Introduction

The root-knot nematode poses an important threat to various crops, and is distributed widely throughout the world [1]. Chemical nematicides are broadly employed for the control of root-knot nematodes, but are restricted in the field due to environmental concerns and the possibilities of residues on agricultural products. Alternative means for nematode control include the use of plant extracts, nematophagous fungi, secondary metabolites of bacteria, and plant growth-promoting rhizobacteria [2–4]. Root-knot nematodes have already been shown to be effectively controllable via the application of the nematophagous fungus, Paecilomyces lilacinus [5]. Combinations of nematicidial microbial agents, including Bradyrhizobium haponicum, Trichoderma pseudokoningii, and Glomus mossease, have been successfully introduced in the field to control the root-knot nematode [6]. A combination of P. lilacinus and Bacillus frimus has also been successfully employed to destroy the egg mass of the root-knot nematode [7].

The results of recent studies have shown that 2,4-diacetylpholoroglucinol and extracellular protease from P. fluorescens CHA0 [8, 9] as well as hydrogen cyanide from P. aeruginosa PAO1 [10] are major factors in the killing of nematodes. In addition, the chitinase and protease of P. lilacinus were identified as major factors in the inhibition of eggshell development and the hatching of juveniles of the root-knot nematode [11]. The two-component GacS/GacA regulation systems in the antagonists of plant pathogenic nematodes, Pseudomonas fluorescens CHA0 and Pseudomonas sp. BG33R, have been identified as key regulators in the production of microbial determinants involved in killing of phytopathogenic nematodes [8, 12].

Pseudomonas chlororaphis O6 promotes plant growth, induces systemic resistance against various pathogens and drought tolerance, and suppresses the mycelial growth of plant pathogenic fungi. Pyrrolnitrin and phenazines are key reactive factors, [13–16] and the sensor kinase, GacS of P. chlororaphis O6, regulates their production [15]. In this study, we determined that the culture filtrate of P. chlororaphis O6 controls the root-knot nematode. We used mutants to assess the functions of the secondary metabolites. Our results showed that P. chlororaphis O6 mutants, deficient in pyrrolnitrin and phenazine production but proficient in HCN production, induce the mortality of the second juvenile stage (J2) of the root-knot nematode, Meloidogyne hapla. In addition, the GacS/GacA global regulator in P. chlororaphis O6 appears to be a key regulator in the biocontrol of root-knot nematodes.

Materials and Methods

Bacteria Strains and Culture Conditions

The wild-type strain of P. chlororaphis O6, the gacS mutant, and its complemented mutant, a phenazine-deficient mutant (phzA −), and a pyrrolnitrin-deficient mutant (prnA −, unpublished data) were constructed [15, 17] and used in these studies. Strains were grown in liquid King’s medium B (KB) [18] and a medium-enhancing HCN production liquid medium (KB amended with 4.5 g, glycine l−1 at pH 7.2) for 36 h at 28°C with shaking at 200 rpm. Escherichia coli DH5α was cultivated for 36 h at 37°C with shaking at 200 rpm. After cultivation, each of the bacterial suspensions were adjusted to OD600nm = 0.8 with sterile water using a UV–Visible Spectrophotometer (Shimadza, Japan).

Nematode Killing Bioassasy

The second juvenile stage (J2) of the root-knot nematode, M. hapla, was obtained from naturally infested soil at depths of 0–30 cm from the Fruit Experimental Station, in Haenam, Jeonnam, Korea. Plant debris and small gravel were removed from the soil, and the soil was dried for 2 days under shaded conditions. Batches of approximately 30 g of the dried soil were passed through a 1.4-mm sieve (Standard sieve, No. 14), and the juveniles were collected using the Sieve and Baermann funnel technique [19]. The suspensions (5 ml) of juveniles were placed in 15-ml tubes for 30 min to allow for settling. Removal of 4 ml of the top layer yielded a suspension of 120 ± 10 organisms ml−1, and this fraction was used to assess the killing effect of the bacterial preparations under light microscope.

In order to evaluate the effects of the bacterial components on the killing of the juveniles, bacterial strains grown for 36 h in KB or KB containing glycine liquid medium were adjusted to an OD600nm = 0.8 (~1 × 108 cfu ml−1) with sterile water, and then diluted 10-fold or 100-fold to evaluate nematode killing effects of different concentrations of bacterial cultures before mixing with an equal volume of J2-stage nematodes. After 1 h of incubation of the mixture at room temperature, J2 mortality was evaluated under a low-power microscope (Carl Zeiss Discovery V12, Germany) by touching the nematodes with a sharp tip. Nematodes that did not respond with movement were considered to have been killed. Assessment of each preparation involved three replicates examining responses with at least 50 nematodes each.

The cultures were separated into cell-free supernatants and cells by 15 min of centrifugation at 7000 rpm. The supernatant was evaluated for its effect on the J2 phase nematodes with and without boiling for 5 min at 100°C. The cells were then examined at a density of OD600nm = 1.4, and three independent studies were conducted.

Greenhouse Bioassay Using Tomato

Tomato seeds (Rapsody, Syngenta seed, Korea) were planted in square pots (115 cm3) with nursery soil (Bio-Sangto, SeminisKorea, Korea) and grown in chambers with 16 h light (2,000 lux, 80 μmol photons m−2 s−1) and 8 h darkness for 5 weeks. The plants were transplanted to pots (9 cm diameter × 8 cm depth) containing 120 g of soil. This soil was obtained from a kiwi fruit farm and was naturally infested with M. hapla (J2 juvenile, 120 ± 10 30 g−1 soil). The bacterial wild type, gacS mutant, and the GacS-complemented strains of P. chlororaphis O6 were grown with shaking at 200 rpm in KB containing glycine liquid medium for 36 h at 28°C. E. coli DH5α was cultivated at 37°C. After adjusting the cell suspensions to OD600nm = 0.8 with sterile water, each pot was drenched with 100 ml of the culture. The nematicide, Fosthiazate GR (active ingredient: 5%, 1,000 m2 6 kg−1) was applied as a positive treatment and the noninoculated medium diluted 10-fold with sterile water was employed as a negative control. The plants were placed in a greenhouse under shaded conditions. After 42 days, the roots of plants were collected, and then the numbers of root-knot galls were counted and root fresh weights were recorded. Each treatment was conducted with three replicates per treatment with 50 tomato plants each treatment−1.

Data Analysis

Data were analyzed by ANOVA using SPSS 12.0 K for Windows software (SPSS institute, NC, USA) The significance of the effect of bacterial treatment was evaluated by Duncan’s multiple range test (P < 0.05).

Results

Nematicidal Activities of P. chlororaphis O6

Cultures of P. chlororaphis O6 reduced the live populations of J2 root-knot nematodes by 95%; the juveniles no longer exhibited motion after 60 min of treatment (Fig. 1a). Cultures of the wild type, the gacS mutant, and the complemented mutant strains grown on KB evidenced similar levels of mortality, where the cultures of E. coli had little effect (Fig. 1b). When KB medium containing glycine was employed as the growth medium, mortality induced by the wild-type culture was increased, to approximately 95%. Mortality caused by the gacS mutant was decreased, and that caused by the complemented-gacS strain remained similar to the results seen with the KB medium (Fig. 1c). E. coli-induced motality was elevated by approximately 30%. Mortality percentages of 10-fold diluted cultures of P. chlororaphis O6 wild type, the gacS mutant, and the complemented-gacS mutant were range of 10–30% (data not shown).

Fig. 1
figure 1

Effect of bacterial cultures of P. chlororaphis O6, the gacS mutant, and the complemented-GacS mutant strain on the killing of the second juveniles of the root-knot nematode. a Dead and live second juveniles of the root-knot nematode, M. hapla, after 60 min incubation with bacterial cultures. Two different growth media, KB medium (b) and KB medium containing glycine (c), were used to evaluate the effects of bacterial cultures on the mortality of the root-knot nematode. The data shown are for bacterial cultures adjusted to OD600nm = 0.8 with sterile water. Pc O6: wild-type P. chlororaphis O6, Pc GacS: gacS mutant, Pc ComGacS: complemented-gacS mutant, E. coli: E. coli DH5α. As a control, juveniles were treated with 10-fold diluted KB. The data are expressed as the means ± standard deviation of three replicates for mortality after treatment with cultures as compared with the controls (mortality 0%). Different letters indicate a statistically significant difference (P < 0.05) according to the results of Duncan’s multiple test

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Nematicidal Compounds in Culture Filtrate

The separation of culture into cells and supernatants demonstrated that the cells evidenced no nematicidal activity (Fig. 2a). However, the culture filtrate, even when heat treated by boiling, was nematicidal to the same level as when cells were present. The use of mutants lacking in phenazine or pyrrolnitrin production demonstrated that these secondary products were not involved in killing. The mortality of the J2 juveniles when treated with cultures lacking phenazines, with a phzA mutant, or pyrollnitrin, with the prnA mutant, was between 91 and 100% (Fig. 2b). Nematicidal activity was eliminated by dilutions of the culture filtrates from the wild type, the gacS mutant, and the gacS-complemented strain of P. chlororaphis O6 from glycine amended KB cultures a 10- or 100-fold dilutions (data not shown).

Fig. 2
figure 2

Effect of bacterial cultures or cell-free culture filtrates of P. chlororaphis O6 wild type and mutant strains lacking production of secondary metabolites on the killing of the root-knot nematodes. Pc O6: P. chlororaphis O6, O6-cell: cell culture of P. chlororaphis O6. O6-CF: culture filtrate from P. chlororaphis O6, CF-Boil: culture filtrate of P. chlororaphis O6 boiled for 5 min at 100°C. Pc PhzA−: lacked phenazine biosynthesis in P. chlororaphis O6, Pc PrnA−: lacked pyrrolnitrin biosynthesis in P. chlororaphis O6; each preparation was adjusted to OD600nm = 0.8 with sterile water before application. Control: King’s B liquid medium autoclaved and diluted to 10-fold with sterile water. The data are expressed as the means ± standard deviation of three replicates. Different letters indicate a statistically significant difference (P < 0.05) according to the results of Duncan’s multiple test

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Greenhouse Evaluation of P. chlororaphis O6 Cultures

The growth of tomato plants in root-knot nematode-infested soil caused extensive galls in the roots (Table 1). Fosthiazate treatment reduced the formation of root-knot galls dramatically. Significant reductions were noted with cultures of the wild-type strain and the complemented-GacS mutant strain. However, treatment with cultures of the gacS mutant reduced the levels of root-knot gall formation to the same extent as was observed with E. coli cultures, but to a lesser reduction extent than that of wild type. When fresh weights of the roots were examined, the highest mass was observed from the plants treated with the wild-type cultures (Table 1).

Table 1 Effect of bacterial cultures of P. chlororaphis O6 strains on formation of root-knot and root fresh weight in tomato plant
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Discussion

This study demonstrated that P. chlororaphis O6 when grown in liquid culture produces secondary metabolites that caused mortality of the J2 stage of root-knot nematode, M. hapla, and reduced significantly root-knot nematode symptoms in roots of greenhouse-grown tomatoes. The effective factors causing mortality were secreted into the growth medium and were stable to boiling in the culture medium. Mutations in gacS reduced the level of control in the greenhouse assay with root-knot nematode compared with the wild-type and the complemented mutant treatments. GacS in conjunction with GacA regulates many aspects of secondary metabolism in P. chlororaphis O6 and other pseudomonads [20]. One GacS-dependent product is hydrogen cyanide (HCN) and this factor has been correlated with the biological control of plant diseases [21] and HCN produced by P. aeruginosa PAO1 killed nematodes directly [10]. Glycine is a precursor to HCN production [22]. The P. chlororaphis O6 also produces HCN [15]. Thus, it is estimated that the HCN produced by P. chlororaphis O6 might affect the killing J2 of M. hapla, and HCN liquid medium evidenced higher mortality rates than KB liquid medium. Indeed, we determined that the presence of glycine in KB medium improved the extent of mortality of the juvenile nematodes for the wild-type strain.

However, our results showing that the GacS mutant strain also exhibited inhibitory activity is at odds with our finding that a mutation in gacS impaired HCN production in P. chlororaphis O6 [15]. Thus, we can conclude that P. chlororaphis O6 produces additional nematicidal factors. We have eliminated via appropriate methods, any roles of phenazines and pyrrolnitrin in nematicidal activity. However, other pseudomonad secondary compounds have been implicated in this process. These include the demonstrated suppression of the J2 phases of M. javanica by 2,4-diacetylphloroglycinol and pyoluteorin from P. fluorescens CHA0 [9]. In addition to antibiotics, a major extracellular EDTA-sensitive protease, which is regulated by the GacS/GacA signal transduction pathway, was reported as a biocontrol factor against the root-knot nematode [8]. Antibiotics and chitinase activities were found to be pivotal in reducing the egg hatching of M. incognita by Paenibacillus illinoisensis KJA-424 [23], and the protease and chitinase of Paecilomyces lilacinus were effective with juveniles of M. javanica [11]. The effective factors causing mortality were secreted into the growth medium and were stable until boiling in the culture medium. The cells were found to be inactive on the in vitro bioassay. Whether such products are involved with P. chlororaphis O6 has to be determined in future studies.

Our observation that the bacterium was effective in reducing root-knot symptoms when used to drench tomato plants raises the question as to whether a more effective culture medium might be derived. Biocontrol with a cyanobacterium, Oscillatoria chlorine, against M. arenaria root-knot formation demonstrated that a high application rate and early timing were critical factors in effective suppression [24]. The effect of timing of P. chlororaphis O6 applications should also be taken into consideration. In addition, in the field, a combination of biocontrol agents with chitin reduced nematode infection more than the effects of single biocontrol treatments [5]. Chitin would function as a selective nutrient base for certain biocontrol agents and would promote the production of chitinases that function as biocontrol effectors. Thus, the integration of P. chlororaphis O6, which cannot grow alone on chitin, with chitin-utilizing biocontrol agents is a matter of great future interest.

In summary, bacterial culture filtrates of P. chlororaphis O6 evidenced strong nematicidal activity. In P. chlororaphis O6, phenazine and pyrrolnitrin production proved not to be a major factor, but the formation of hydrogen cyanide from the glycine added to culture medium was an important factor in this nematicidal activity. P. chlororaphis O6 produces secondary metabolites that induced mortality in the J2 stage of the root-knot nematode, M. hapla, and significantly reduced root-knot nematode symptoms in the roots of greenhouse-grown tomatoes. The results of our study showed that P. chlororaphis O6 could be employed as a biocontrol agent for the control of root-knot nematodes, and the global regulator, GacS, functions as a positive regulator of the expression of nematicidal compounds and enzymes in P. chlororaphis O6.