Bacillus velezensis JK-XZ8 prevents and controls crown gall disease on Prunus subhirtella by colonizing and inducing resistance

Xitang Zhang·Weiliang Kong ·Xiaoqin Wu ·Jianren Ye

Abstract Crown gall of Prunus subhirtella Miq.,flowering cherry,is a soil-borne bacterial disease caused by Agrobacterium tumefaciens Smith &Townsend and has caused serious damage to the species in recent years in China.In this study,the colonization of sapling roots,the biocontrol efficacy,the expression of defense-related genes and the enzyme activity in roots of P.subhirtella inoculated with the Bacillus velezensis JK-XZ8 against A.tumefaciens were determined under greenhouse conditions.The results showed that the JK-XZ8 strain colonized in the rhizosphere and root surfaces of the saplings.Sole application of the strain increased activities of polyphenol oxidase (PPO) and phenylalanine ammonia lyase (PAL) without affecting superoxide dismutase (SOD).SOD and PPO activities increased in the prevention group,and the activity of PAL increased in both the prevention and cure groups.The expression of the defense-related genes PAL and PR-1 of salicylic acid pathway in roots increased in both the prevention and cure groups.Simultaneously on day 3,the expression of both COI and MYC2 genes of the jasmonic acid pathway significantly increased in the two groups.The incidence of crown gall in the prevention and the cure groups were 48.1% and 66.7%,respectively,significantly lower compared to the pathogens alone (87.5%).This study showed that the application of the JK-XZ8 strain reduced the incidence of crown gall in P.subhirtella saplings in the two groups,and the prevention group had better control efficacy.In addition,the JK-XZ8 strain protects against crown gall by developing induced systemic resistance and systemic acquired resistance in the roots of the cherry saplings.

Keywords Bacillus velezensis · Prunus subhirtella ·Crown gall · Plant-induced resistance · Control effect

Introduction

In the last decades,crown gall has become a major bacterial disease in tree nurseries and plantations,and an early comprehensive review about theAgrobacterium tumefacienshost range reported 643 susceptible hosts belonging to 93 families,including gymnosperms and dicotyledons (Zhu et al.2000;Tolba and Soliman 2013).According to reports in China,crown gall can occur in species such as flowering cherry,peach,apricot,plum,apple,pear,grape,eucalyptus,poplar and soapberries (Liang 2007;Wang 2007;Wang et al.2013;Yu 2016).As a popular garden species,Prunus subhirtellaMiq.,flowering cherry,is naturally distributed throughout China such as in Qingdao Zhongshan Park,Nanjing Jiming Temple,Beijing Yuyuantan Park,Shanghai Botanical Garden,Wuxi Shantou Temple,and Wuhan Donghu Cherry Garden (Wang 2010).However,crown gall has seriously affected its cultivation.According to a preliminary investigation,the disease occurs in provinces such as Shandong,Beijing and others (Zhan et al.2005).

Researchers have been studying different methods to control crown gall.Chemical controls have a limited effectiveness and failed to effectively control the disease (Sun et al.2009).Biological control has received widespread attention due to its environmentally friendly nature and non-polluting of the environment.Kerr (1980) reported that bacteriocin-producingAgrobacterium radiobacterK84 had a beneficial effect on the control of peach crown gall in 1974.Then,numerous biocontrol strains have been discovered,such asPseudomonas aureofaciensB-4117 andP.fluorescensCR330D,which control crown gall caused byA.tumefaciens(Khmel et al.1998).In addition,Rahnella aquatilisHX2 can inhibit grape crown gall caused byA.vitisK308 (Chen 2007),andRhizobium vitisVAR03-1 is a biological control agent that prevents and treats apple crown gall (Kawaguchi et al.2012).

Bacillusis the most widely used genus of biocontrol bacteria to prevent various types of plant diseases and harmful insects.There are a limited number of reports on the biological control of crown gall disease.Abdallah et al.(2015)evaluatedB.amyloliquefaciens32a using carrot discs to prevent crown gall by producing lipopeptide bacteriostatic substances.Similarly,Frikha-Gargouri et al.(2017) reported thatB.methylotrophicus39b reduced tomato crown gall by producing bacteriostatic surfactants.In addition to the production of bacteriostatic substances,the induction of plant resistance byBacillusis also an important mechanism of defense (Wang 2012).Plant-induced disease resistance is divided into two categories:systemic acquired resistance (SAR) and induced systemic resistance (ISR).SAR is induced by pathogenic microorganisms,physical factors and chemical factors,and ISR is induced by nonpathogenic microorganisms.In both cases,the resistance of the plant is related to differential gene expression,which is closely related to hormonal or enzyme activities (Conrath 2011).For example,Tan (2013) found that the application ofB.amyloliquefaciensT-5 increased the activity of polyphenol oxidase (PPO),phenylalanine ammonia lyase (PAL) and peroxidase (POD) in tomato seedlings infected withRalstonia solanacearum.Expression of the phytohormones salicylic acid,ethylene,and jasmonic acid was significantly higher in these seedlings.Beris et al.(2018) found that theB.amyloliquefaciensMBI600 strain induced tomato salicylic acid pathway resistance to tomato spotted wilt virus(TSWV) and potato virus Y.Li et al.(2015) showed thatB.amyloliquefaciensLJ02 increased the activities of SOD,POD,PPO and PAL in cucumber seedlings and stimulated the salicylic acid-mediated defense response.

B.velezensisis a species in the operational groupB.amyloliquefaciens(Fan et al.2017a;Wang et al.2008).AlthoughB.velezensisas a biocontrol bacterium has an effect on many diseases,its control of flowering cherry crown gall has not been reported.In preliminary research in our laboratory,theB.velezensisJK-XZ8 strain with an inhibitory effect onA.tumefaciensof cherry crown gall was screened from the rhizosphere of flowering cherry trees,and its controlling effect was proven by field experiments (Yan et al.2020).However,after the application ofB.velezensisJK-XZ8,the activities of defense-related enzymes (PAL,PPO,and SOD) and the expression of related genes of the salicylic acid and jasmine pathways (PAL,PR-1,COI1,MYC2) inP.subhirtellaroots are remain unknown.It is also beneficial to study the biocontrol mechanism of this strain.This study has three objectives:(1) to examine the colonization ofB.velezensisJK-XZ8 onP.subhirtellaroots;(2) to evaluate whether the JK-XZ8 strain induces systemic resistance againstA.tumefaciensPX-1,and to identify one or more signaling pathways involved in JK-XZ8-mediated induced systemic resistance by analyzing the expression patterns of the SA-responsivePR1andPALgenes and the JA-responsiveMYC2andCOI1genes inP.subhirtellatrees;and,(3) to determine the biocontrol effect of the JK-XZ8 strain.

Materials and methods

Plants,bacterial strains,and growth conditions

One-year-oldP.subhirtellasaplings were acquired from Qingdao,Shandong and planted in 24-cm diameter×18-cm high plastic pots.Natural soil was collected from Nandashan,Nanjing Forestry University.The plant growth-promoting rhizobacterium (PGPR) wasBacillus velezensisJK-XZ8,originally isolated from the rhizosphere ofP.subhirtellain Xuzhou City,Jiangsu Province.TheA.tumefaciensPX-1 pathogen was isolated from the rhizosphere of a diseased tree (Yan et al.2020).The above two strains were stored at? 80 °C.In the greenhouse biocontrol test,the strains were inoculated into a Luria–Bertani (LB) liquid medium and cultured in a shaker at 200 rpm for 24 h at 28 °C.The bacterial cells were then collected by centrifugation at 10,000 rpm for 10 min and re-suspended in sterilized distilled water and the cell concentration adjusted to 108cfu/mL (colony-forming units per mL).

Detection of the antibacterial activity of the biocontrol strain

The biocontrol strains JK-XZ8,JK-XZ8 Rif andA.tumefaciensPX-1 were cultured in 25 mL liquid shaker flasks for 24 h.Subsequently,100 uL ofA.tumefaciensPX-1 was evenly coated on an LB plate,three filter papers sheet with a diameter of 6 mm were placed on the plate,and 10 uL of candidate biocontrol strains were added to the filter papers.The plate was placed in an incubator at 28 °C for 24 h to observe any antagonistic effect.

Colonization of JK-XZ8 in the rhizosphere and root surfaces of saplings

The JK-XZ8 strain was inoculated into a liquid medium at 28 °C containing rifampicin,an antibiotic used to treat several types of bacterial infections,and shaken at 200 rpm in an incubator.A sample of this culture was spread onto a LB solid medium containing rifampicin.The growing bacteria were transferred onto a LB liquid medium containing higher concentrations of rifampicin,the concentrations increased incrementally to 200 μg/mL for three generations,and then the bacteria were again transferred onto LB medium containing 200 μg/mL rifampicin.Therefore,the JK-XZ8 strain had a stable inheritance of rifampicin resistance.The strains with resistance inheritance were cultured in LB solid medium ten times,and strains with similar growth rates were selected.As a result,the marker strain JK-XZ8 Rif with rifampicin resistant (200 μg/mL) was obtained.In the colonization test,the number of bacterial colonies ofB.velezensiswas measured on a plate with a concentration of 100 μg/mL rifampicin.The growth curves of the original JK-XZ8 strain and the labeled JK-XZ8 Rif strain were determined by a Bio Screener automatic microbial growth curve analyzer (FP-1100-C,Finland).

In the colonization test,the JK-XZ8 and JK-XZ8 Rif numbers were measured in the presence of 100 μg/mL rifampicin.Root systems were sampled five days after inoculation and then every 10 days for a total of five times;i.e.,the rhizosphere,root surface soil and root tissues were sampled 5,15,25,35 and 45 days after inoculation.When sampling,the saplings were removed carefully from the soil and sampling repeated for three saplings.Suspensions of the treated soil samples and root tissues were treated according to the dilution plate method,and then uniformly coated on an LB plate with 150 μg/mL rifampicin at 28 °C for 24 h,and the number of colonies counted.The formula for calculating colonization density was:colonization density log10(cfu/g)=log10 {(average number of colonies repeated three times in the same dilution gradient×dilution multiple)/sample weight} (Walker et al.2002).

Enzyme activity assay

Roots were collected on the 3rd and 5th days after treatments,and three samples were selected and stored at? 80 °C.The activities of SOD,PPO,and PAL were measured using an enzyme activity assay kit from Keming Biotechnology Co.,Ltd.,(Suzhou,China).

Analysis of gene expression by qPCR

RNA was extracted from root tissues using the polyphenolic polysaccharide plant total RNA extraction kit (Beijing Zhuangmeng International Biogene Technology Co.,Ltd.,Beijing).The extracted RNA was synthesized into cDNA for qPCR using a reverse transcription kit from Thermo Fisher Scientific (ZP411C-2,Beijing).All primers are shown in Table 1.The conditions for qPCR were:initial denaturation at 94 °C for 5 min,95 °C for 10 s,60 °C for 34 s (40 cycles),and dissociation at 95 °C for 15 s.Each sample contained 10 ng of cDNA and 0.2 mM of each primer in a final volume of 20 mL.Each target gene was calibrated against the 18S reference gene (Zhang 2010),and the quantification of transcripts was presented asin accordance with the CT method.All genes were analyzed in three technical and four biological replicates under a similar detection system.

Table 1 Primers used in realtime RT-qPCR analysis

Greenhouse experiments

This experiment was carried out in an open-air greenhouse at the College of Forestry,Nanjing Forestry University in April 2019.P.subhirtellasaplings were planted in pots,and one month after the saplings sprouted,five treatments were applied:(1) Controls were not inoculated withB.velezensisJK-XZ8 orA.tumefaciensPX-1;(2) The prevention group was first treated withB.velezensisJK-XZ8 andA.tumefaciensPX-1 was introduced on the sixth day;(3) The cure group was first inoculated withA.tumefaciensPX-1 and on the sixth day,the root systems were treated withB.velezensisJKXZ8.The controls,prevention and cure groups consisted of 24 saplings each;(4)B.velezensisJK-XZ8 was inoculated;and,(5)A.tumefaciensPX-1 was inoculated alone.Ten saplings were inoculated individually for these treatments.Cultivation and management conditions were the same for all saplings.All the strains tested were centrifuged and re-suspended in sterile water and cell concentration adjusted to 108cfu/mL and applied to the roots by irrigation.A 15 mL mixture of biocontrol bacteria and pathogenic bacteria was applied in the prevention group and cure group,and the same amount of distilled water was applied in the controls.

Growth assay

Height and diameter at the base of the saplings were measured with a tape measure and a vernier caliper.Net height growth rate (%)=[(height after defoliation?height before treatment)/height before treatment]×100,and net growth of ground diameter (%)=[(ground diameter after defoliation?ground diameter before bacteria application)/ground diameter before bacteria application]×100.

Evaluation of the biocontrol efficacy of JK-XZ8 on crown gall

After complete defoliation of the saplings,the biocontrol effect was evaluated.The number and size of tumors and the incidence of crown gall were counted.The incidence rate was calculated according to the grading status (Table 2).

Table 2 Classification criteria for crown gall of flowering cherry saplings

Statistical analysis

Experiments were carried out using a completely randomized design.All statistical analyses were performed with SPSS 16.0 (SPSS Inc.,Chicago,IL,USA).The data were analyzed by one-way ANOVA and mean separations were performed using Duncan′s multiple range tests.The significance level was defined asP<0.05.

Results

Detection of the rifampicin resistance marker and the stability of B.velezensis JZ-XZ8

The wild-type biocontrol strain JK-XZ8 was cultured under different concentrations of rifampicin (Rif),and the labeled strain JK-XZ8Rif was obtained at a concentration of 200 μg/mL (Fig.1 a).According to the measurements collected by the growth curve instrument,there was no difference in growth between the biocontrol strain JK-XZ8 and the labeled strain JK-XZ8Rif,indicating that rifampicin labeling did not affect the normal growth of the strain (Fig.1 b).The filter disk antagonistic experiment showed that the labeled strain JK-XZ8Rif still had an antagonistic effect onA.tumefaciensPX-1,the average diameter of the inhibition zone was 13.3 mm (Fig.1 c).

Fig.1 Detection of the rifampicin resistance marker and the stability of B.velezensis JK-XZ8.a Growth of labeled strains with different concentrations of rifampicin.The numbers represent the concentration of rifampicin; b Growth curves of the wild-type strain JK-XZ8 and marker strain JK-XZ8Rif; c Labeled strain JK-XZ8Rif antagonizing A.tumefaciens PX-1

Dynamics of JK-XZ8Rif in the rhizosphere

After the saplings were growing for one month,each was inoculated with the marker JK-XZ8Rif strain,and the root soil was collected in 5,15,25,35 and 45 days after inoculation.The marker strain was detected in the rhizosphere and on the root surfaces continuously.The results showed that the colonization density of the labeled strain in the rhizosphere decreased from 4.4 lg cfu/g on the 5th day to 3.8 lg cfu/g on the 45th day.The colonization density on the root surfaces tended to stabilize from 4.2 lg cfu/g on the 5th day to 3.5 lg cfu/g after the 25th day.No labeled JK-XZ8Rif strain was detected in the roots (Fig.2).

Fig.2 Number of colonization of the marker strain JK-XZ8Rif in the roots of flowering cherry saplings.Bars are standard errors of triplicate experiments.Different letters indicate statistically significant differences (P <0.05) among treatments according to the least significant difference test

Effects of different treatments on the activities of defense-related enzymes

The activities of defense-related enzymes in the roots were different in different treatments.The activities of PAL and PPO in saplings treated with the biocontrol bacteriumB.velezensisJK-XZ8 were not different from the controls on the third day but increased on the fifth day (Fig.3 a,b).SOD activity in roots treated with JK-XZ8 alone was not different from the controls on the third or fifth day(Fig.3 c),indicating that the JK-XZ8 strain induced enzymatic activities of PAL and PPO in the saplings.PAL activity was inhibited on the third and fifth days of treatment of the roots withA.tumefaciensPX-1 (Fig.3 a),and SOD activity was always higher in the group treated withA.tumefaciensPX-1 than in the controls (Fig.3 c).By day 3,SOD activity was 3 times higher in the saplings treated withA.tumefaciensPX-1 than in the controls.On day 5,SOD activity was 1.1 times higher than that of the controls(Fig.3 c).PPO activity in roots treated withA.tumefaciensPX-1 increased on the third day but was no different from the controls on day 5 (Fig.3 b).The results show that inoculation with the pathogenic bacterium inhibited the activity of defense enzyme PAL in the root and it was speculated that the free radical-scavenging activity of the SOD enzyme increased rapidly because of the large number of free radicals after infection with the bacterium PX-1.

Fig.3 Expression of different defense-related enzymes in sapling roots under different treatments.a–c PAL,PPO and SOD enzyme activities in single bacterial application; d–f PAL,PPO and SOD enzyme activities in different bacterial applications.Bars are standard errors of triplicate experiments.Lowercase letters and uppercase letters indicate statistically significant differences (P <0.05) among treatments in 3 days or 5 days according to the least significant difference test

By day 6,saplings treated with the bacterium JK-XZ8 were inoculated withA.tumefaciensPX-1 as the prevention group.In contrast,saplings inoculated in advance with the pathogen PX-1 were treated with the biocontrol JK-XZ8 on the sixth day as the cure group.The results by day 3 showed that PPO activities in the roots in the prevention and cure groups did not differ from those in the controls.By day 5,PPO activity in the cure saplings was lower than in the prevention and control groups,indicating that an early application of JK-XZ8 could help normalize the activity of PPO (Fig.3 e).PAL activities in the prevention and the cure groups were significantly lower than those in the controls by day 3.However,by day 5,PAL activities in the prevention and cure groups were not different from the controls(Fig.3 d),indicating that the bacterium JK-XZ8 applied in both treatments could induce PAL activity in cherry saplings roots.SOD activity in the prevention group was lower than the controls by the third day but increased to a normal level by day 5.In the cure group,SOD activity increased to normal levels after the third day but it was lower than the controls after the fifth day,indicating that early application of biocontrol bacteria can help stabilize root SOD activities at normal levels (Fig.3 f).

Expression of defense-related genes under different treatments

The expression of genes related to the salicylic acid pathway (PAL,PR-1) and jasmonic acid pathway (COI1andMYC2) was determined on day 3 and 5 in different treatments.PALgene expression increased by day 3 in roots treated withB.velezensisJK-XZ8 alone,and the expression did not differ from that in the controls on the 5th day(Fig.4 a).PALgene expression in cherry roots inoculated withA.tumefaciensPX-1 was inhibited by day 3.On day 5,the flowering cherry defense system was initiated andPALgene expression increased (Fig.4 a).ThePR-1gene in the saplings decreased on day 3 regardless of inoculation with the biocontrol bacterium or pathogen.By day 5,the biocontrol bacterium had induced the upregulation ofPR-1expression.PR-1gene expression was inhibited in saplings treated with the pathogen (Fig.4 b).In saplings treated with JK-XZ8 alone,the expression of theCOI1gene,which is related to the jasmonic acid pathway in the roots,was lower than in the controls by the third or fifth day.The COI1gene was lower in saplings inoculated with PX-1 alone than in the controls by day 3 but increased to normal by day 5 (Fig.4 c).TheMYC2gene was highly expressed on the third and fifth days in saplings treated with JK-XZ8.In saplings treated with the pathogenic bacterium,the expression of theMYC2gene increased by day 3 and returned to normal by day 5 (Fig.4 d).These results indicate that the biocontrol bacterium JK-XZ8 can activate the salicylic acid and jasmonic acid pathways of flowering cherry saplings by inducing the expression of thePALandMYC2genes.

ThePALgene was significantly inhibited on day 3 in the prevention group but increased to normal on the fifth day(Fig.4 e).At the same time,thePR-1gene of the prevention group was significantly improved by the 3rd and 5th day(Fig.4 f).These results indicate that when the roots were infected with the pathogenic bacterium,early application of the biocontrol bacterium helped the plants to restore normal defense expression.Expression of theCOI1andMYC2genes was upregulated on the third day in the roots in the prevention group.By day 5,theCOI1gene expression was lower in the prevention group than that in the controls,and theMYC2gene was normally expressed (Fig.4 g,h).In the cure group,in which the pathogen PX-1 was first inoculated and then the biocontrol bacterium JK-XZ8 was administered,the expression of theCOI1gene was upregulated and the expression of theMYC2gene was the same as that of CK on the third day.By day 5,both theCOI1andMYC2genes were upregulated (Fig.4g,h).These results indicate that the biocontrol bacterium can induce the expression of the salicylic acid pathway-related genesPALandPR-1and the jasmonic acid pathway-related genesCOI1andMYC2in cherry saplings regardless of whether the biocontrol bacterium was applied before or after pathogen inoculation.

Fig.4 Defense-related gene expression under different treatments.a–d PAL,PR-1,COI1 and MYC2 genes in single bacterial application;e– h PAL,PR-1,COI1 and MYC2 genes under different application strain sequences.Bars are standard errors of triplicate experiments.Lowercase letters and uppercase letters indicate statistically significant differences (P <0.05) among treatments in 3 days or 5 days according to the least significant difference test

Growth profile of P.subhirtella saplings in different treatments

Height and ground diameter of saplings were improved in different treatments.There was no significant difference in height growth in the group treated with biocontrol bacteriumB.velezensisJK-XZ8 alone,the saplings treated with pathogen PX-1 alone and the controls.The growth rate in the prevention and cure groups was significantly higher than in the other three treatments,with an increase of 20–21 cm.For ground diameters,the controls were significantly lower than other treatments,and growth in the prevention and the cure saplings was similar at approximately 3 mm.The net growth of saplings treated with the biocontrol bacterium JK-XZ8 alone and the pathogen PX-1 alone was higher than that of the controls,but also significantly lower than that of the cure and prevention groups (Fig.5).This indicates that the biocontrol bacterium was beneficial to growth and development.PX-1 also affected height and ground diameter growth,which is related to its specific pathogenesis.

Fig.5 Net growth of plant height (black) and basal diameter (gray) of different treatments.Bars are standard errors of triplicate experiments.Lowercase letters and uppercase letters indicate statistically significant differences (P < 0.05)among treatments according to the least significant difference test

Biocontrol effects of the different treatments

The occurrence of crown gall in cherry saplings under different treatments is shown in Fig.6.The tumors on saplings treated with the pathogen alone were the largest and located in the main and lateral roots.Roots of saplings treated with the biocontrol bacterium JK-XZ8 alone were the most prolific.The size of the tumors in the prevention group was smaller than in the cure group (Fig.6 a,c).The index of the disease incidence of different treatments is shown in Fig.6 b.The disease incidence of the prevention group was 48.1%,significantly lower than the cure group (66.7%) and the group treated with the pathogen alone (87.5%).Therefore,these findings indicate that the application of the biocontrol bacterium prior to pathogen infection is more conducive to the control of cherry crown gall (Fig.6 d),although the cure saplings also exhibited a reduction in the incidence of cherry crown gall to some extent.

Fig.6 Effect of different treatments on crown gall of flowering cherry saplings.a tumors of the flowering cherry saplings,the red circle indicates a tumor; b disease incidence (black) and disease index (gray); c mean tumors size and d control efficiency

Discussion

The colonization of rhizosphere microorganisms in roots is not only an important parameter to evaluate diverse bacteriaplant systems but also an indispensable research focus to reveal the ecological characteristics and functions of bacteria in the rhizosphere (Tan et al.2013).In particular,for biocontrol bacteria,their ability to survive,reproduce and colonize the plant rhizosphere is the basis and key to controlling soil borne plant diseases (Li et al.2006;Ramos et al.2011).Several researchers have noted that the colonization of antagonistic strains in the host rhizosphere is closely related to their biocontrol efficiency.By labelingR.vitisVAR03-1 andA.radiobacterK84 with rifampicin antibiotics,Kawaguchi et al.(2012) found that these biocontrol bacteria persisted in the rhizosphere of apple within one year,and although the number of bacteria decreased,it was still effective in controlling crown gall.Hu et al.(2012) identified rifampicin resistance in theB.amyloliquefaciensAF11 strain,inhibiting the banana wilt pathogenFusarium oxysporumf.sp.cubenseRace 4 (FOC Race 4).The AF11Rif strain colonized banana roots and stems with 118 cfu mg?1and 32 cfu mg?1,respectively,and the wilt was reduced by 71.5% in 50 days after inoculation.In this study,B.velezensisJK-XZ8Rif survived in the rhizosphere and on the root surfaces of cherry saplings,and the bacterial concentration was 3.81 lg cfu/g and 3.51 lg cfu/g by the 45th day.Colonization withB.velezensisJK-XZ8Rif might be beneficial for controlling crown gall.

B.velezensishas a highly efficient and extensive biocontrol effect and is harmless to the environment;thus,it has been widely studied and applied (Fan et al.2017b,2018;Huang et al.2017).Meng et al.(2016) demonstrated thatB.velezensispromotes the growth of sugar beets,carrots,cucumbers,peppers,potatoes,pumpkin,tomatoes,corn and radish (Liu et al.2010).Zhu (2014) appliedB.amyloliquefaciensBEB33 to banana pot experiments and reported that it significantly promoted plant growth and that the effect of the strain on banana wilt (F.oxysporum) was 73.5%.Tan (2013)also found through a pot experiment thatB.amyloliquefaciensT-5 and CM-2 significantly promoted tomato growth and effectively reduced the tomato bacterial wilt disease,and the biocontrol effect was 79.4% and 70.1%,respectively.However,there are few studies on the control of crown gall disease usingB.velezensis.In this study,the greenhouse experiment showed thatB.velezensisJK-XZ8 effectively prevented crown gall disease in flowering cherry.The biocontrol efficiency in the prevention group was 51.8%,significantly higher than that in the cure group (33.3%),indicating that early application can reduce the incidence of crown gall.

In this study,the growth in the prevention and cure saplings were significantly higher than that treated with the biocontrol bacterium alone.The growth and development of cherry saplings infected with the pathogen PX-1 alone was also superior to that of the control,which proved thatA.tumefacienscould,to some extent,promote growth and development.This may be due to its unique pathogenesis.A.tumefaciensintegrates T-DNA by transferring extrachromosomal genetic material,(a fragment of a plasmid),of the bacterium into the plant genome,including two growth agents(auxin and cytokinin) and a group of amino acid derivatives.Gene expression of phytohormone synthesis leads to plant tumorigenesis,the formation of tumors (Tzf ira and Citovsky 2008),so plant hormones produced by pathogens also promote growth and development to some extent.

When plants are infected with pathogenic bacteria,changes in enzyme activities related to resisting the infection are used as an important index to measure the defense response (Yu and Hao 2014).Liang et al.(2018) sprayedB.amyloliquefaciensTWC2 on sugarcane leaves and the results showed that CAT,POD,SOD,PAL and PPO activities were significantly higher than in untreated leaves.Qu(2016) appliedB.amyloliquefaciensBP30 to pear leaves,showing that it induced PPO,POD and PAL activities.PAL,an enzyme found in various plants and a small number of microorganisms,links primary and phenylpropanoid metabolism and catalyzes the first step of phenylpropanoid metabolism,generating phenylpropane.It is a key rate-limiting metabolic enzyme and also one of the most studied in the phenylpropanoid metabolic pathway.PAL is involved in the synthesis of substances such as salicylic acid and also in the synthesis of lignin phytoalexin;thus,it is an essential enzyme in the resistance enzyme system.PPO is also an important enzyme that creates resistance to plants.It catalyzes the formation of lignin and other phenolic oxidation products,forming a protective barrier against invasive pathogens.Catalyzed α-quinone is toxic and acts as a disease inhibiting bacterial growth (Wang et al.2005).In this study,the application ofB.velezensisJK-XZ8 alone increased PPO and PAL activities.WhenA.tumefaciensPX-1 was applied to the saplings,the expression of PAL was significantly inhibited but not PPO.These results show that the pathogen PX-1 can promote self-infection by inhibiting PAL enzyme activity.In the prevention group,although the activity of PAL was suppressed by the third day,it increased to a normal level by the fifth day.PPO activity was induced for the longest period of time in the prevention group,demonstrating that early application of the biocontrol bacterium is more beneficial for PPO activities.SOD is an important enzyme in the antioxidant defense system which functions in the catalysis of the disproportionation or dismutation of superoxide and the reduction in the toxicity of reactive oxygen species (ROS).In this study,SOD activity was not affected in the saplings treated with JK-XZ8 alone.However,JK-XZ8 helped plants increase SOD activity when saplings were infected with the pathogenic bacterium.This result is consistent with the findings of Elanchezhiyan et al.(2018)that PAL,PPO,and SOD activities in tomato treated withB.amyloliquefaciensFBZ24 increased.In the present study,when saplings were infected with the pathogenic bacterium,SOD and PPO activities increased to resist pathogens,but PAL activity was inhibited.This result indicated that the infection of flowering cherry saplings withA.tumefaciensaffected the salicylic acid pathway and antioxidant system of plants,which is consistent with the results of Zhao and Wen (2012).They reported that whenP.subhirtellaseedlings were infected with root crown gall,POD and SOD activities were enhanced,while CAT activity was reduced.

In plants,the disease resistance signal transduction pathways,including the SA pathway,the JA pathway and the ethylene (ET) pathway,are complex and major signal transduction pathways.SA is an endogenous phenolic substance with low levels in plants and a key signaling molecule for SAR.SAR is characterized by systemicity,persistence,a broad spectrum and security.Two markers that significantly distinguish SAR from other disease-resistance responses are broad-spectrum resistance to pathogens and the expression of pathogenesis-related (PR) proteins (Qu 2016).In this experiment,the application of the biocontrol bacterium JK-XZ8 alone significantly increased the expression ofPALandPR-1genes in cherry saplings,althoughPALincreased by the third day andPR-1increased by the fifth day.A.tumefacienscan significantly inhibit the expression of thePALandPR-1genes in cherry saplings.The results of the prevention group and the cure group showed that the expression of thePALandPR-1genes were induced inP. subhirtellaregardless of inoculation before or after pathogen exposure,and the induction of thePALgene was later than the induction of thePR-1gene.The results of this study indicate that the application ofB.velezensisJK-XZ8 can induce the salicylic acid pathway inP.subhirtellato enhance resistance toA.tumefaciensinfection.The jasmonic acid pathway is an important defense pathway and plays a vital role in abiotic/biological stress responses and in plant growth.COI1is a key receptor in the pathway,which was originally isolated fromArabidopsis thaliana,and regulates growth,hardiness,disease resistance and insect resistance (Xu and Yao 2009).Therefore,the early response ofCOI1after abiotic or biotic stress is important for the activation of the jasmonic acid pathway.MYC2is also a core regulator of a large number of jasmonic acid signaling pathway branches (Liu et al.2018).If the pathway is blocked,endogenous jasmonates that regulate development will not accumulate.Resistance genes fail to activate the pathway which hinders plant responses to pathogens and ultimately leads to susceptibility (Lu 2015).In this study,the application of the biocontrol bacterium JK-XZ8 and the pathogen PX-1 alone significantly inhibited the expression of theCOI1gene.AlthoughCOI1was inhibited,theMYC2gene significantly increased,indicating that the saplings induced the gene to initiate the jasmonic acid pathway.From the results of the prevention and the cure groups,the jasmonic acid signaling pathway was induced by applying JK-XZ8 (Dombrecht et al.2007;Li et al.2019).Therefore,the antagonistic bacteriumB.velezensisJK-XZ8 induces systemic resistance (ISR) inP.subhirtellaby simultaneously activating salicylate-and jasmonate-dependent signaling pathways.

ISR is an adaptive strategy that improves the defensive capacity of plants,and is marked by an enhanced activation of induced defense mechanisms.Plant growth-promoting rhizobacteria and fungi,both of which induce systemic resistance,can also trigger defense priming (Zamioudis et al.2013).This induction of resistance to plant is known as ISR.B.velezensisJK-XZ8 acts as a beneficial rhizosphere bacterium that has the ability to induce plant resistance.In addition,studies have shown that ISR does not involve the accumulation of proteins or salicylic acid associated with the disease process but rather the pathway regulated by jasmonate and ethylene.In this study,JK-XZ8 increased the expression of the jasmonic acid pathway-related genesCOI1andMYC2,indicating that this biocontrol bacterium may induce systemic resistance inP.subhirtella.At the same time,Wu et al.(2018) showed that starch-free SQR9 can induce plant resistance to pathogens but that bacterial antibiotics are essential for induction.Therefore,it is suspected that JK-XZ8 may also produce some substances to help induce systemic resistance inP.subhirtella.Systemic acquired resistance can be triggered by exposing plants to toxic,nontoxic and pathogenic microorganisms related to ISR.Depending on the plant and the cause,it takes time to establish systemic acquired resistance,in which the accumulation of salicylic acid associated with the disease occurs(Mandal and Ray 2011;María et al.2017).In this study,JK-XZ8 induced an increase in the activities of defenserelated enzymes PPO,PAL,and SOD as well as increased the expression of the key genesPALandPR-1of the salicylic acid pathway.The antibacterial strain JK-XZ8 can also induce systemic acquired resistance inP.subhirtella.According to Beneduzi et al.(2012),the protective effect of the induction of resistance is significantly less than that obtained by systemic acquired resistance,and the dependence on plant genotype is also observed during the production of ISR.Moreover,ISR and SAR provide better protection when combined,than use alone,indicating that they play a role in inducing resistance to pathogens.Based on the results of this study,the biocontrol bacteriumB.velezensisJK-XZ8 can create ISR and SAR inP.subhirtellasaplings.

Conclusion

Biological control is safe,effective and environmentally friendly,andB.velezensisJK-XZ8 is a biocontrol strain that effectively controls crown gall onP.subhirtella.The mechanism of prevention by the strain was studied through antibiotic labeling,greenhouse biocontrol detection,and the detection of defense-related enzymes and genes.The following observations can be made:

(1)B.velezensisJK-XZ8 survives in the rhizosphere and on root surfaces ofP.subhirtellasaplings.Under greenhouse conditions,the bacteria number on root surfaces was stable 35 days after application.

(2) When the saplings were threatened by pathogenic bacteria,the biocontrol bacteria JK-XZ8 increased the phenylalanine and polyphenol oxidase pathways and superoxide dismutase enzymes.B.velezensisJK-XZ8 increased the expression ofPALandPR-1genes in the salicylic acid pathway andCOI1andMYC2genes in the jasmonic acid pathway.It was shown thatB.velezensisJK-XZ8 could stimulate both induced and systemic acquired resistance to crown gall.

(3) The biocontrol bacteria JK-XZ8 promoted the growth ofP.subhirtellasaplings and effectively reduced the incidence of crown gall.The prevention group had better control efficacy than the cure group.