Suppressing a plant-parasitic nematode with fungivorous behavior by fungal transformation of a Bt cry gene

Chihang Cheng,Yongjun Wang,Jialing Qin,Choufei Wu,Mengying Lei,Liqin Zhang

doi:10.1186/s12934-018-0960-5

Chihang Cheng, Yongjun Wang + Show 4 more

Open Access

https://doi.org/10.1186/s12934-018-0960-5

Copy DOI

Abstract

BackgroundPine wilt disease, caused by the pinewood nematode Bursaphelenchus xylophilus (PWN), is an important destructive disease of pine forests worldwide. In addition to behaving as a plant-parasitic nematode that feeds on epithelial cells of pines, this pest relies on fungal associates for completing its life cycle inside pine trees. Manipulating microbial symbionts to block pest transmission has exhibited an exciting prospect in recent years; however, transforming the fungal mutualists to toxin delivery agents for suppressing PWN growth has received little attention.ResultsIn the present study, a nematicidal gene cry5Ba3, originally from a soil Bacillus thuringiensis (Bt) strain, was codon-preferred as cry5Ba3Φ and integrated into the genome of a fungus eaten by PWN, Botrytis cinerea, using Agrobacterium tumefaciens-mediated transformation. Supplementing wild-type B. cinerea extract with that from the cry5Ba3Φ transformant significantly suppressed PWN growth; moreover, the nematodes lost fitness significantly when feeding on the mycelia of the cry5Ba3Φ transformant. N-terminal deletion of Cry5Ba3Φ protein weakened the nematicidal activity more dramatically than did the C-terminal deletion, indicating that domain I (endotoxin-N) plays a more important role in its nematicidal function than domain III (endotoxin-C), which is similar to certain insecticidal Cry proteins.ConclusionsTransformation of Bt nematicidal cry genes in fungi can alter the fungivorous performance of B. xylophilus and reduce nematode fitness. This finding provides a new prospect of developing strategies for breaking the life cycle of this pest in pines and controlling pine wilt disease.

Highlights

Pine wilt disease, caused by the pinewood nematode Bursaphelenchus xylophilus (PWN), is an important destructive disease of pine forests worldwide
Transformation and expression of cry5Ba3Φ in Botrytis cinerea, the diet fungus of PWN in laboratory The plasmid pUC57-cry5Ba3Φ, prepared by wholesequence synthesis, was validated by digestion with the restriction enzymes XhoI and SpeI, resulting in an approximately 2600 bp DNA fragment (PtrpC, cry5Ba3Φ, and TtrpC), which was absent from the gel in case of the plasmid pUC57 (Fig. 1b)
Polymerase chain reaction (PCR) amplification using primers cry-F and cry-R showed that plasmid pTFCM-cry5Ba3Φ was successfully carried by A. tumefaciens AGL-1 (Fig. 1d)

Summary

Introduction

Pine wilt disease, caused by the pinewood nematode Bursaphelenchus xylophilus (PWN), is an important destructive disease of pine forests worldwide. The plant pathogenic pinewood nematode Bursaphelenchus xylophilus (PWN) is in ecological balance with native pine species in North America [13], but it has become an invasive alien species in Japan, South Korea, and China, and spread into Portugal and Spain [4, 14]. This nematode is the major causal agent of pine wilt disease, which has devastated more than one million hectares of pine forests in China [15]. Compounds originally produced by Streptomyces species (such as avermectin, emamectin, milbemycin, and their derivatives) [16,17,18], nematicidal constituents screened from plants [19, 20], and endoparasitic fungi of nematode [21], were found applicable to be biopesticides for controlling PWN

Methods

Results

Discussion

Conclusion