RKNs used in these experiments were isolated from root galls of ginseng cultivated in Jinan, Jeon-buk Province, Korea, and identified as Meloidogyne hapla based on analysis of 28S rRNA sequences following the method described by Oh et al. (2009; Kim, unpublished data). Four-week-old tomato plants (Solanum lycopersicum cv. Rutgers) grown in a growth chamber were inoculated with second-stage juveniles (J2) of M. hapla and cultivated at approximately 25±2°C in a greenhouse. Seven weeks following inoculation, whenever inoculums were required, tomato plants were uprooted and root systems were carefully washed with running tap water to remove adhered soil. Egg masses of M. hapla were handpicked with the help of the forceps and were placed on a Baermann funnel for 3-5 days to obtain hatched out J2 (Southey, 1986). The inoculum concentrations of J2 were adjusted to required densities using sterile distilled water (SDW), and were employed for in vitro and pot experiments in our study.

Soil samples were collected from various fields and mountainous areas, from which bacteria were isolated through the dilution plating method (Dhingra and Sinclair, 1985), resulting in a total of 523 bacterial isolates. These bacterial isolates and 19 Paenibacillus strains solated from the 4-year-old Korean ginseng roots with rot symptoms (Jeon et al., 2003); in total, 542 bacteria were subjected to in vitro nematicidal activity screening.

For the first screening bacterial isolates were cultured in brain heart infusion (BHI) broth (BHIB; Conda S.A., Madrid, Spain) at 28°C for 2 days agitating at 200 rpm in a shaking incubator. The bacterial cultures were diluted with SDW to concentrations of 2% and 1%, and then 100 μL of the diluted bacterial culture was transferred to each well of a 96-well Microtest™ Tissue Culture Plate (Becton Dickinson Labware, Franklin Lakes, NJ, USA) with three replications, into which 50-70 M. hapla J2 were placed. After 24 h of exposure, M. hapla J2 were examined under a low power stereoscopic microscope, and nematodes were considered dead if they were straight, stiffened, and did not move when probed with a fine needle, compared to flexible and responsive (to the fine needle touch) nematodes that were considered alive (Cayrol et al., 1989).

For the second screening, the bacterial isolates selected in the first screening (based on their high nematicidal activities) were screened again for nematicidal activity using the bacterial cell suspensions without culture filtrate (culture filtrate-free bacterial cell suspensions) that may contain nematicidal compounds produced during culture. For this, the bacterial cultures grown in BHIB at 28°C for 48 h in a shaking incubator (at 200 rpm) were centrifuged at 5,162 × g to collect bacterial cells in a pellet by removing supernatant from bacterial cultures. The collected bacterial cells in the pellet were suspended in SDW and diluted to the proper concentrations, for which their colony-forming units (CFU) were examined through the dilution plating method (Dhingra and Sinclair, 1985). Nematicidal activity of the bacterial suspensions was examined using 100 μL of the bacterial suspensions with concentrations of 1×10⁶ CFU/mL and 1×10⁸ CFU/mL using the same methods mentioned in the first screening of bacterial cultures for nematicidal activity.

One bacterium (isolate C1-7) out of 542 bacterial isolates was selected in the first and second screening tests as a potential candidate for the biological control of M. hapla. The bacterium C1-7 was identified based on colony morphology, gram staining, carbon source assimilation, and 16S rRNA gene sequencing analysis. Colony morphology was examined by visualizing the colonies formed after 3 days of culturing on BHI agar. Gram staining was conducted following the method previously described by Schaad et al. (2001). Bacterial morphology was examined under an energy-filtering transmission electron microscope (Libra 120, Carl Zeiss, Oberkochen, Germany) operated at an accelerating voltage of 120 kV following negative staining with 2% phosphotungstic acid (PTA). Carbon source assimilation of the bacterial isolate was examined with a Biolog GN test kit (Biolog Inc., Hayward, CA, USA). For 16S rRNA gene sequencing analysis, chromosomal DNA was extracted from the 2-day-old bacterial colony on BHI agar using a FastDNA spin kit (MP Biomedicals, Santa Ana, CA, USA). In addition a polymerase chain reaction (PCR) was performed using universal primers 27mF (5′-GAGTTTGATCMTG GCTCAG-3′) and 1492mR (5′-GGYTACCTTGTTACG ACTT-3′) in a MyCycler Thermal Cycler system (Bio-Rad, Hemel Hempstead, Herts, UK) for the amplification of the 16S rRNA gene (Yoon et al., 1998). The amplified PCR products were purified using a Wizard SV Gel and PCR cleanup system (Promega, Madison, WI, USA), and were then used to analyze for 16S rRNA gene sequences with an ABI PRISM BigDye™ Terminator Cycle Sequencing Kit (Applied Biosystems, Foster City, CA, USA). Results were compared with ribosomal DNA sequences registered in GenBank using a BLASTN program for bacterial identification (Thompson et al., 1994).

To determine the optimal population density and culture media for the most efficient application of isolate C1-7, growth curves were examined every 6 h for 114 h after inoculation on four different media including nutrient broth (NB; Conda S.A.), BHIB, Luria-Bertani (LB) broth (LBB; Difco, Detroit, MI, USA), and Bacto™ tryptic soy (TS) broth (TSB; Becton, Dickinson and Company, Le Pont de Claix Cedex, France). For this step, the numbers of CFU were examined by the dilution plating method (Dhingra and Sinclair, 1985).

Four different culture media (NB, BHIB, LBB, and TSB) were used to examine bacterial growths and to test for the nematicidal activity of C1-7. One milliliter of bacterial solution was taken from the bacterial culture at intervals of 6 h after the initial inoculation. Samples were diluted with SDW to various concentrations (0.5%, 1.0%, 2.0%, and 5.0%), and then 100 μL of diluted bacterial solution was transferred to each well of a 96-well Cell Culture Plate (F-Type; SPL Life Sciences, Pocheon-si, Gyeonggi-do, Korea) into which 50-70 J2 of M. hapla were placed. After 24 h of exposure, dead and living M. hapla J2 were counted under a low-power stereoscopic microscope as mentioned above in the in vitro screening.

The culture filtrate of isolate C1-7 was also examined for nematicidal activity. The bacterial isolate was grown in BHIB at 28°C in a shaking incubator (200 rpm) for 18 h (just after the end of the exponential growth phase) and 48 h (in stationary phase). The bacterial cultures were centrifuged at 5,162×g to separate the culture supernatant from the bacterial cells condensed in pellet, and the culture supernatant was filtered through a sterilized 0.22-μL Millex Filter Unit syringe (Millipore, Bedford, MA, USA). The culture filtrate was subjected to three heat treatment conditions including a no-heat treatment, heat treatment at 80°C for 30 min, and heat treatment at 121°C for 20 min. Then, each sterilized culture filtrate was diluted with SDW to various concentrations, and 100 μL of the diluted culture filtrate was transferred to each well of a 96-well Cell Culture Plate (F-Type) into which 50-70 M. hapla J2 were placed. After each 24 h and 48 h of exposure, J2 mortality was examined as above.

C1-7 was tested for biological control of M. hapla on host plants, carrot (Daucus carota subsp. sativus) cv. Shinheukjeon-Ochon and tomato, both of which are highly susceptible to the RKNs (Bélair, 1984; Hartman and Sasser, 1985). The carrot is a representative host crop of M. hapla, and control is urgently required for crop cultivation. The tomato cv. Rutgers is also susceptible to M. hapla and other Meloidogyne species (Hartman and Sasser, 1985), and was used for comparison in our study. Four-week-old seedlings were planted in 8-cm-diameter plastic pots that were filled with a sterilized sand-bed soil (1:1) mixture, and each plant was inoculated with M. hapla by dispending 5 mL of the nematode suspension containing 200 J2/mL per pot. For the biological control of the nematode, 10 mL of C1-7 culture grown in BHIB at 28°C in a shaking incubator (200 rpm) for 3 days and diluted with SDW to the concentration of 1×10⁸ CFU/mL was poured around the plant rhizosphere at the same time as nematode inoculation. Plants with neither nematode inoculation nor bacterial treatment served as controls. Fresh BHIB (10 mL; diluted as much as the bacterial culture) was poured around the plant rhizosphere to examine the effect of the medium on nematode control. Each treatment was replicated five times and the pot experiments were conducted at 25±2°C under greenhouse conditions. Plants were watered sufficiently every other day. Four weeks after treatments, plants were carefully uprooted, and root systems were gently washed with running tap water to remove adhered soil. The severity of root galling on plants infected with M. hapla was assessed on a 0-5 rating scale according to the percentage of galled tissues, in which a rating of 0 = 0-10% galled roots; 1 = 11-20%, 2 = 21-50%, 3 = 51-80%, 4 = 81-90%, and 5 = 91-100% (Barker, 1985). Egg masses of M. hapla were handpicked from infected roots using forceps, and an egg mass index was assigned to each count using a rating scale of 0 = no egg mass, 1 = 1-3 egg masses, 2 = 4-10 egg masses, 3 = 11-30 egg masses, 4 = 31-100 egg masses, 5 = >100 egg masses per root system (Roberts et al., 1990).

Root segments with various degrees of galling were cut off from carrot and tomato roots inoculated with M. hapla, both with and without the bacterial treatment. The root segments were fixed in Karnovsky's fixative consisting of 2% glutaraldehyde and 2% paraformaldehyde in 0.05 M cacodylate buffer (pH 7.2) for 48 h at 4°C (Karnovsky, 1965). The fixed specimens were washed in 0.05 M cacodylate buffer (pH 7.2) at 4°C three times each for 10 min. The specimens were then post-fixed in 1% osmium tetroxide and 0.05 M cacodylate buffer for 1.5 h at 4°C before brief rinsing with distilled water. After en bloc staining in 0.5% uranyl acetate for 30 min at 4°C, the specimens were dehydrated in an ethanol series of 30%, 50%, 70%, 80%, and 90% each for 10 min, and finally three times in 100% ethanol for 10 min each. For further dehydration, the specimens were treated with propylene oxide at room temperature two times each for 15 min. The specimens were infiltrated with a 2:1 mixture of propylene oxide and Spurr's epoxy resin for 4 h, then a 1:1 mixture overnight, followed by 100% Spurr's epoxy resin under a vacuum for 4 h in a desiccator. The specimens were embedded in fresh Spurr’s epoxy resin in a dental mold, which was placed at 70°C for 8 h in a dry-oven for polymerization. The specimens embedded in the resin were cut to slices of 1,000 nm thickness with a glass knife on an ultramicrotome (MT-X, RMC, Tucson, AZ, USA). The sections were stained using decuple diluted toluidine blue working solution consisting of 1% toluidine blue in 2% sodium borax, and observed under a compound light microscope (Axiophot, Carl Zeiss, Oberkochen, Germany).

Under in vitro conditions, the bacterial cultures of a total of 542 bacterial isolates grown in BHIB for 48 h were diluted to 1.0% and 2.0%, and screened for nematicidal activity against M. hapla. Among these bacterial cultures, 23 bacterial isolates had a high nematicidal activity, providing near to 100% mortality at 2.0% concentration (except for two isolates: S-2 and S-4) and was still effective at 1.0% bacterial isolate (except for three isolates: S-2, S-4, and S-7; Fig. 1A). In the second in vitro screening experiment with culture filtrate-free bacterial cell suspensions at concentrations of 1×10⁶ CFU/mL and 1×10⁸ CFU/mL, all isolates except for C4-19 and S-5 showed 100% nematode mortality at a concentration of 1×10⁸ CFU/mL; however, only three isolates (C1-7, S-2, and S-4) provided 100% mortality at 1×10⁶ CFU/mL (Fig. 1B), indicating that isolate C1-7 had the highest nematicidal activity of 100% morality at the lowest concentrations of both the bacterial culture and suspension .

C1-7 formed large circular, cream-colored, flat colonies with an undulate margin after 3 days culture on BHI agar (Fig. 2A), and it was gram-positive, rod-shaped, and had more than four peritrichous flagella (Fig. 2B), showing the most similarities with the genus Bacillus (Schaad et al., 2001). The carbon source assimilation of the bacterial isolate examined using a Biolog assay revealed the utilization of 15 carbon sources including glycerol, but not 34 other carbon sources including mannitol, revealing the highest similarity (92.6%) with Bacillus cereus (data not shown). The analysis of 16S rRNA gene sequences for C1-7 revealed a maximum similarity (100%) to those of B. cereus strains listed in GenBank with accession numbers KC113613.1, JX848326.1, JX994144.1, and JX994107.1 (data not shown), including an E-value of 0.0, confirming the bacterium’s identity as B. cereus.

For all media examined, the lag phase observed in population growth was hardly noted in the culture of the bacterial isolate C1-7; however, its population increased rapidly up to 18 h after inoculation in all media examined, revealing an exponential phase with populations reaching concentrations of 1×10⁸ CFU/mL ~1×10⁹ CFU/mL (Fig. 3). Population growth rates were differentiated after the exponential phase depending on the media, revealing a stationary phase from 36 h through 60 h after inoculation (or to the end of culturing with no death phase) in all but NB, maintaining a bacterial density of approximately 0.5×10⁹ CFU/mL in BHIB and approximately 1.0×10⁸ CFU/mL in LBB and TSB. In NB, the bacterial population decreased significantly after the exponential growth phase, with no noticeable duration of the stationary phase, to a final population of less than 1.0×10⁶ CFU/mL.

No nematicidal activity was observed for the bacterial cultures at all concentrations of NB and at the concentration of 0.5% of all culture media used in this study (Table 1). Only 5.0% of the bacterial cultures in LBB and 2.0% and 5.0% in TSB showed sufficiently high nematicidal activity throughout the culturing period, while all concentrations except for 0.5% in BHIB showed high nematicidal activity throughout the culturing period, indicating that BHIB is the optimal medium for the growth of C1-7 for the nematicidal activity.

The bacterial culture filtrate displayed much higher nematicidal activity against M. hapla than either BHI or the control (SDW), regardless of culture time, heating temperature, and time exposed to heating (Table 2). The culture filtrate of the bacterial isolate using an 18-h culture time showed no significant difference in nematicidal activity against M. hapla J2 between the exposure times of 24 h and 48 h, regardless of heating temperatures, displaying nearly total nematode mortality at concentrations of 2% and greater. However, the nematicidal activity of the bacterial culture filtrate using a 48-h culture time was higher using an exposure time of 48 h compared to one of 24 h. Total nematode mortality was observed at a concentration of 1% using a treatment time of 48 h, but not with a treatment time of 24 h, for both of which no significant differences in nematicidal activity were detected among the heating temperatures (Table 2).

In the pot experiments under greenhouse conditions, root-knot galls and egg masses formed on carrot and tomato plants inoculated with M. hapla were significantly suppressed by treatment with the C1-7 bacterial culture (1×10⁸ CFU/mL), illustrating that the size of root-knot galls and the number of root-knot galls and egg masses significantly decreased in the C1-7 treatment compared to the nontreatment control (Table 3, Fig. 4). For root-knot gall formation, complete suppression happened in tomato, while for egg mass formation, complete suppression occurred only in carrot; however, 90% suppression of root galling was observed in carrot and 75% suppression of egg mass formation was observed in tomato (Table 3). BHIB treatment also reduced root galling and egg mass formation, especially significantly egg mass formation in tomato; however, its control efficacy was far below that of the bacterial culture.

In the control plants with no bacterial treatment, numerous large giant cells were formed in carrot and tomato roots infected with M. hapla, which is characterized by cellular hypertrophy with the formation of multiple nuclei and sometimes cell wall ingrowths (Fig. 5). The proliferation of gall cells was noted around the giant cells formed in the roots with no treatment control. Giant cells were also formed in the root tissues infected with the nematode and treated with BHIB and the bacterial culture; however, they were small in size with no cell wall ingrowths and often appeared degenerative with vacuolar cytoplasm and electron-dense materials (presumed to be chromatin materials) scattered in the giant cells (Fig. 5C, D, G). No giant cell formation was found in the nematode-infected tomato root tissues treated with the bacterial culture (Fig. 5H). Gall cells proliferated in BHIB to the same extent as the untreated control, but did not proliferate with the bacterial treatment, illustrating that giant cell formation was confined in the stele (Fig. 5D).