Klebsiella pneumoniae strain SnebYK, subsequently referred to as SnebYK, is a Gram-negative facultatively anaerobic bacterium that was isolated from mud at the outlet of a pesticide manufacturing factory in Liaoning Province, China. SnebYK was identified by Luo (2010) and stored at -80°C at the Nematology Institute of Northern China at Shenyang Agricultural University. Before use, the strain was streaked onto a nutrient agar medium and incubated at 28°C to confirm purity. A single colony was selected, transferred to nutrient agar medium, and incubated for 48 h at 28°C. The SnebYK fermentation broth (1 × 10⁹ CFU ml^-1) was prepared according to the method previously described by Chen et al. (2014).

The population of H. glycines (race 3, HG type 0) was maintained on soybean [Glycine max (L.) Merr. ‘Liaodou15’] grown in the experimental field of the Nematology Institute of Northern China. Heterodera glycines specimens were morphologically identified following the method previously described by Subbotin et al. (2010) prior to use in the experiment. Cysts were extracted from soybean rhizosphere soil in the experimental field using a floating sieve, and then surface-sterilized with 0.5% sodium hypochlorite. Second-stage juveniles (J2) were incubated as described by Tian et al. (2014). The hatched J2 were assessed for survival and collected in a beaker for inoculation.

The siderophore production of SnebYK was determined as described by Schwyn and Neilands (1987). SnebYK was inoculated in the center of a chrome azurol S agar plate and incubated at 28°C for 7 days; plates were observed to determine whether a yellow–orange halo appeared around the colony. Assays of inorganic and organic phosphate solubilization by SnebYK were performed using the National Botanical Research Institute’s phosphate growth medium and Mongina organic culture medium with lecithin, respectively (Hao et al., 2012; Ji et al., 2014). SnebYK was spot-inoculated onto the plate and incubated at 28°C for at least 7 days. A clear zone around the colony indicated that the strain tested had phosphate solubilization activity. The phosphate-solubilizing ability of SnebYK was evaluated using the phosphomolybdate blue colorimetric method (Murphy and Riley, 1962). Ammonia production was detected with Nessler’s reagent (Singh et al., 2015).

The nitrogen-fixing ability of SnebYK was tested using ACCC55 agar plates (Gao et al., 2015). The nitrogenase activity of SnebYK was evaluated through an acetylene reduction assay, following the protocol of Boddey and Knowles (1987). To analyze the nifH gene of SnebYK, genomic DNA was extracted from the SnebYK strain using a bacterial genomic DNA extraction kit (Tiangen Biotech, China). The primers nifH1 (5′-ADNGCCATCATYTCNCC-3′) and nifH2 (5′-TGYGAYCCNAARGCNGA-3′) were used for polymerase chain reaction (PCR) amplification of the nifH gene (Gaby and Buckley, 2012). The PCR amplification conditions were 5 min at 94°C for initial denaturation; 35 cycles of 94°C for 1 min, 57°C for 1 min, and 72°C for 1 min, followed by a 72°C extension for 10 min (Liu et al., 2011). PCR products were analyzed by electrophoresis on 1.2% agarose gels and then sequenced by Sangon Biotech Co. Ltd., Shanghai, China. The obtained sequences were compared with those in the GenBank database (RRID: SCR_002760) using the NCBI BLAST algorithm (Pinto-Tomás et al., 2009; Liu et al., 2011; Lin et al., 2012). The phylogenetic tree was established using the neighbor-joining method in Mega 7.0.26. To detect the expression of nifH, reverse transcription (RT)-PCR analysis was conducted. SnebYK colonies grown on ACCC55 agar plates were collected, and colonies grown on nutrient agar plates were also collected as controls. Total RNA was extracted from the samples using a bacterial total RNA extraction kit (Tiangen Biotech, China), and the PrimeScript RT Reagent kit (TaKaRa, China) was used for cDNA synthesis following the manufacturer’s instructions. The primers used to amplify nifH were PolF (5′-TGCGAYCCSAARGCBGACTC-3′) and PolR (5′-ATSGCCATCATYTCRCCGGA-3′) (Poly et al., 2001). The 16S rRNA gene was used as an internal control. For the PCR, 100 ng cDNA was used, with the following program: 94°C for 4 min; 30 cycles of 94°C for 30 s, 60°C for 30 s, and 72°C for 30 s, followed by an extension at 72°C for 5 min. PCR products were visualized using the GoldView DNA staining reagent (Aidlab Biotech, China) following agarose gel electrophoresis and analysis.

Soybean seeds were surface-sterilized with 0.5% sodium hypochlorite, washed with sterilized water at least three times, and then dried at room temperature. Treated soybean seeds were coated with the SnebYK fermentation broth at a 70:1 mass ratio. Control soybean seeds were coated with sterilized water. All seeds were sown in 10 cm × 10 cm plastic pots containing a sterilized mixture of soil and sand (1:1, v/v) and grown in a greenhouse at 26/21°C with a 16/8 h photoperiod. The soybean seedlings were thinned to one plant per pot once two euphylla were observed. Each treatment included 10 replicates and was arranged in a completely randomized design. Thirty days after sowing, soybean seedlings were harvested from all pots, the soil adhered to the roots was carefully removed with tap water, and the shoot length, taproot length, and fresh weight of the plants in each treatment were measured. Root morphology images were obtained using a scanner (Expression 10000 XL, Epson, Japan) and then analyzed using the WinRHIZO image analysis system (V4.1c, Regent Instruments, Canada).

Soybean seeds were surface-sterilized as described and sown in pots containing the sterilized soil mixture, with one seed per pot. When soybean seedlings had two euphylla, the roots were removed from the mixture and washed thoroughly with running tap water. Roots of approximately the same size were selected and divided into two equal parts with a scalpel: one part was designated the inducer root system, and the other part was designated the responder root system (Adam et al., 2014). The inducer and responder roots were transplanted into two plastic pots, with 200 ml of sterilized mixture in each. When the two parts of the root systems had been successfully fixed, the inducer root system was inoculated either with SnebYK fermentation broth (10 ml, 1 × 10⁹ CFU ml^-1) or with sterile water as a control. After 5 days of treatment, all responder roots were inoculated with 2,000 J2 of H. glycines as shown in Figure 1A. Fifteen days after nematode inoculation, the responder roots were stained with NaClO-acid fuchsin (Bybd et al., 1983). The number of H. glycines in the roots was counted, and the developmental stages of H. glycines were recorded simultaneously. Each treatment had four repetitions, and the entire experiment was repeated twice.

To verify the validity of the split root system (SRS), BOX-PCR analysis and 16S rRNA gene sequencing were performed to test the colonization of the root system by SnebYK. Twenty days after the inducer roots were inoculated with SnebYK or sterile water, the responder and inducer root systems were removed. Immediately, 0.5 g of each root was collected and ground with a tissue grinder to release the rhizosphere and endophytic bacteria. A bacterial suspension was obtained after adding 1 ml of sterile water to the tissue grinder. After further dilution with sterile water (1,000×), 100 μl of the suspension was spread onto an ACCC55 agar plate and incubated at 28°C. Each treatment included three replications, and all steps were conducted under sterile conditions. After 72 h, morphologically distinct colonies of bacteria were selected and cultured separately. The genomic DNA of each strain was extracted with a bacterial genomic DNA extraction kit (Tiangen Biotech, China). The electrophoretograms of the BOX-PCR products were used to screen out different bacterial strains and could also be used to investigate the colonization of SnebYK by comparing them with the SnebYK electrophoretogram. The primer used for the BOX-PCR was BOXA1R (5′-CTACGGCAAGGCGACGCTGACG-3′) (Versalovic et al., 1994). The amplification was conducted following the method reported by Ateba and Mbewe (2013) in a 25 μl reaction mixture with the DNA samples obtained from the bacterial strains found in the SRS. Strains with different fingerprinting profiles were identified by 16S rRNA gene sequencing as described by Sarma and Saikia (2014).

Soybeans were treated with SnebYK and grown as previously described for the pot experiment. Sterilized water was used as control. When soybean seedlings reached the two-euphylla stage, their roots were inoculated with 2,000 J2 of H. glycines. To measure the transcript levels of defense-related genes in real time, soybean roots were collected at 0, 24, and 48 h after nematode inoculation, froze immediately in liquid nitrogen and stored at -80°C until use. Total RNA was extracted from the soybean roots using the MiniBEST Universal RNA Extraction kit (TaKaRa, China). Total RNA from each sample (1 μg) was reverse-transcribed using the PrimeScript RT Reagent kit (TaKaRa, China). Quantitative real-time PCR (qRT-PCR) was performed in a CFX Connect Real-Time PCR Detection System (Bio-Rad, United States) using a SYBR green I quantitative PCR master mix (TaKaRa, China). The reaction conditions were 95°C for 30 s followed by 40 cycles at 95°C for 5 s, and 60°C for 30 s (fluorescence measurement step). After 40 cycles, a melting-curve analysis was performed (15 s at 95°C, 30 s at 60°C, and 15 s at 95°C, fluorescence measurement step). Each sample included three replicates. Expression of the soybean gene Actin11 was used as an internal standard for normalization, and the data were quantified using the 2^-ΔΔCt method (Livak and Schmittgen, 2001).

All data were analyzed using SPSS Statistics 19.0 (RRID: SCR_002865) and expressed as the mean ± standard deviation. Significant differences among treatments in the in vitro juvenile mortality assay were determined according to Duncan’s multiple range test (P < 0.05). Significant differences between treatments in other experiments were evaluated using t-tests (P < 0.05).

Klebsiella pneumoniae has been reported to possess many plant growth-promoting properties. Therefore, several such properties, including nitrogen fixation, ammonia production, phosphate solubilization, and siderophore production, were analyzed in SnebYK (Table 1). An orange halo zone was observed on the chrome azurol S agar plate, indicating that SnebYK produced siderophores. The ability of SnebYK to solubilize inorganic phosphate was confirmed by the clear zone around SnebYK on the National Botanical Research Institute’s phosphate agar plate. However, nothing was observed on the Mongina agar plate, which indicates that SnebYK could not solubilize organic phosphate. SnebYK grown on the nitrogen-free medium showed high nitrogenase activity. The yellow color observed following the addition of Nessler’s reagent to culture filtrate indicated that SnebYK could produce ammonia. A 363 bp amplicon specific to nifH (GenBank accession number: MF580385), a functioning gene encoding nitrogenase iron protein, was obtained from the genomic DNA of SnebYK (Figure 2A). The RT-PCR results showed that the nifH gene was expressed (1.24-fold the level of the internal control) when SnebYK was cultured under nitrogen-free conditions but was not expressed when SnebYK was cultured in a medium with sufficient nitrogen (Figure 2B). Phylogenetic analysis revealed that the nifH sequence of SnebYK was closely related to that of the K. pneumoniae strain NG14 and other strains in the genus Klebsiella (Figure 2C).

Since SnebYK exhibits a variety of growth-promoting properties, the effect of SnebYK on soybean growth was evaluated. SnebYK-treated soybean plants had well-developed second compound leaves and roots (Figure 3). The second compound leaves of untreated soybean were not fully expanded, and their roots were sparse. In the presence of SnebYK, soybean seedlings exhibited increased biomass (Table 2). SnebYK caused significant increases in the shoot length and fresh weight, which were 9.18% and 39.51% higher, respectively, than those in the control. However, SnebYK did not affect taproot length or the root–shoot ratio. SnebYK also had a considerable impact on root growth. SnebYK increased the total root length, total root surface area, and total root volume by 52.10%, 47.73%, and 42.72%, respectively, compared to those of the untreated control.

SnebYK had low contact toxicity to H. glycines, as the mortality rate after 24 h was only 18.23% (Figure 4A). To evaluate whether coating seeds with SnebYK provided protection against H. glycines, seeds were sown in pots after coating and then the soybeans were inoculated with H. glycines. The number of females significantly influences the nematode population. Thus, we evaluated the effect of SnebYK on the control of both the adult female and the total population of H. glycines. Thirty days after H. glycines inoculation, juveniles and adults, especially adult females, in the soil and the soybean roots were counted. The nematode population of SnebYK-treated soybean was significantly smaller than that of the control, with inhibition rates reaching 47.32% (Figure 4B). Analysis of the population revealed that SnebYK dramatically decreased the numbers of both juveniles and adult males, as well as adult females. Interestingly, the proportion of adult females in the H. glycines population was 40.88% (±4.77%) after treatment with SnebYK, while that in the control was 52.42% (±2.19%). This indicates that SnebYK significantly decreased the proportion of adult females, demonstrating that SnebYK effectively restrained the population of H. glycines and prevented their development into females. In the presence of H. glycines, SnebYK-treated soybean maintained a higher fresh root weight than the untreated control, concomitantly with a significantly increased root–shoot ratio (Table 3). After analysis of the root system, we found that the total root volume of SnebYK-treated soybean was significantly higher, by 28.74%, than that in the control (Table 3).

To determine the effect of SnebYK on the control of H. glycines in the field, experiments were conducted at the experimental test site in Kangping. We observed that SnebYK effectively reduced the number of H. glycines in per root by 53.23% and 33.75% in 2016 and 2017, respectively (Figure 5A). The 2016 data showed that SnebYK did not reduce the number of adult females on the soybean roots but did reduce their abundance in the soil by 40.11%. The 2017 data showed that SnebYK greatly reduced the number of adult females both on the roots and in the soil (Figures 5B,C). Figures 5D–F summarize the effect of SnebYK on soybean growth under H. glycines infection in the field trials. The results indicated that, although SnebYK-treated soybeans had a higher fresh weight than that of the untreated soybeans, the difference was not significant.

While SnebYK resulted in low H. glycines mortality in the in vitro assay, it exhibited the capacity to control H. glycines in both the potting and field experiments when used to coat soybean seeds. Therefore, we conducted experiments to verify whether SnebYK could elicit systemic resistance in soybean against H. glycines. The SRS is considered an effective tool for demonstrating induced resistance, and in this study, we confirmed the validity of the SRS before using it. Rhizosphere and endophytic bacteria from the SRS were cultured on ACCC55 agar plates. After 1 day, several bacterial colonies (with morphologies similar to that of SnebYK) were observed on only the agar plate that was inoculated with the bacteria from the inducer root treated with SnebYK. More bacterial colonies appeared after 2 days. When comparing the BOX-PCR fingerprints and 16S rRNA identifications of rhizosphere and endophytic bacteria from the SRS, we found K. pneumoniae in only the inducer roots treated with SnebYK (Figure 1B). Since the responder roots were not colonized by SnebYK, the SRS used in this experiment was valid. Next, we conducted a split root experiment to determine whether SnebYK elicited ISR to H. glycines in soybean. Penetration of the responder roots of the SRS, assessed 15 days after H. glycines inoculation, was significantly reduced when the inducer roots of the soybean were treated with SnebYK. This treatment caused a 61.78% reduction in the total nematode penetration when compared with that in the untreated control. Specifically, the numbers of J2, third-stage juveniles (J3), and fourth-stage juveniles (J4) were significantly lower in the responder roots than in the control group (Figure 6A). The proportion of H. glycines juveniles of each stage in the responder roots is summarized in Figure 6B. The proportions of J2, J3, and J4 in the control group were 60.59%, 34.62%, and 4.8%, respectively. Meanwhile, the proportions in the SnebYK-treated group were 73.27%, 25.1%, and 1.63%, respectively. Therefore, inoculation with SnebYK significantly increased the proportion of J2 by 12.68% and decreased the proportions of J3 and J4 by 9.52% and 3.17%, respectively.

To investigate whether SnebYK-mediated ISR was accompanied by primed defense responses to H. glycines in the roots, the expression levels of PR1, PR2, PR3, PR5, PR9, PR10, PDF1.2, NPR1-1, and NPR1-2 in soybeans treated or not treated with SnebYK were assessed using qRT-PCR. Transcript levels of the defense genes analyzed did not differ in the non-challenged soybeans regardless of the presence of SnebYK. After H. glycines inoculation, elevated PR5 expression was detected in control roots, indicating that soybean responds rapidly to H. glycines infection. Moreover, transcription of PR5 in soybeans pretreated with SnebYK was 5.04-fold and 3.23-fold higher at 24 and 48 h after inoculation, respectively, than those in control soybeans. Differences were also found in the expression levels of PR1, PR2, PR3, PR9, PR10, PDF1.2, and NPR1-2 between untreated soybeans and soybeans pretreated with SnebYK upon challenge with H. glycines. Twenty-four hours after H. glycines inoculation, these genes were induced in control soybeans, whereas the increase upon H. glycines challenge was lower in soybeans previously treated with SnebYK. However, 48 h after inoculation of H. glycines, the expression levels of PR1, PR2 and PDF1.2 were significantly higher in soybeans pretreated with SnebYK than in control soybeans; the transcript levels of PR9, PR10 and NPR1-2 did not differ significantly between the treatments; and the expression of PR3 was much lower in SnebYK-treated soybean roots than in untreated soybean roots (Figure 7). The transcript levels of NPR1-1 did not differ between soybeans pretreated with SnebYK and soybeans not treated with SnebYK following the inoculation of H. glycines.