Applied Microbiology and Biotechnology

, Volume 102, Issue 7, pp 3301–3314 | Cite as

Nematicidal protease genes screened from a soil metagenomic library to control Radopholus similis mediated by Pseudomonas fluorescens pf36

  • Deqiang Chen
  • Dongwei Wang
  • Chunling Xu
  • Chun Chen
  • Junyi Li
  • Wenjia Wu
  • Xin Huang
  • Hui Xie
Applied genetics and molecular biotechnology


Controlling Radopholus similis, an important phytopathogenic nematode, is a challenge worldwide. Herein, we constructed a metagenomic fosmid library from the rhizosphere soil of banana plants, and six clones with protease activity were obtained by functionally screening the library. Furthermore, subclones were constructed using the six clones, and three protease genes with nematicidal activity were identified: pase1, pase4, and pase6. The pase4 gene was successfully cloned and expressed, demonstrating that the protease PASE4 could effectively degrade R. similis tissues and result in nematode death. Additionally, we isolated a predominant R. similis-associated bacterium, Pseudomonas fluorescens (pf36), from 10 R. similis populations with different hosts. The pase4 gene was successfully introduced into the pf36 strain by vector transformation and conjugative transposition, and two genetically modified strains were obtained: p4MCS-pf36 and p4Tn5-pf36. p4MCS-pf36 had significantly higher protease expression and nematicidal activity (p < 0.05) than p4Tn5-pf36 in a microtiter plate assay, whereas p4Tn5-pf36 was superior to p4MCS-pf36 in terms of genetic stability and controlling R. similis in growth pot tests. This study confirmed that R. similis is inhibited by the associated bacterium pf36-mediated expression of nematicidal proteases. Herein, a novel approach is provided for the study and development of efficient, environmentally friendly, and sustainable biocontrol techniques against phytonematodes.


Metagenomic library Protease gene Radopholus similis Nematode-associated bacteria Pseudomonas fluorescens Biological control 



This study was funded by the National Foundation of Natural Science of China (grant number 30671366 and 31071665).

Compliance with ethical standards

Conflict of interest

The authors declare that they have no conflict of interest.

Ethical approval

This article does not contain any studies with human participants or animals performed by any of the authors.

Supplementary material

253_2018_8869_MOESM1_ESM.pdf (652 kb)
ESM 1 (PDF 651 kb)


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Copyright information

© Springer-Verlag GmbH Germany, part of Springer Nature 2018

Authors and Affiliations

  1. 1.Laboratory of Plant Nematology and Research Center of Nematodes of Plant Quarantine, Department of Plant Pathology, College of AgricultureSouth China Agricultural UniversityGuangzhouPeople’s Republic of China
  2. 2.Guangdong Province Key Laboratory of Microbial Signals and Disease Control, College of AgricultureSouth China Agricultural UniversityGuangzhouPeople’s Republic of China

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