Colonization of Bacillus cereus NJSZ-13, a species with nematicidal activity in Masson pine (Pinus massoniana Lamb.)

Liangliang Li · Jiajin Tan · Fengmao Chen · Dejun Hao

Abstract Bacillus cereus NJSZ-13, an endophytic bacterium with nematicidal activity, was isolated from stems of healthy Pinus elliottii Engelm. Colonization of P. massoniana Lamb. by endophytic B. cereus was studied using scanning electron microscopy and confocal laser scanning microscopy. After the plasmid pGFP78 containing the green fluorescent protein(GFP)gene was transformed into the NJSZ-13 strain, the NJSZ-13:gfp showed the same nematicidal activity and growth curve as the wild-type strain, and the plasmid pGFP78 was stably maintained in strain NJSZ-13 for at least 96 h of bacterial cultivation on medium without antibiotics.After inoculation into Masson pine roots, colonization of the NJSZ-13:gfp strain in plant roots and stems was visualized using confocal laser scanning and the strain was enumerated in inoculated roots and stems. These results suggest that NJSZ-13:gfp is an efficient colonizer of Masson pine and can transfer vertically from roots to stems.

Keywords Bacillus cereus · Endophytes · Green fluorescent protein · Plasmid stability · Colonization

Introduction

Pine wilt disease (PWD), caused by infection with the pinewood nematode Bursaphelenchus xylophilus (Steiner& Buhrer) Nickle, is perhaps one of the most serious threats to pine forests worldwide. The prevalence of PWD is driven by interactions among factors of the pine, the pinewood nematode, a long-horned beetle, fungi, bacteria,and the environment (Dwinell 1997). Over the past few years,mass application of chemical pesticides has not only polluted the environment and upset ecological equilibria,but has also caused pesticide resistance in the nematode,leading to diminished control efficiency(Ryss et al.2011).Therefore, many researchers have turned to biological controls as alternatives to pesticides.Increasing numbers of bacteria with nematicidal activities have been isolated,including Bacillus thuringiensis (Wei et al. 2003), Pseudomonas spp. (Siddiqui et al. 2005), Pasteuria penetrans(Davies and Opperman 2006), and B. cereus (Gao et al.2016). These strains have shown great potential in the biological control of nematodes.

Often microorganisms selected for biocontrol have favorable effects on disease control in vitro, but their practical application in the wild is not ideal. One of the most important reasons for this difference is that the bacteria cannot colonize permanently due to complicated field conditions, which affects their functioning. This is also a primary reason for the large fluctuation and poor repeatability of some microbial biocontrol agents released in forests (Hallmann et al. 2011). However, endophytes can overcome this constraint as they colonize within the tissues of healthy or symptomless hosts without causing apparent harm (Kloepper et al. 1992; Mcinroy and Kloepper 1995).Endophytes are ubiquitous in various vegetative parts of plants, including roots, stems, leaves and tubers, and have been isolated from numerous species (Andrews 1992;Lodewyck et al. 2002). Endophytic bacteria fall into a variety of bacterial phyla and certain bacteria play important roles in increasing plant tolerance, promoting plant growth, and controlling plant diseases (Sturz et al. 2000;Zehnder et al. 2001; Newman and Reynolds 2005; Ryan et al. 2008; Zheng et al. 2008; Wu et al. 2014). Unfortunately, only a very small number of endophytic bacteria with nematicidal activity have been isolated to date, and there is scarce literature on the colonization and distribution of nematicidal endophytes within plant tissues.

For nearly a decade, green fluorescent protein (GFP)isolated from a jellyfish (Aequorea victoria Murbach &Shearer) has been considered a credible gene marker and has been widely employed in studies of the interactions between microorganisms and their hosts (Errampalli et al.1999; Compant et al. 2005). As early as 1997, plasmids have been constructed with the gfp gene and successfully expressed in Pseudomonas spp. (Bloemberg et al. 1997).Von der Weid et al. (2005) evaluated the potential of the Paenibacillus brasilensis strain PB177,which is capable of inhibiting phytopathogenic fungi, to colonize maize plants by labeling it with the gfp gene. Ren et al. (2015) studied the colonization and enumeration of Sinorhizobium meliloti CHW10B,which has the capacity to solubilize phosphorus,in Taxus chinensis var.mairei Lemee&Lev.(Cheng&Fu)using a GFP technique. However, to date few studies are available about the utility of the gfp gene marker in tracking the colonization and distribution of endophytes with nematicidal activity in pines.

In a previous study, we isolated the endophytic bacterium NJSZ-13 from the inner tissues of a surface-sterilized stem of healthy Pinus elliottii in Nanjing Zhongshan Botanical Garden and identified it as B.cereus.The results of a nematicidal bioassay showed that strain NJSZ-13 had significant nematicidal activity against the pinewood nematode, and 81.5% nematode mortality was achieved after 48-h treatment with ～105colony forming units(cfu)/mL strain NJSZ-13 cells in a buffer suspension (Li et al. 2017). The growth-promotion and disease-resistance roles of biocontrol bacteria depend heavily on the ability to stably colonize plants (Lugtenberg et al. 2001). Therefore,in this paper, we investigate the colonization patterns of one gfp-tagged strain NJSZ-13 in Masson pine (P. massoniana), a susceptible species found in China (Yang and Wang 1989). After gfp tagging, colonization in Masson pine was visualized, and NJSZ-13:gfp was enumerated in root and stem tissues.

Materials and methods

Bacterial strains, plasmid, nematode, and culture conditions

The endophytic strain B. cereus NJSZ-13, which is antagonistic to the pinewood nematode, was deposited as No. M2016660 in the China Center for Type Culture Collection (Wuhan, China). Strain NJSZ-13 was grown in Luria broth (LB) medium composed of 1% tryptone, 0.5%yeast extract, 1% sodium chloride, and adjusted to pH 7.2-7.4 followed by sterilization via autoclaving at 121 °C for 20 min. Escherichia coli strain DH5α carrying the pGFP78 plasmid was grown in LB medium supplemented with tetracycline (10 μg/mL) and ampicillin (100 μg/mL)at 37 °C. The pGFP78 plasmid was constructed by Qi(2006) from the China Agricultural University, and carries the constitutively expressed gfpmut3a gene and co-resistance to ampicillin and tetracycline.

The nematode B. xylophilus isolated from dead Pinus thunbergii Parl. in Lianyungang, Jiangsu Province, was cultured in Botrytis cinerea at 25 °C for 7 days, collected via the Baermann funnel technique (Gray 1984), and then adjusted to an appropriate suspension with sterile phosphate-buffered saline(PBS)containing 1.42 g/L Na2HPO4,0.27 g/L KH2PO4, 0.2 g/L KCl, 8 g/L NaCl, at pH 7.4.

Preparation of the aseptic seedlings of Masson pine

Seeds of Masson pine were immersed in 70% ethanol for 30 s and 30% H2O2for 30 min, and then rinsed six times with sterile water.After surface sterilization,aliquots of the sterile water used in the final rinse were plated on LB medium at 28 °C overnight to check for contamination.The seeds were then placed in half-strength Murashige-Skoog (MS) culture medium (HOPE Bio-Technology Co.,Ltd.,Qingdao,China)containing 1%agar and cultivated in a tissue culture room (25 °C, 16-h light period, 8-h dark period). After 3 months of cultivation, the seedlings were used for colonization with the endophytic strain.

SEM examination of endophytic strain NJSZ-13 in plant tissues

The colonization of endophytic strain NJSZ-13 in Masson pine was observed in a gnotobiotic system through SEM.Strain NJSZ-13 was grown in LB medium at 28 °C and 200 r/min for 24 h. The bacterial cells were collected through centrifugation at 4 °C and 10,000 r/min for 20 m,and then diluted to ～105cfu/mL using sterile PBS for inoculation.Roots of the aseptic seedlings described above were inoculated by dipping them into bacterial suspension of strain NJSZ-13 for 2 h. The seedlings were then rinsed with sterile water, replanted in half-strength MS medium,and incubated in a tissue culture room. Seven days after inoculation, the seedlings were treated with 70% ethanol for 3 min and re-rinsed with sterile water six times. The roots and stems of the seedlings were cut into 0.5-1.5 cm pieces using a sterilized razor blade. These sections were first pre-fixed in glutaraldehyde (2%) plus formaldehyde(2%)in sterile PBS at 4 °C for 12 h,and then post-fixed in osmium tetroxide (2%) for 30 min. The samples were subsequently dehydrated with serial concentrations of ethanol at room temperature. Specifically, the samples were dehydrated for 15 min in 30% ethanol once, 15 min in 50%ethanol once,15 min in 70%ethanol once,15 min in 90% ethanol twice, and 15 min in 100% ethanol three times. The dehydrated samples were dried using a critical point drying instrument(K850;Emitech,East Sussex,UK).The dried samples were dissected using a sterilized blade,sputter-coated with Au-Pd using ion-sputtering equipment(E-1010;Hitachi,Tokyo,Japan),and observed by scanning electron microscopy(SEM) (NeoScope JCM-5000;Nikon,Tokyo, Japan). Aseptic seedlings were inoculated with sterile PBS as controls.

Construction of gfp-tagged B. cereus NJSZ-13(strain NJSZ-13:gfp)

Plasmid pGFP78 was transformed into B. cereus NJSZ-13 through the modified hyperosmotic approach (Xue et al.1999). E. coli DH5α containing plasmid pGFP78 were grown in LB medium supplemented with tetracycline(10 μg/mL) and ampicillin (100 μg/mL) at 37 °C and 200 r/min for 24 h in a rotary shaker. The cells were harvested through centrifugation at 4 °C and 10,000 r/min for 20 m, and the plasmid was extracted using the Plasmid Midi kit (TIANGEN Biotech Co., Ltd., Beijing, China).

NJSZ-13 was transferred into 5 mL LB medium and cultured overnight. A 2.5 mL sample of the culture was then inoculated into 40 mL LB medium containing 0.5 M sorbitol, and cultured at 28 °C and 200 r/min until its OD600was ～0.9. The bacterial cells were harvested through centrifugation at 4 °C and 10,000 r/min for 5 min and re-suspended three times in 50 mL pre-chilled sterile water containing 0.5 M sorbitol,0.5 M mannitol,and 10%glucose. Finally, the cell suspension was concentrated to 1 mL and pipetted into Eppendorf tubes (60 μL per tube)for electroporation. A concentration of 50 ng plasmid(1-8 μL) was added to 60 μL competent cells. After incubation on ice for 2 min, the mixture was transferred into a pre-cooled electroporation cuvette (Bio-Rad, Hercules, CA, USA), and transformation was conducted with an electroporator(ECM 630;BTX, San Diego, CA,USA).The parameters for electroporation were 2.0 kV, 25 μf,200 Ω,and 5.0 ms.One mL LB medium containing 0.5 M sorbitol and 0.38 M mannitol was added to the electroporation cuvette and cultured at 28 °C and 200 r/min for 3 h.The resulting bacterial cells were grown on LB plates containing 10 μg/mL tetracycline and 100 μg/mL ampicillin at 28 °C overnight to screen for positive transconjugants. These were fixed on glass slides, and their expression levels of gfp were examined under a fluorescence microscope (Axio Imager M2; Zeiss, Oberkochen,Germany). The transconjugant with the highest brightness was identified, and the presence of the gfp gene was confirmed through PCR using gene-specific primers. The sequences of the primers were as follows:

The primers were synthesized by GenScript Co., Ltd.(Nanjing, China).

The PCR reaction was performed with 5 min pre-denaturation at 95 °C,followed by 33 cycles of 30 s at 94 °C,30 s at 50 °C,50 s at 72 °C and a final extension of 3 m at 72 °C, and then cooled to 4 °C. The resultant PCR products were detected using 2% agarose gel electrophoresis.

Detection of in vitro plasmid stability of NJSZ-13:gfp

The stability of plasmid pGFP78 was determined throughout the 96 h cultivation in LB medium in the absence of antibiotic at 28 °C and 200 r/min. Every 12 h,50 μL bacterial culture was inoculated into 50 mL fresh medium and cultured under the conditions described above.The same inoculation process was successively performed eight times. For each inoculation, aliquots were plated on LB medium and over 200 single colonies were transferred using a toothpick to LB agar medium supplemented with the appropriate antibiotics. Colonies were counted and the percentage of clones carrying the plasmid was estimated.All experiments were repeated three times.

Growth kinetics and nematicidal activity of NJSZ-13:gfp

The gfp-labeled strain NJSZ-13:gfp and wild-type strain NJSZ-13 were grown on LB agar plates for 24 h, and the colony morphology of the two strains was compared. The two strains were then inoculated into liquid LB medium,the former with added antibiotics, at 28 °C and 200 r/min for 24 h. Bacterial cells of the two strains were harvested by centrifugation, washed three times in sterile water to remove antibiotics and bacterial metabolites,and then were adjusted to an approximate suspension of 105cfu/mL. A volume of 1 mL bacterial suspension was used to inoculate 100 mL fresh LB medium at 28 °C and 200 r/min in a rotary shaker. Every 2 h, the bacterial densities of the two strains were measured with a spectrophotometer(LAMBDA 365; PerkinElmer, Waltham, MA, USA) at A600 nm to enable plotting of growth curves.

The nematicidal activities of the labeled and wild-type strains were determined using bacterial cell suspensions in sterile water. From each bacterial suspension (～105cfu/mL), 200 μL was transferred into a 1.5 mL Eppendorf tube. A 200 μL nematode suspension, (containing about 1000 nematodes), was then added and the solution was mixed. Next, the Eppendorf tubes were placed in an incubator at 25 °C. Every 24 h, a 20 μL mixture, containing about 50 nematodes, was transferred onto a glass slide and examined under a light microscope to record the mortality of the nematodes. Controls were incubated with sterile PBS. Each treatment was repeated three times. The nematodes were considered dead when no movement was observed under a light microscope,and when slight tapping of the nematodes with a needle did not cause movement.The mortality rate was defined as the ratio of dead nematodes to total nematodes tested.

Enumeration of the gfp-labeled strain in plant tissues

Strain NJSZ-13:gfp was cultured in LB medium at 28 °C and 200 r/min for 24 h. Cells were harvested through centrifugation at 4 °C and 10,000 r/min for 20 min, and then adjusted to an approximate suspension of 105cfu/mL using sterile PBS for inoculation. The inoculation procedure was the same as that used for SEM. The inoculated seedlings were then planted into pots containing unsterilized natural soil in an environmental chamber (22-28 °C,14 h/day light).On 4,6,9,12,15,20,25,30,and 40 days after inoculation,the quantities of endophytic strain NJSZ-13:gfp in roots and stems were determined by counting the CFU on resistance LB plates.Plant samples were collected and the substrate was carefully removed from the roots.After the stems and roots were weighed, surface sterilization was performed by treating the samples with 70%ethanol for 30 s and 0.1%sodium hypochlorite for 20 min,and then removing the sterilizing agents from the samples through rinsing with a sterile water.Samples were naturally dried under sterile conditions and ground into 5 mL sterile water. The supernatant solution was diluted and grown on LB plates containing 10 μg/mL tetracycline and 100 μg/mL ampicillin at 28 °C.Green fluorescent colony counts of strain NJSZ-13:gfp were performed under UV light. Each treatment was repeated three times.

In planta visualization of strain NJSZ-13:gfp using CLSM

The colonization and distribution of NJSZ-13:gfp in the roots and stems of Masson pine were visualized with confocal laser scanning microscopy (CLSM) (LSM710;Zeiss). After inoculation for 10 days, the seedlings were collected and washed with sterile water.Hand-cut sections of root and stem tissues were fixed on glass slides using 0.25% agarose and observed using an excitation laser of 488 nm and emission wavelength of 500-550 nm. Images were acquired and merged using Zeiss Confocal Software(ZEN 2.3). Seedlings inoculated with sterile PBS were observed under the same conditions as controls.

Results

SEM examination of endophytic strain NJSZ-13 in Masson pine

Seven days after the aseptic seedlings were inoculated,SEM confirmed the presence of strain NJSZ-13 in root and stem tissues.A large number of bacterial cells had gathered in the roots (Fig. 1a), which indicates that strain NJSZ-13 had entered roots through the surfaces.NJSZ-13 cells were also visualized in the stems (Fig. 1b), which suggests that the endophytic strain NJSZ-13 could be vertically transferred from roots to stems. Plants inoculated with sterile PBS served as the control, and no bacterial cells were observed in their root (Fig. 1c) or stem tissues (Fig. 1d).

Gfp labeling of endophytic bacterium NJSZ-13 and in vitro plasmid stability

The pGFP78 plasmid was successfully transformed into the endophytic bacterium B. cereus NJSZ-13, and the strain NJSZ-13 tagged with the gfp gene(NJSZ-13:gfp)exhibited stable gfp activity.Fluorescence of strain NJSZ-13:gfp was detected using fluorescence microscopy (Fig. 2). The presence of plasmid pGFP78 in the transconjugant was confirmed through PCR and agarose gel electrophoresis.When plasmids of the transconjugants were extracted and amplified with specific primers, a band was observed at～750 bp (Fig. 3), which was consistent with our expectation. To investigate plasmid stability in strain NJSZ-13:gfp,the strain was successively grown in LB medium in the absence of antibiotics for 96 h, resulting at least 50 generations, and then examined for fluorescence. The results showed that 91.0% of NJSZ-13:gfp cells were still carrying the appropriate plasmid (Fig. 4).

Fig. 1 SEM of B.cereus NJSZ-13 colonizing Masson pine in a gnotobiotic system. After inoculation 7 days, the colonization of NJSZ-13 was observed in root (a) and stem(b). No bacteria were observed in the root (c) and the stem(d) of plants inoculated with sterile phosphate-buffered saline (PBS)

Fig. 2 Fluorescence of strain NJSZ-13:gfp in culture solution.The wild endophytic bacterium B. cereus NJSZ-13 tagged with gfp(NJSZ-13:gfp)was observed under fluorescence microscopy.Bacterial cells with green fluorescence were displayed as bright green rods on a glass microscope slide. Scale bar = 10 μm

Growth kinetics and nematicidal activity of the gfplabeled strain

The colony morphologies of the wild-type strain NJSZ-13 and the gfp-labeled strain NJSZ-13:gfp on LB plates differed slightly(Fig. 5). The colonies of the wild-type strain were white, round, and waxy with a smooth surface,whereas the colonies of the labeled strain were white,round, waxy and protuberant. The growth kinetics curves of NJSZ-13 and NJSZ-13:gfp in LB liquid medium are shown in Fig. 6. The data showed that the two strains had the same growth rates in LB medium, and entered the logarithmic growth phase at nearly the same time. In addition, the labeled strain NJSZ-13:gfp exhibited similar nematicidal activity to the wild-type strain NJSZ-13, and the mortality rates of nematodes were 30.6%,78.4%,100%and 32.1%,83.2%,100%after treatment for 24 h,48 h and 72 h, respectively. These results showed that GFP expression in NJSZ-13:gfp does not have a detectable negative effect on its growth or nematicidal activity.

Fig. 3 PCR amplification of the gfp gene in NJSZ-13:gfp and NJSZ-13. M: 2000 kb DNA ladder+ marker; 1: NJSZ-13:gfp; 2: NJSZ-13

Fig. 4 Plasmid stability of engineered strain NJSZ-13:gfp.Error bars are standard deviations (n = 3)

Fig. 5 The colony morphologies of labeled strain NJSZ-13:gfp and wild-type strain NJSZ-13 on LB medium

Fig. 6 Growth curves of strains NJSZ-13 and NJSZ-13:gfp. Error bars are standard deviations (n = 3); in some case, these are not visible as they are smaller than the graph points

Fig. 7 Populations of NJSZ-13:gfp in Masson pine roots and stems as determined by counting the CFU on plates. After inoculation for 4-40 days, the quantification of NJSZ-13:gfp within roots and stems was performed by counting the CFU on plates with two antibiotics(10 μg/mL tetracycline and 100 μg/mL ampicillin),and cell counts of strain NJSZ-13:gfp were taken under UV light (green fluorescent colonies)

Fig. 8 Visualization of inoculated endophyte NJSZ-13:gfp within tissues by confocal laser scanning microscopy (CLSM). Initially,the root surface was covered by NJSZ-13:gfp bacterial aggregates or biofilms(a).After inoculation for 10 days,bacterial cells with green fluorescence were displayed as bright green rods inside roots(b) and stems (c, d). The roots(e) and stems (f) of the plant inoculated with sterile PBS served as the control.Images ae were longitudinal sections.Images d, f were transverse sections. Scale bar = 100 μm

Enumeration of the gfp-labeled strain in plant tissues

Enumeration of the labeled strain NJSZ-13:gfp within plant roots and stems was performed by counting the colonies formed on resistance plates (Fig. 7). Cell counts of NJSZ-13:gfp within roots fell continuously during the monitoring period. Forty days after initial inoculation, the cell counts of colonizing bacteria were 0.53 × 102cfu/g in roots. In stems, the cell counts of NJSZ-13:gfp initially decreased over the first 20 days, and then gradually increased from days 21-40. After 40 days, the cell counts of NJSZ-13:gfp in stems were 1.71 × 102cfu/g.Overall,NJSZ-13:gfp cell counts increased progressively from roots to stems,and cell counts in the stems were significantly higher than those in the roots. In addition, we did not observe endophytes naturally resistant to tetracycline and ampicillin recovered from plants inoculated with sterile PBS.

In planta visualization of strain NJSZ-13:gfp using CLSM

When Masson pine roots were soaked with a bacterial suspension of the labeled strain for 2 h, the root surface was covered by strain NJSZ-13:gfp bacterial aggregates or biofilms (Fig. 8a). NJSZ-13:gfp cells then proceeded to enter into the root tissues (Fig. 8b) from the surface. As time progressed, cells were also observed in stem tissues(Fig. 8c, d). SEM and CLSM observations demonstrated that the endophyte NJSZ-13 was a good colonizer of Masson pine, and could colonize and transfer in planta.

Discussion

Bacillus could represent a useful tool for the protection of pine species from pinewood nematodes. The pine endophyte, B. amyloliquefaciens JK-JS3, exhibited significant nematicidal activity toward pinewood nematodes, with mortality reaching 100% after the nematodes were treated with fermentation filtrates of strain JK-JS3 for 24 h (Zhu et al. 2009). However, few previous investigations have tracked or visualized endophytes with nematicidal activity within plants. We demonstrated that the nematicidal endophytic bacterium B. cereus NJSZ-13 could colonize plant tissues and transfer in planta, providing the basis for utilization of this endophytic strain in the biocontrol of pinewood nematodes.

Compared to other methods, the gfp gene marker offers many advantages, including good stability, in situ monitoring, and particularly, a minimal influence on the strain(Errampalli et al. 1999). Even so, it is necessary to determine the growth kinetics and plasmid stability of a gfplabeled strain before studying the population dynamics of such microorganisms within plant tissues. For example, it was found that gfp-labeled Ralstonia solanacearum had a growth lag compared to the wild-type strain (Che et al.2008). In this study, the gfp shuttle vector pGFP78 was delivered into B. cereus NJSZ-13 through electroporation,and was expressed successfully. The gfp-tagged strain NJSZ-13:gfp exhibits similar nematicidal activity and growth curve to the wild-type strain NJSZ-13. Furthermore, the pGFP78 plasmid was stably maintained in strain NJSZ-13 for at least 96 h of bacterial cultivation on medium without antibiotics,which suggests that the engineered strain NJSZ-13:gfp could be used to evaluate population dynamics in Masson pine.

After Masson pine was inoculated with a suspension of strain NJSZ-13:gfp for 40 days, large quantities of labeled endophytes were recovered from roots and stems, and higher densities of strain NJSZ-13:gfp were detected in the stem than in root tissues.This result suggests that the stems were the preferred sites for bacterial colonization. By contrast, Li et al. (2006) enumerated of B. cereus B47 in tomato roots and stems, and found that its preferred colonization site was the root. This difference likely occurred because the endophytic bacterium NJSZ-13 was isolated from the stem, and thus stems are more suitable for the survival and reproduction of the labeled strain. Furthermore, because pinewood nematodes usually infect the stems of pine, this result provided a strong benefit of development and application of the antagonistic bacterium NJSZ-13. In addition, the cell counts of NJSZ-13:gfp in stems first decreased and then increased. This changing trend could probably be attributed to the fact that after being transferred to the stem,the labeled strain needed time to acclimatize to the stem environment,leading to death of some cells. After a period of acclimation, the labeled endophytes began to reproduce in the stems, and their quantity gradually increased and then stabilized.

In this study, we evaluated the ability of the endophyte NJSZ-13, which exhibits nematicidal activity, to colonize Masson pine tissues.No apparent harm was observed to the inoculated seedlings, which suggests that strain NJSZ-13 has good potential as a biocontrol agent.

AcknowledgementsWe are grateful for the assistance of all staff and students in the Institute of Forest Protection, Nanjing Forestry University, Nanjing, People’s Republic of China.