The M. incognita used in this study was obtained from the Institute of Vegetables and Flowers, Chinese Academy of Agricultural Sciences (IVF-CAAS; Beijing, China). The second-stage M. incognita juveniles (J2s) were inoculated on pepper (Capsicum annuum cv. Qiemen) plants and maintained in the greenhouses of the IVF-CAAS for 40 days until egg masses formed. The egg masses were treated with 2% NaOCl and incubated at 28°C for 24 h in sterile water to hatch J2s.

Bacillus velezensis strain Bv-25 was isolated from the rhizosphere soil of cucumber plants. The soil was sampled from a greenhouse in Langfang city (Hebei, China) during the fruiting period (May 2) and used to isolate bacteria using the gradient dilution method on Luria–Bertani (LB) agar medium at 28°C for 48 h. For molecular identification, the 16S ribosomal DNA was amplified by polymerase chain reaction (PCR) with primers 27F and 1492R (Alberoni et al., 2019). The sequence was analyzed using NCBI BLAST¹ The bacterium was preserved in the Provincial Common Microbe Center of the Chinese Committee for Microbial Culture Conservation (preservation no. CGMCCNO.16523).

Zhongnong no. 6 cucumber seed was obtained from IVF-CAAS. The cucumber seeds were surface-sterilized in 0.5% NaOCl, washed thoroughly with sterile water, and germinated on sterile, moist filter paper in a Petri dish for 3 days at 28°C under darkness. Then, the germinated cucumber seeds were maintained in a sterile mixture of peat:vermiculite (2:1, vol/vol) in a growth chamber with a 15-h light and 9-h dark cycle at 28° for 2 weeks to get cucumber seedlings.

Bacillus velezensis strain Bv-25 was cultured for 48 h in 100 mL of LB liquid medium (10 g tryptone, 5 g yeast extract, and 10 g NaCl in 1 L sterilized water) at 28°C on a shaking incubator (200 revolutions/min [rpm]) and adjusted to a density of 1.0 × 10⁸ colony-forming units (CFUs) per milliliter with sterile water. To collect the supernatant, the Bv-25 fermentation broth was centrifuged at 12,000 rpm for 3 min. Each test for nematicidal activity involved six treatments: 10% and 100% fermentation broth, 10% and 100% supernatant from the broth, abamectin (1 μg/mL) as positive control, and sterile water as negative control. To reduce errors, each treatment had 10 replicates, and each test was repeated three times.

The tests were conducted in 24-well plates incubated at 28°C for 24 h. The J2s were considered dead when the bodies of nematodes were straight and did not move upon stimulation with 0.5 M NaOH (Harada and Yoshiga, 2016). Nematode mortality rates in each treatment were recorded at 24 h after treatment. The adjusted mortality rate was calculated by the following formula: [(Mortality rate of nematodes treated by Bv-25 − Mortality rate of nematodes treated by water)/(1 − Mortality rate of nematodes treated by water)] × 100.

To investigate the inhibition effect of strain BV-25 on M. incognita egg hatching, we conducted experiments with four treatments: Bv-25 fermentation broth at concentration 1.0 × 10⁸ CFUs/mL, the supernatant, 10% LB medium, and sterilized water as negative control. Each treatment had 10 replicates, and each experiment was repeated three times. A total of 150 eggs were mixed with each treatment in separate wells. The 24-well plates were incubated at 28°C for 72 h in a growth chamber (Mendoza et al., 2008). The numbers of hatched J2s were counted at 72 h after incubation (Saikia et al., 2013). The egg-hatching rate was calculated according to the following formula: [(Number of hatched J2/Total eggs) × 100].

To understand the molecular effect of Bv-25 volatile compounds on the J2s, we conducted fumigation experiments in three-well culture dishes according to a previously published method (Gu et al., 2007). Five milliliters of either fermentation broth (1.0 × 10⁸ CFUs/mL) or sterile water as a control was put in one well of a dish, and 1 mL of a suspension containing 5,000 J2s added to another, separate well. The dishes were sealed with two layers of sealing film and placed in an incubator at 28°C for 48 h. Three experiments of 15 dishes per treatment were performed. The mortality rate and status of J2s were checked every 24 h by spot checks. The death of J2s was verified as described previously (Harada and Yoshiga, 2016).

Total RNA was extracted from J2s using a tissue cell RNA microextraction kit (Beijing Zhongchuang) every 24 h. The converted first-strand cDNA was obtained using the premix for two-step reverse transcriptase (RT)–quantitative PCR (qPCR)–type reverse transcription reagent (Eric Bio) and subjected to gradient dilution of at least five to six gradients as a template for real-time PCR. qPCR was performed for two essential nematode genes mpk-1 and flp-18, and the chemosensory gene ord-1, using the primers in Supplementary Table 1 (Dong et al., 2014, 2016; Shivakumara et al., 2019). The calculation formula for the standard curve was automatically obtained through the BIORAD system: Y = aX + b, where Y is the expression amount and X is the Ct value (Cottyn et al., 2011). Time at 0 h served as the control.

The roots of cucumber seedlings were immersed in strain Bv-25 fermentation broth (1.0 × 10⁸ CFUs/mL) for 0, 0.5, 1, 2, 3, 6, 9, 12, 24, or 48 h. At each sampled time, the roots of 10 cucumber seedlings were harvested, dried with absorbent paper, cut with sterile scissors, and macerated. Three samples of 0.2 g each were used to extract DNA using the TIANamp DNA Kit (TIANGEN, Beijing, China).

The quantities of Bv-25 on the cucumber roots were determined by real-time PCR. The specific primers were BV-25-F (5′-GTGAGGTAACGGCTCACCAA-3′) and BV-25-R (5′-TGCTCCGTCAGACTTTCGTC-3′), which amplified a 142-bp fragment. Then, real-time PCR SYBR Green I reaction system was prepared in a 20-μL volume, consisting of 10 μL of 2 × Super Real PreMix Plus (TIANGEN), 1 μL each of BV-25-F and BV-25-R primer (0.1 μmol/μL), 1 μL of genomic DNA (diluted 10-fold with double distilled [dd] water) and 7 μL of dd water. The PCR reaction procedure used was as follows: 95°C for 15 min and then 40 cycles of 95°C for 10 s and 60°C for 32 s. From 65 to 95°C, the fluorescence signal was recorded every 0.5°C. The Ct values and regression equation were determined as per a previous method (Cottyn et al., 2011) and used to estimate the quantities of BV-25 on cucumber roots.

To visually observe the infection, cucumber seedlings roots were immersed in Bv-25 fermentation broth (1.0 × 10⁸ CFUs/mL) for 3 h to allow the bacteria to attach to the roots or sterile water as a negative control. Then, the cucumber seedlings were inoculated with approximately 300 J2s in a Petri dish and incubated in a chamber at 28°C for 48 h. To maintain moisture during the incubation period, 3 mL sterile water was added to each Petri dish. After 48 h of incubation, the roots were stained with fuchsin (Bybd et al., 1983) and observed under a microscope.

The cucumber seedling roots were soaked in strain Bv-25 fermentation broth and sampled as previously mentioned. Three root samples were collected at each time point, frozen in liquid nitrogen, and stored at −80°C. To analyze gene expression, total RNA extraction from root samples was conducted using the RNAprep Pure Plant Kit (TIANGEN), and cDNA synthesis was performed using the FastKing gDNA Dispelling RT SuperMix (TIANGEN). The expression characteristic of actin (internal control) and four defense signal pathway genes (pr1, pr2, lox1, and ctr1) were analyzed by a real-time PCR SYBR Green I reaction system with gene-specific primers (Supplementary Table 1) (Shoresh et al., 2005; Wan et al., 2010; Migocka and Papierniak, 2011). The expression level of the four genes was normalized and compared with the expression level of actin quantified by the ΔΔCt method (Beaubois et al., 2007).

To evaluate the induction of resistance against M. incognita mediated by strain Bv-25 on cucumber seedlings, we conducted a split-root experiment. The cucumber seedlings at the two-leaf stage were transferred to sterilized peat:vermiculite mixture (2:1, vol/vol) in the split-root setup (Martínez-Medina et al., 2017), consisting of two 500-mL adjacent pot compartments. Forty-eight hours after transplanting, approximately 300 J2s were inoculated to the roots in the pots on the right side (Xing et al., 2017). Ten milliliters of Bv-25 fermentation broth (1.0 × 10⁸ CFUs/mL) or sterile water as negative control was inoculated into the pot on the left side. Three experiments were done with 10 replicates per treatment. The experiments were conducted in a greenhouse under conditions of 28°C and 9-h light/15-h dark. Thirty days after J2s inoculation, the numbers of galls on the cucumber roots treated by Bv-25 and the controls were counted. Control efficiency was calculated according to formula: [(Number of galls on cucumber root under control − Number of galls on cucumber root under Bv-25 treatment)/Number of galls on cucumber root under control] × 100.

The cucumber seedlings at the two-leaf stage were transplanted to pot (three cucumber seedlings per pot) filled with 2,000 mL sterile peat: vermiculite mixture (2:1, vol/vol). The pots were located in the greenhouse under conditions of 28°C and 9-h light/15-h dark. One week after transplanting, an experiment was conducted consisting of three treatments: Bv-25 fermentation broth (1.0 × 10⁸ CFUs/mL), a 1/1,000 dilution of 1.8% avermectin (Green Agricultural Science and Technology Group, Beijing), and sterile water as control. A total of 300 J2s were inoculated to each pot. Three such experiments of 10 replicates per treatment were performed. Galls on cucumber roots were counted at 30 days after J2 inoculation. The control efficiency was calculated as before.

Field experiments were performed in a greenhouse with heavy M. incognita infestation in Langfang City (Hebei, China) in April 2019. The greenhouse was divided into nine blocks (9 m²) arranged using randomized block design with three replications. Each block consisted of 2 rows and 15 cucumber plants planted per row. The row space was 50 cm and plant-to-plant spacing was 40 cm. Before cucumber seedling transplantation, the population densities of M. incognita were counted using the method of Yin et al. (2021).

Cucumber seedlings (cultivar Zhongnong 6) at the two-leaf stage were transplanted into the greenhouse. For Bv-25 treatment, each cucumber seedling was irrigated with a mixture of 100 mL of Bv-25 fermentation broth (1.0 × 10⁸ CFUs/mL) and 400 mL of water. In the nematicide control, 10% fosthiazate granules (Ishihara Industrial Co., Ltd., Japan) were applied to the soil at the depth of 15 to 20 cm in line with the state standards (22.5 kg/ha) before transplanting. The untreated control was irrigated with water. Each treatment had three replicates. The shoot length of the cucumber plants and fruit weight were measured three times. Regular management was carried out in the greenhouse during the experimental period. The gall index was scored on a 1- to 5-point scale: 1 = no galls; 2 = 1–25% galled roots; 3 = 26–50% galled roots; 4 = 51–75% galled roots; 5 = > 75% galled roots (Dube and Smart, 1987). The disease index was calculated according to the following formula: Disease index = [Σ (Number of cucumbers in the gall index × Gall index)/(Number of total cucumbers × Highest gall index)] × 100. The managing effect of strain Bv-25 against M. incognita in the field was assessed with the formula: Managing effect (%) = [(Disease index of control − Disease index of Bv-25 treatment)/Disease index of control] × 100.

The statistical analyses were performed using one-way analysis of variance (ANOVA) with Duncan multiple-range test (p < 0.05) and Tukey multiple-comparisons test (p < 0.001) (SPSS software, v.220; SPSS Inc., Chicago, IL, United States). Regression analysis of Bv-25 quantification was also carried out with SPSS. All data are presented as mean ± standard error.

The mortality rates of M. incognita after treatment with abamectin, the Bv-25 fermentation broth, and 10% fermentation broth were 100%, 100%, and 96.1%, respectively, which were significantly higher than those of other treatments (Figure 1). The mortality rates for the supernatant broth and 10% supernatant were 78.3% and 59.5%, respectively (Figure 1). The mortality rates for water and 10% LB medium were the lowest. Strain Bv-25 thus had strong nematicidal activity against M. incognita.

The egg-hatching rate of M. incognita eggs 72 h after treatment with strain Bv-25 fermentation broth was the lowest (2.1%), followed by the Bv-25 supernatant (4.6%), 10% fermentation broth (12.3%), 10% supernatant (14.4%), 10% LB medium (37.2%), and sterile water (44.2%) (Figure 2). Bv-25 treatment thus markedly suppressed egg hatching of M. incognita.

Under Bv-25 fumigation treatment, most J2s (89.2%) gradually reduced movement within 24 h but recovered mobility with NaOH treatment, suggesting that the J2s were paralyzed by volatile organic compounds produced by Bv-25 within 24 h. At 48 h, nearly all J2s (98.2%) could not move in response to NaOH and were regarded as dead. The paralysis or mortality rates of J2s treated with Bv-25 fumigation were much higher than the water controls (Figure 3A).

To further assess the effect of Bv-25 fumigation on J2s, three key M. incognita genes were further analyzed by qPCR. The results show that a good linear relationship existed between the Ct value of the three-gene amplification reaction cycle numbers and the logarithmic value of the DNA copy number (flp-18: y = 4.945x + 17.865, R² = 0.9994; ord-1: y = 3.2051x + 22.681, R² = 0.9964. mpk-1: y = 3.5399x + 19.066, R² = 0.9944). The expression patterns of the three genes were significantly different between the water control and the Bv-25 fumigation treatment, being relatively steady with water but decreasing sharply with Bv-25 fumigation from 0 to 48 h. The expression of ord-1 decreased 122.1 times in the treated nematodes compared with control (Figure 3B), mpk-1 decreased 67.8 times from 122.1 to 1.8 (Figure 3C), and flp-18 decreased 32.1 times from 122.1 to 3.8 (Figure 3D).

The Ct value of the amplification reaction cycle had a linear relationship with the logarithmic value of the Bv-25 concentration (y = −3.03x + 34.968; R² = 0.9909; p < 0.01). The Ct value decreased with increasing time, demonstrating an increase in Bv-25 colonization on cucumber roots (Table 1). From 24 to 36 h, the concentration of Bv-25 markedly increased from 5.94 × 10⁷ CFUs/g to 3.26 × 10⁸ CFUs/g, suggesting that strain Bv-25 was able to colonize cucumber roots and reproduce rapidly.

Forty-eight hours after inoculation, the number of J2s in cucumber roots treated by strain Bv-25 was 4.3 per plant and markedly less than that in control roots with 83.0 (Figure 4A). Under control treatment, plenty of J2s entered into the vascular bundles and grouped together (Figures 4B–E). The results indicate that strain Bv-25 could inhibit M. incognita invasion into cucumber roots.

The expression pattern of pr1 was significantly up-regulated, reaching the highest level at 12 h (Figure 5A), whereas pr3 reached the highest expression level at 48 h, 140 times higher than that of the control group (Figure 5B). The expression of lox1 increased at first and then decreased after 1 h, increased and reached the peak at 6 h (Figure 5C), and then declined until 48 h. There was no significant difference in ctr1 gene expression between control and Bv-25–treated group (Figure 5D). These results indicated that strain Bv-25 activates the salicylic acid (SA) and jasmonic acid (JA) defense signaling pathways in cucumber roots. In the split-root experiment, the average number of root galls on cucumber roots treated by strain Bv-25 (53.5 per plant) was significantly lower than that on control roots (85.3 per plant) (Figures 6A–C). The results together showed that Bv-25 could enhance resistance of cucumber to reduce M. incognita infection.

In the pot experiment, cucumber roots treated with strain Bv-25 fermentation broth and abamectin had fewer root nodes (4.2 and 16.4, respectively) than those treated with water (62.8) (Figure 7). For strain Bv-25, this amounted to a 73.8% decrease in the number of cucumber root nodes.

The soil in the greenhouse had many M. incognita (43 J2s/100 g of soil). Cucumber plants in the control plots had significantly more root galls, shorter heights, and lower yields than treated plants. The disease index was markedly higher in control plants (88.4) than in the Bv-25 treatment (33.9) and fosthiazate treatment (23.4) plants. The control effect of Bv-25 treatment was 61.6%, and it increased heights and yielded 14.4 and 36.5%, respectively (Table 2).