Kasumi Takeuchi¹ , Naomi Noda¹, Yuichi Katayose², Yoshiyuki Mukai², Hisataka Numa²

¹Plant-Microbe Interactions Research Unit, ²Agrogenomics Research Center

［Abstract］

We have isolated three strains of pseudomonads, namely, Cab57, Os17 and St29 that suppress plant diseases in the rhizosphere that could facilitate effective and ecological applications of biocontrol agents. The strain Cab57 was identified as Pseudomonas protegens, whereas strains Os17 and St29 were found to be the closest to P. protegens although they showed different 16S rRNA pattern. Comparative genome analysis also revealed the biocontrol factors which are important for the plant protection efficacy of the strain Os17.

［Keywords］

Keywords: plant protection, biocontrol strains, comparative genome analysis

［Background］

Pseudomonas protegens and some other root-colonizing pseudomonads classified into the Pseudomonas fluorescens group are effective biocontrol strains, which suppress plant diseases in the rhizosphere. Screening for plant beneficial pseudomonads has led to advances in biocontrol research particularly in effective and ecological application of biocontrol agents. In this study, we aimed to screen biocontrol pseudomonad strains relative to P. protegens in the fields in Japan. We also identified biocontrol factors which are important for the plant protection efficacy through the genomic comparison and characterization of each strain.

［Results and Discussion］

1. Approximately 2,800 fluorescent pseudomonads were obtained from plant roots. Based on PCR analysis, a total of 48 isolates were selected as candidates of 2,4-diacetylphloroglucinol-producing (phlD⁺) strains,. Among them, Cab57 was identified as P. protegens based on 16S rRNA gene analysis (with 100% identity) and whole-genome analysis. The genome is organized into a single circular chromosome with 6.8 Mbp (Table 1).
   
2. Strains Os17 and St29, which organized into a single circular chromosome with 6.9 Mbp and 6.8 Mbp, respectively, were found to belong to the same species. Although these strains were relatively closest to P. protegens, they were also found to be different based on 16S rRNA gene and whole-genome analyses (Table 1).
   
3. The genes associated with Gac/Rsm signal transduction pathway are fully conserved in these strains as reported in P. protegens. Strain Cab57 was found to exhibit typical Gac/Rsm activities and antibiotic production, which were enhanced by knocking-out the retS gene, a sensor kinase acting as an antagonist of GacS (Fig. 1A).
   
4. Strains Cab57 and Os17 showed prominent plant protection efficacy at the same level of strain P. protegens CHA0, whereas strain St29 was less effective (Table 1).
   
5. Comparative genome analysis revealed that the complete rhizoxin analog biosynthesis gene cluster (ca. 79 kb) found in the Os17 genome was absent in the St29 genome. In an rzxB mutant, which lacks the polyketide synthase essential for the production of rhizoxin analogs, the growth inhibition activity against fungal and oomycete pathogens (Fig. 1B) and the plant protection efficacy were attenuated as compared with those of wild-type Os17. These findings suggest that rhizoxin analogs are important biocontrol factors of this strain.

1. Strains Cab57 and Os17 are effective for the application as biocontrol agents.
   
2. These strains are also useful as antibiotic producers.

Table 1. General genomic features and comparison of plant protection efficacy of Pseudomonas protegens CHA0, P. protegens Cab57, Pseudomonas sp. Os17, and Pseudomonas sp. St29.

 

Fig. 1. Antibiotic activities of Pseudomonas strains toward Pythium ultimum. (A) Antibiotic activity of P. protegens Cab57 wild type was enhanced by knocking-out the retS gene. (B) Antibiotic activity of Pseudomonas sp. Os17 wild type was attenuated by knocking-out the rzxB gene.

 

 

[Collaborators]

Kosumi Yamada (University of Tsukuba), Nobutaka Someya (National Agriculture and Food Research Organization)

[Reference]

1. Takeuchi K, Noda N, Someya N (2014) Complete genome sequence of the biocontrol strain Pseudomonas protegens Cab57 discovered in Japan reveals strain-specific diversity of this species PLoS ONE 9 (4):e93683
   
2. Takeuchi K, Noda N, Katayose Y, Mukai Y, Numa H, Yamada K, Someya N (2015) Rhizoxin analogs contribute to the biocontrol activity of newly isolated Pseudomonas strain Molecular Plant-Microbe Interactions 28 (3):333-342
   
3. Patent application #JP-2015-118395 (Japan)