects of natamycin, chitinase, and glucanases on B. cinerea, this study transformed theb-1,3-1,4-glucanase gene from P. polymyxa A21 to S. lydicus A01. The engineered S. lydicus AG01 showedsubstantially high glucanase activity, and had similar natamycin production and chitinase activity as thewild-type strain A01. Compared to the wild-type strain A01, the antifungal effects of S. lydicus AG01 on B.cinerea, including inhibition of spore germination and mycelial growth, were highly improved. Theimproved biocontrol effect of S. lydicus AG01 is likely attributed to the heterologous expression of glu-canase from P. polymyxa, which acted synergistically with natamycin and chitinase to increase the anti-fungal activity of the strain.� 2015 The Authors. Published by Elsevier Inc. This is an open access article under the CC BY-NC-ND license

(http://creativecommons.org/licenses/by-nc-nd/4.0/).

1. Introduction and has a significant inhibition effect on B. cinerea (Lu et al., 2008;

Botrytis cinerea is a widespread phytopathogenic fungus thatcan cause gray mold rot or Botrytis blight of many vegetables, orna-mentals, fruits, and even field crops throughout the world (Alfonsoet al., 2000). Chemical control of Botrytis is not effective and oftenassociated with the appearance of resistant Botrytis sp. strains tothese chemicals (Rosslenbroich and Stuebler, 2000). Streptomyceslydicus strain A01 isolated from the soil of suburban vegetable fieldin Beijing, China, is capable of producing natamycin and chitinase,

Wu et al., 2013a,b). Natamycin is a tetraene macrolide antibioticwidely used as a food preservative and fungicide in food, medicine,and veterinary products, and it has the potential to control plantfungal diseases in agricultural production (Ture et al., 2011;Currie et al., 1990; Lu et al., 2008; Wu et al., 2014a).Pathogenesis-related (PR) proteins such as chitinases and glu-canases can degrade fungal cell walls and play important roles inplant defence against pathogens, and are widely used as antifungalagents in plant protection (Errakhi et al., 2007; Shi et al., 2010; Parket al., 2012). Paenibacillus polymyxa strains produce several antibi-otics and hydrolytic enzymes, which play an important role in thebiocontrol of plant pathogens (Deng et al., 2011; Ito and Koyama,1972; Raza et al., 2009; Beatty and Jensen, 2002).

Table 1Physiological, biochemical characteristics and carbon source utilization of strain A21.

Physiological and biochemical property Result Carbon source Result

Gelaune liquefaction + D-glucose +

Casein + D-mannitol +

Starch + D-lactose +

Formation of indole � D-galactose +

Citrate utilization � D-Maltose +

Nitrate reduction + I-inositol �Catalase + D-fructose �Oxidase � D-xylose �Chitinase � D-rhamnose �Cellulase + D-Glucitol +

V-P test + D-Cellobiose +

MR test + L-arabinose -

2% Nacl + D-Melibiose +

5% Nacl � Sucrose +pH 5.7 + Glycerol +50 �C + Raffinose +60 �C � Trehalose +

+, positive; �, negative.

A21(KP268494)P. polymyxa DSM 36T(AJ320493)

P. peoriae strain DSM 8320T(AJ320494) P. kribbensis AM49T( AF391123) P. terrae strain AM141T(AF391124)

P. hunanensis strain FeL05T(EU741036) P. timonensis 2301032T(AY323612)

P. barcinonensis type strain BP-23T(AJ716019) P. kobensis DSM10249T(AB073363)

P. nanensis MX2-3T(AB265206)

P. granivorans (AF237682) P. daejeonensis strain AP-20T( AF290916)

Bacillus subtilis strain DSM 10T(NR027552)

100

100

7381

6862

59100

9868

4533

27

0.01

P. validus JCM 9077T(AB073203)

P. amylolyticus NRRL NRS-290T (D85396)

P. camelliae strain b11s-2T (EU400621)

Fig. 1. Phylogenetic tree derived from nearly complete 16S rDNA gene sequencesshowing relationships between the strain A21 and the related type species of thegenus Paenibacillus.

142 J. Li et al. / Biological Control 90 (2015) 141–147

In this study, we isolated a bacterial strain (A21) from the snowcovered high altitude area in Tibet, China, that showed high antag-onistic activity against several plant fungal diseases, especiallygray mold diseases caused by B. cinerea. It displayed an obvious

Fig. 2. Dual culture plates of control and inhibition of B. cinerea by P. polymyxa A21. HypPDA. Each plug was placed at the center of a PDA plate and incubated at 25 �C for 1 day. Sa single streak was inoculated onto both sides of the plug at a distance of 2 cm apart. T

halo on lichen polysaccharides plates by congo red staining, indi-cating a strong glucanase activity. However, glucanase activityhas not been detected in S. lydicus A01. Combining two biocontrolfactors can have synergistic antifungal effects and thus enhancefungal resistance in plants or microorganisms. For example, com-bined expression of chitinase and b-1,3-glucanase genes in riceincreased resistance against Rhizoctonia solani (Sridevi et al.,2008). Heterologous expression of chitinase from Trichoderma har-zianum in natamycin-producing strain S. lydicus A01 increased itsantagonistic effect on B. cinerea and Fusarium oxysporum f. spp(Wu et al., 2013a,b).

To improve the antifungal activity of S. lydicus A01, we clonedand expressed the glucanase gene from strain A21 under the S. ery-thraea ermE⁄ promoter in S. lydicus A01, generating the recombi-nant strain S. lydicus AG01. We also investigated the glucanaseactivity and antifungal capacity of S. lydicus AG01, as well as itsinhibitory effects on spore germination and mycelial growth of B.cinerea.

2. Materials and methods

2.1. Strains and plasmids

S. lydicus A01 (CGMCC 1653) was isolated from suburban veg-etable field soil, and identified as S. lydicus based on the 16SrDNA sequences and phenotypic comparison (Lu et al., 2008).The GenBank accession number for 16S rDNA of S. lydicus strainA01 is EF532323. Strain A21 (CGMCC 10708) was isolated fromthe snow covered high altitude area in Tibet, China. The fungalpathogen B. cinerea was deposited in the Institute of Plant andEnvironment Protection, Beijing Academy of Agriculture andForestry Sciences, Beijing, China (Lu et al., 2008). Escherichia colistrains DH5a and ET12567 (pUZ8002) were used as the cloninghost and donor for conjugal transformation, respectively. The inte-gration vector pIB139, which harbored the ermE⁄ promoter fromvector pSET152 containing U31 int and attP (Wilkinson et al.,2002), was used to introduce glucanase gene into S. lydicus.Plasmid pUC19 was used for routine cloning and sub cloningexperiments. Conjugation and regeneration were performedaccording to Kitani et al. (2000) and Paranthaman andDharmalingam (2003). The transformants were selected by60 lg mL�1 apramycin and further confirmed by PCR analysis withthe specific primers of glucanase gene. Synthesis of oligonucleotideprimers and DNA sequencing of PCR products were performed byInvitrogen Biotechnology (China).

hal plugs (7 mm in diameter) were cut from a 3-day-old culturation of B. cinerea ontrain A21 was cultured on King’s B modified medium agar at 25 �C for 36 h, and thenhe plug was further incubated in the dark at 28 �C for 7 days.

A21 -1,3-1,4glucanase (KP268493)

P. polymyxa SC2 endo- -(1,3)(1,4)glucanase (ABZ80848)P. terrae -glucanase (WP014278271)

P. peoriae -glucanase (WP010346864) P. macerans -13-14-glucanase (AAO66468)

P. polymyxa CP7 -13-14-glucanase (ABY41241) P. barcinonensis -13-14-glucanase (AIU68830)

P. curdlanolyticus -glucanase (WP006039960) P. mucilaginosus -glucanase (WP014368820)

P. borealis -glucanae (AIQ60347) P. wynnii -glucanase (KGE18644) 99

100

71

98

41

54

2498

0.02

A

A21

B

Fig. 3. Detection of glucanase activity and sequence alignment of PglA with relatedb-1,3-1,4-glucanases. (A). Detection of glucanase activity on lichen polysaccharidesplates with congo red staining. (B). Neighbor-joining tree showing the phylogeneticposition of PglA with related b-1,3-1,4-glucanases from Paenibacillus species basedon amino acid sequences. The percentages of bootstrap values (based on 1,000bootstraps) are shown at the internal nodes.

A01 AG01

B

A M P

717 bp

2 kb

1 kb 750 bp 500 bp

C

0

20

40

60

80

100

120

3 6 12 18 24 48 72 96 120

Cultivation time (h)

Rel

ativ

e ac

tivity

(%)

Fig. 4. Gel image of pIBg139 positive plasmid identified by double-enzymedigestion and glucanase activity analysis of strain S. lydicus AG01. (A). A gel imageof pIBg139 positive plasmid identified by double-enzyme digestion; Lane M, DNAMarker; Lane P, pIBg139 positive plasmid. (B). Detection of glucanase activity onlichen polysaccharides plates with congo red staining. (C). Time course of the endo-glucanase production by S. lydicus AG01. The maximum value was set as100% = 1.72 U/mL. Data are mean ± SD of three independent experiments.

J. Li et al. / Biological Control 90 (2015) 141–147 143

2.2. Physiology and biochemistry

The physiological and biochemical features of A21 weredetermined using standard methods (Sneath 1986; Dong et al.2001). Utilization of different carbon sources and the assessmentof saccharolytic activity were determined by standard methods(Dong et al. 2001). The sources of carbon used were D-glucose,

D-mannitol, D-lactose, D-galactose, D-Maltose, I-inositol, D-fructose,

D-xylose, L-rhamnose, D-Glucitol, D-Cellobiose, L-arabinose, D-Melibiose, Sucrose, Glycerol, Raffinose and Trehalose. Carbon sourcessterilized will be added to the basal medium to give a final concen-tration of 1% according to Shirling and Gottlieb (1966).

2.3. PCR amplification of 16S rDNA from strain A21

Genomic DNA was prepared with a TIANamp Bacteria DNA Kit(Tiangen, Beijing, China). The primer sequences for the amplifica-tion and sequencing of the 16S rDNA genes were: 27F (50-AGAGTTTGATCCTGGCTCAG-30) and 1525R (50-AAAGGAGGTGATCCAGCC-30) (Devereux and Willis, 1995), which amplified a fragment ofapproximately 1458 bp. Fragments obtained were purified andsequenced.

2.4. Analysis of the antagonistic activity of strain A21 against B.cinerea

The antagonistic activity of strain A21 against B. cinerea wasdetermined using a dual culture assay described by Ferreira et al.(1991) and Huang et al. (2014). Hyphal plugs (7 mm in diameter)were cut from a 3-day-old cultivation of plant pathogen fungi onPDA. Each plug was placed at the center of a PDA plate and incu-bated at 25 �C for 1 day. Strain A21 was cultured on King’s B mod-ified medium (Atlas, 1995) agar at 25 �C for 36 h, and then a singlestreak was inoculated onto both sides of the plug at a distance of2 cm apart. The plug was further incubated in the dark at 28 �Cfor 7 days. Plates were checked for the presence of inhibiting zones,which were measured. Plates without inoculated bacteria were

used as controls and each assay was performed with three plates,and all tests were repeated three times.

2.5. Construction of expression vector of Streptomyces with the b-1,3-1,4-glucanase gene from P. polymyxa A21

The primers gluF (50-GGAATTCCATATGATGATGAAGAAGAAGTC

TTGG-30) and gluR (50-GCTCTAGACTAATTGCTCGTATATTTTACC-30)designed according to the sequence of endo-beta-1,3-1,4 glucanasegene of P. polymyxa strain SC2 (GenBank accession number:EU410619) were used to clone glucanase gene from P. polymyxaA21 (underlined nucleotides were added to introduce NdeI andXbaI sites for cloning). A 717 bp segment was verified by DNAsequencing and the PCR fragment was digested with NdeI/XbaI and

144 J. Li et al. / Biological Control 90 (2015) 141–147

ligated into NdeI/XbaI-digested pIB139 to generate pIBg139. Therecombinant vector was introduced into S. lydicus A01 by inter-generic conjugation. The transformants were selected by60 lg mL�1 apramycin and further confirmed by PCR analysis withthe specific primers for glucanase gene.

2.6. Glucanase activity assay

Relative glucanase activity was examined by measuring thehalo diameter of enzyme diffusion on agar plates containing sub-strate as described previously by Wu et al. (2014b) and Nguyenand Quyen (2010). The fermentation for glucanase productionand glucanase activity assay was conducted with 0.5% (w/v) lichenpolysaccharides in 0.2 M acetic acid buffer (pH 5.0) according toWu et al. (2014b) and Mandels et al. (1976) with minor modifica-tion. The amount of reducing sugars released in the reagent solu-tion at 50� C for 10 min was measured at the wavelength of540 nm (spectrophotometer UV-2500, Hewlett Package, USA).Glucose was used as the standard for the estimation of reducingsugars. One unit (U) of the glucanase activity was defined as theamount of enzyme required to release 1 lmol glucose per minuteunder experimental conditions.

2.7. Fermentation and analysis of natamycin

Spores (5 � 107) of S. lydicus A01 and AG01 from PDA agar slantwere inoculated into 50 ml of a seed culture medium (Wu et al.,2014a), and incubated at 29 �C for 24 h on a rotary shaker(250 rpm). The YSG medium (2.8% soybean flour, 0.7% yeastextract, 6% glucose) (Du et al., 2009) was used for natamycin pro-duction analysis according to Wu et al. (2014a). Natamycin extrac-tion and HPLC (Japan Analytical Industry Co., Ltd., Japan) analysiswith C18 reverse phase column (LC-9101-JAIGEL-ODS, Japan) wascarried out as described by Wu et al. (2014a).

A01 AG01

A

Dia

met

er o

f in

hib

itio

n z

on

e(cm

)

B

C

AG01

Fig. 5. Control effects of S. lydicus AG01 and the wild-type A01 on spore germination of Bincubation at 28 �C for 5 days. Each assay was performed with three plates. B. Diameterstreated with fermentation supernatants of S. lydicus AG01, the wild-type A01 after 3 d incCK. Data are means of three determinations ± standard deviation (error bars). Bars with

2.8. Chitinase hydrolysis activity assay

Chitinase hydrolysis activity was measured by the halo diame-ter of enzyme diffusion on the chitinase production medium (0.2%colloidal chitin, 0.4% NH4NO3, 0.05% K2HPO4, 0.05% MgSO4�7H2O,0.05% KCl, 0.001% FeSO4�7H2O, and 2% agar). S. lydicus strains A01and AG01 were cultured in the plate for 10 days and the diametersof the chitin hydrolysis zones were measured. Each treatment wasperformed in triplicate.

2.9. Assay of antagonistic effect on spore germination and mycelialgrowth of B. cinerea

Two Oxford cups were placed on an agar plate containing 25 mlof PDA inoculated with B. cinerea spore suspension (5 � 107) andindividually loaded with 100 lL (100 � diluted) 3-day-old fermen-tation supernatant of S. lydicus in YEME medium (0.3% yeast extract,0.5% peptone, 0.3% malt extract, 1% glucose) (Aparicio et al., 2000)without sucrose. After incubation at 28 �C for 5 days, the inhibitiondiameters of B. cinerea were measured. Each assay was performedthree times. The antagonistic effects on spore germination andmycelial growth of B. cinerea were determined according to Wuet al. (2013a) with some modifications. Twenty-five milliliters ofPDA medium, containing 2 ml 3-day-old fermentation supernatantsof S. lydicus AG01 and the wild-type A01 in YEME medium withoutsucrose, were added into a 9 cm diameter Petri dish, while anotherpetri dish contained 25 ml of PDA medium with 2 ml supernatantof un-inoculated fermentation broth as control (CK). The edges ofthe colonies after 3 d incubation were cut with a sterile scalpeland observed under the light microscope (Leica DMIRE2 microscope,Germany). Each treatment was performed in triplicate.

2.10. Statistical analysis

All experiments were conducted in triplicate. Data were pre-sented as mean ± standard deviation (error bars) and analyzed

b

a

0.0

1.0

2.0

3.0

4.0

A01 AG01

A01 CK

. cinerea. A. Inhibition zones produced by S. lydicus AG01 and the wild-type A01 afterof the inhibition zones. C. Micrographs showing the spore germination of B. cinerea

ubation, and the un-inoculated fermentation broth with corresponding treatment asthe same letters are not significantly different according to LSD test at P < 0.05.

J. Li et al. / Biological Control 90 (2015) 141–147 145

using SPSS statistical software. The comparisons between differenttreatments were tested by ANOVA and bars with the same lettersare not significantly different according to LSD test at P < 0.05.

3. Results

3.1. Taxonomic classification of strain A21

Strain A21 was positive for catalase, cellulose, methyl red, andVoges–Proskauer reactions. It could liquefy gelatin, hydrolyzecasein and starch, and reduce nitrate to nitrite. It was negativefor the production of indole, citrate utilization, oxidase and chiti-nase activity. It utilized most of tested carbon sources exceptI-inositol, D-fructose, D-xylose, L-rhamnose, and L-arabinose.Growth ranges for temperature, pH, and NaCl concentration arepresented in Table 1.

The cultural, biochemical and physiological characteristics ofstrain A21 are typical properties of species P. polymyxa. A1458 bp 16S rDNA partial sequence of strain A21 was amplifiedand sequence comparison using BLAST suggested a close relation-ship with P. polymyxa. Phylogenetic analysis (Fig. 1) indicated that

c cb

ba

a

0

1

2

3

4

5

6

7

8

2 3

Duration of i

Co

lon

y d

iam

eter

(cm

)

AG01 A01

A

C

B

Fig. 6. Control effects of S. lydicus AG01 and the wild-type A01 on mycelial growth of B. csupernatants of S. lydicus AG01, the wild-type A01 and CK, respectively. B. Photos showfermentation supernatants of S. lydicus AG01, the wild-type A01 and CK. C. Micrographsupernatants of S. lydicus AG01, the wild-type A01 and CK. Data are means of three detebetween different treatments on the same day. Bars with the same letters are not signi

strain A21 was similar to P. peoriae, P. terrae AM141T, and P.kribbensis AM49, and was most closely related to P. polymyxaDSM 36T (99 % similarity). The consensus sequence of 16S rDNAwas deposited in GenBank under accession No. KP268494.

3.2. Antagonistic activity of P. polymyxa A21 against B. cinerea

P. polymyxa A21 showed high antagonistic activity against graymold diseases caused by B. cinerea. After 7 days, the mean diameterof B. cinerea colonies inoculated with strain A21 was 17 mm and78 mm in parallel for the control (Fig. 2). Dual culture plates wereincubated for a further 10 days, and B. cinerea did not overwhelmthe bacterial colony, indicating a persistent inhibition.

3.3. Cloning and characterization of the glucanase gene in P. polymyxaA21

P. polymyxa strain A21 displayed an obvious halo on lichenpolysaccharides plates by congo red staining, indicating a strongglucanase activity (Fig. 3(A)). A 717 bp ORF encoding 239 aminoacids was obtained from strain A21, with primers designed

cc

bb

a

a

4 5

ncubation(days)

CK

inerea. A. Colony diameters of B. cinerea incubated on PDA plates with fermentationing the colony diameters of B. cinerea after a 3 d incubation on PDA plates with

s showing the mycelial morphology of B. cinerea on PDA plates with fermentationrminations ± standard deviation (error bars). The comparisons were conducted justficantly different according to LSD test at P < 0.05.

146 J. Li et al. / Biological Control 90 (2015) 141–147

according to the endo-b-1,3-1,4-glucanase gene from P. polymyxaSC2 (ABZ80848). BLAST analysis revealed that the ORF (239 aa),designated PglA, showed a 99% sequence identity to theendo-b-1,3-1,4-glucanase from P. polymyxa SC2 (ABZ80848), 85%sequence identity to the endo-b-1,3-1,4-glucanase from P. macer-ans (AY221029), and 84% sequence identity to that from B. macer-ans CICC-20649 (X55959). Phylogenetic analysis showed the closerelationship between PglA and Paenibacillus genus b-1,3-1,4-glucanases (Fig. 3(B)). The sequences of pglA was submitted toGenBank with the accession No. KP268493.

3.4. Expression of the glucanase gene from P. polymyxa A21 onglucanase activity, natamycin production, and chitinase activity in therecombinant S. lydicus strain

The endo-b-1,3-1,4-glucanase gene from P. polymyxa strain A21was cloned into the pIB139 vector to generate pIBg139, and thepIBg139 positive plasmid was identified via double-enzyme diges-tion. As shown in Fig. 3(A), the pIB139 vector was 5922 bp inlength, and the fragment of pglA gene was 717 bp (Fig. 4(A)).Plasmid pIBg139 was transformed into S. lydicus A01, and positivetransformants were selected by 60 lg mL�1 apramycin and furtherconfirmed by PCR analysis with primers specific for pglA gene of P.polymyxa A21. The recombinant strain of S. lydicus A01 was namedAG01. The initial glucanase activity verification of S. lydicus AG01was assessed on lichen polysaccharides plates, and AG01 displayedan obvious halo compared to the wild-type strain A01 by congo redstaining (Fig. 4(B)). Glucanase activity was further detected in cul-ture, and AG01 showed detectable activity at 3 h and the highestactivity at 24 h (Fig. 4(C)). AG01 maintained a relatively steadyhigh activity from 24 h (1.72 U/mL, 100%) to 120 h (1.52 U/mL,88%). These findings suggested that the pglA gene from P. polymyxaA21 driven by ermE⁄ promoter was expressed well in S. lydicusAG01.

On YSG medium, strain AG01 grew similarly as the wild-typestain A01, and it also produced similar level of natamycin(2.09 g/L) as A01 (1.99 g/L) after 72 h-fermentation (Fig. S1). Thetwo S. lydicus strains also had comparable chitin hydrolysis zones,with 0.47 ± 0.1 cm for AG01 and 0.45 ± 0.1 cm for A01 after 10 daysof culturation on the chitinase production medium (Fig. S2). Theseresults indicated that expression of pglA gene had no significantchanges on cell growth, natamycin production, and chitinase activ-ity of S. lydicus.

3.5. Control effect of S. lydicus on spore germination of B. cinerea

Control effect on spore germination of B. cinerea by the fermen-tation broth produced by strains AG01 and A01 under the sameconditions were studied and the diameters of inhibition-zoneswere measured (Fig. 5(A) and (B)). The diameters of B. cinerea-inhi-bition zones produced by the fermentation supernatants of S. lydi-cus AG01 and A01 were 3.4 cm ± 0.26 cm and 2.2 cm ± 0.15 cm,respectively, and statistical analysis showed that the diameters ofinhibition-zones for the fermentation supernatant of S. lydicusAG01 was significantly larger than that of the wild-type strain A01.

In addition, the B. cinerea spore germination was significantlyinhibited when treated with fermentation supernatant of S. lydicusAG01. Nearly no B. cinerea mycelia were produced after incubationfor 36 h with S. lydicus AG01, whereas B. cinerea spores with S. lydi-cus A01 treatment had a greater germination rate with longermycelia. The B. cinerea spores of CK had a germination rate ofalmost 100% with long mycelia (Fig. 5(C)). These results indicatedthat, comparing with the wild-type A01, S. lydicus AG01 had signif-icantly improved the inhibition effect on spore germination of B.cinerea.

3.6. Control effect of S. lydicus on mycelial growth of B. cinerea

The colony diameters of B. cinerea cultured on PDA plates con-taining fermentation supernatants of S. lydicus AG01, A01 and CK,respectively, were measured on days 2–5. The colony diameter ofB. cinerea on PDA plates containing the fermentation supernatantof S. lydicus AG01 was only 1.8 ± 0.17 cm, whereas that on platescontaining the fermentation supernatant of S. lydicus A01 and con-trol broth were 3.8 ± 0.16 and 5.4 ± 0.24 cm after incubation for3 d, respectively (Fig. 6(A) and (B)). The density of B. cinerea myce-lia treated with fermentation supernatant of S. lydicus AG01 wasmuch higher than that treated with S. lydicus A01 (Fig. 6(C)). Incomparison, the B. cinerea mycelia in the CK treatment were foundto be sparse and free. These observations indicated that the inhibi-tion to mycelial growth of B. cinerea by S. lydicus AG01 wasstrengthened as compared with the wild-type A01.

4. Discussion

S. lydicus is a potential biocontrol strain against several plantfungal diseases due to the production of natamycin (Lu et al.,2008; Wu et al., 2013a,b,, 2014a,b). The wild-type strain A01 pro-duces natamycin and exhibits chitinase activity, but shows no glu-canase activity on lichen polysaccharides plates when tested bycongo red staining. Combining different biocontrol factors canenhance fungal resistance in plants or microorganisms (Srideviet al., 2008; Jongedijk et al., 1995; Wu et al., 2013a,b). The trans-genic tomato plants with both chitinase and glucanase showedenhanced resistance against infection with F. oxysporum f. sp.lycopersici, whereas transgenic tomato plants constitutivelyexpressing either one of these genes are not significantly protected(Jongedijk et al., 1995).

P. polymyxa is the plant growth-promoting rhizobacteria (PGPR)(Khan et al., 2008) that can produce antimicrobial metabolites tocontrol plant pathogens (Kajimura and Kaneda, 1996).Antimicrobial effects in P. polymyxa are dependent on productionof antibiotics and hydrolytic enzymes (Deng et al., 2011; Ito andKoyama, 1972; Raza et al., 2009; Beatty and Jensen, 2002). A newstrain A21, isolated from the snow covered high altitude area inTibet, China, was identified as P. polymyxa by physiological, bio-chemical characteristics and 16S rDNA gene analysis. It has a sig-nificant inhibitory effect on gray mold diseases caused by B.cinerea and a strong glucanase activity. In this study, we expressedthe endo-b-1,3-1,4-glucanase gene of P. polymyxa A21 in industrialstrain S. lydicus A01, and the results showed that the recombinantstrain AG01 had significantly enhanced the antifungal activity of S.lydicus. Our work demonstrates that it is a feasible way to develophigh-quality antifungal bioagents by transforming exogenousresistance genes into industrial strain.

The endo-b-1,3-1,4-glucanase of P. polymyxa had been investi-gated and shown special industrial applications in brewing indus-try and poultry husbandry (Buliga et al., 1986; Choct, 1997;Bamforth and Martin, 1983). Few researches about the antimicro-bial activity by endo-b-1,3-1,4-glucanase of P. polymyxa werereported. For example, Yao et al. (2004) reported that P. polymyxab-1,3-1,4-glucanase has inhibitory effect against Pyricularia oryzae.Wen et al. (2010) expressed the antimicrobial b-1,3-1,4-glucanaseof P. polymyxa in E. coli, and the recombinant b-1,3-1,4-glucanasesdisplayed significant inhibition on the mycelium growth of testedfungus, such as Colletotrichum musae and F. oxysporum Schl. f. sp.Cubense. Recombinant b-1,3-1,4-glucanase of Bacillus Mojavensis1A00437 had no activity on Magnaporthe oryzae, but could inhibitR. solani and R. solani kühn efficiently (Zuo et al., 2014). The differ-ent antagonist activity to pathogenic fungi may be due to thespecificity for enzyme constitution and substrate of

J. Li et al. / Biological Control 90 (2015) 141–147 147

b-1,3-1,4-glucanase of different strains. However, there are noreports on the heterologous expression of P. polymyxaendo-b-1,3-1,4-glucanase in industrial production strains as wellas its antagonist activity against B. cinerea. Thus, this study pro-vides a new application for endo-b-1,3-1,4-glucanase of P.polymyxa in the field of biological control.

5. Conclusions

To improve the antifungal activity of S. lydicus A01, theb-1,3-1,4-glucanase gene from P. polymyxa A21 isolated from thesnow covered high altitude area in Tibet, China, was cloned andexpress efficiently in S. lydicus A01. The engineered S. lydicusAG01 showed high glucanase activity and improved antagonisticeffect on B. cinerea compared with the wild-type strain A01, includ-ing enhanced inhibition to B. cinerea spore germination and myce-lial growth. The stronger antifungal activities of S. lydicus AG01likely resulted from the combination of natamycin production, glu-canase activity, and chitinase activity of the strain.

Acknowledgments

This work was supported by Science and Technology InnovationFund from Beijing Academy of Agriculture and Forestry Sciences(No. QNJJ201411), funding for training talents in Beijing City(2014000020060G180), Science and Technology Plan Project ofBeijing (No. D151100003915003).

Appendix A. Supplementary data

Supplementary data associated with this article can be found, inthe online version, at http://dx.doi.org/10.1016/j.biocontrol.2015.06.008.

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