[논문]Hydrogen Cyanide Produced by Pseudomonas chlororaphis O6 Exhibits Nematicidal Activity against Meloidogyne hapla

Kang, Beom Ryong; Anderson, Anne J.; Kim, Young Cheol

doi:10.5423/ppj.oa.06.2017.0115

Hydrogen Cyanide Produced by Pseudomonas chlororaphis O6 Exhibits Nematicidal Activity against Meloidogyne hapla 원문보기

The plant pathology journal, v.34 no.1, 2018년, pp.35 - 43

Kang, Beom Ryong (Institute of Environmentally-Friendly Agriculture, Chonnam National University) , Anderson, Anne J. (Department of Biology, Utah State University) , Kim, Young Cheol (Institute of Environmentally-Friendly Agriculture, Chonnam National University)

Abstract ▼ AI-Helper

Root-knot nematodes (Meloidogyne spp.) are parasites that attack many field crops and orchard trees, and affect both the quantity and quality of the products. A root-colonizing bacterium, Pseudomonas chlororaphis O6, possesses beneficial traits including strong nematicidal activity. To determine the molecular mechanisms involved in the nematicidal activity of P. chlororaphis O6, we constructed two mutants; one lacking hydrogen cyanide production, and a second lacking an insecticidal toxin, FitD. Root drenching with wild-type P. chlororaphis O6 cells caused juvenile mortality in vitro and in planta. Efficacy was not altered in the fitD mutant compared to the wild-type but was reduced in both bioassays for the mutant lacking hydrogen cyanide production. The reduced number of galls on tomato plants caused by the wild-type strain was comparable to that of a standard chemical nematicide. These findings suggest that hydrogen cyanide-producing root colonizers, such as P. chlororaphis O6, could be formulated as "green" nematicides that are compatible with many crops and offer agricultural sustainability.

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문제 정의

The objective of this study was to confirm the protective role of HCN produced by P. chlororaphis O6 and to explore whether a cross-kingdom effect exists for FitD against the root knot nematode, M. hapla. The approach involved engineering mutants of isolate O6 lacking HCN or FitD production to probe the roles of these compounds in second juvenile stage (J2) mortality, which initiate infection of the host plant roots.

제안 방법

The potential fitD or hcn mutants cloned into the pEX18Tc plasmid possessing tetracycline resistance were selected on LB agar plates containing tetracycline and kanamycin. The recombinant plasmids were prepared and used to determine the flanking sequence of the EZ-Tn5 transposon using the KAN-2 FP-1 forward primer and RP-1 reverse primers provided in the EZ::Tn5 [KAN2] transposon kit from Epicentre.
After 2 weeks, the roots of plants were collected, and the numbers of root-knot galls were counted, and root fresh weights were recorded. The study was conducted with three replicates per treatment with three tomato plants per treatment.
, 1997). To confirm the mutation of both genes, PCR products were amplified from both mutants using gene specific primers, and the PCR products were sequenced to determine flanking sequences of EZTn5 insertions in the mutants.

대상 데이터

coli DH5α as a negative control. Approximately 100 J2 juveniles were used for each experiment. Data are expressed as the percent mean nematode survival after microbial treatment compared with the controls.
4). The plant infective J2 juveniles were used as the inoculum and galls were assessed in 14 day-old plants. Under the preventative regime, when nematodes and bacterial preparations were applied at the same time, and application of the nematicide, Fosthiazate, eliminated gall formation.
The wild-type strain of P. chlororaphis O6, mutant strains defective in gacS, hcnA, and fitD, and the complemented gacS and hcnA mutants (Kang et al., 2007; Spencer et al., 2003) were used in the studies. Cells were grown to stationary phase for 36 h at 22℃ with shaking at 200 rpm in liquid King’s medium B (KB) (King et al.
Approximately 500 J2 juveniles of Meloidogyne hapla in 10 ml of sterile water were used per box as inoculum and 10 ml of sterile water was used as a negative control (water). Three replicates were used for each treatment, with three tomato plants each treatment. Data are expressed as the mean and standard deviation of three replicates.
Two pEX18Tc recombinant clones, containing the EZ::Tn5 inserts within the structural genes of fitD and hcnA (Fig. 1), were selected and transferred into P. chlororaphis O6 through tri-parental mating. The marker-exchange mutant of each gene was selected on LB agar medium (Becton Dickinson GmbH, Heidelberg, Germany) supplemented with 5% sucrose and kanamycin in LB agar, as described previously (Miller et al.

데이터처리

Different letters indicate a statistically significant difference (P < 0.05) by Duncan’s multiple test.
The significance of the effect of bacterial treatment was evaluated by Duncan’s multiple range test (P < 0.05).

성능/효과

The finding that both the gacS and the hcnA mutant retained the ability to induce larval mortality, could be explained by a role of the fluorescent siderophore in nematode death. Both these mutants produce the fluorescent siderophore, pyoverdine, which disturbs Fe homeostasis in the host nematode.
The approach involved engineering mutants of isolate O6 lacking HCN or FitD production to probe the roles of these compounds in second juvenile stage (J2) mortality, which initiate infection of the host plant roots. The findings from this in vitro assay were compared to the results of an in vivo assay, in which changes in the number of galls formed on tomato due to root colonization by wild-type O6 cells were compared with the effects of the mutant strains. Another mutant, gacS, which lacks the global Gac/Rsm regulatory system (Anderson et al.
The reduction of juvenile mortality with the hcnA mutant, and restoration of this trait by complementation with the complete hcn operon, suggested a major role of HCN-induced damage for the root knot nematode. The observation that a gacS mutant induced juvenile mortality to a similar extent as the single hcnA mutant under the bioassay conditions indicated that the loss of HCN, rather than other factors in the Gac/Rsm regulatory pathway, was involved in nematode lethality. Similarly, the reduced number of root knot galls in tomato plants when treated with the wildtype or the complemented hcnA mutant, but not with the hcnA mutant, confirmed a major role of HCN in the control of gall formation.
To establish whether the loss of protection against gall formation by the hcn mutant was due to a lack of root colonization, colonization of this mutant was compared with that of the wild-type strain. The same number of cells was recovered from the tomato roots in the wild-type and mutant when assayed at 3, 5 and 7 days after inoculation (Supplementary Fig.

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