Pathogenicity of Aseptic Bursaphelenchus xylophilus

Li-Hua Zhu,Sapna Negi,Jianren Ye,Zhang-Li Wang,Xu-Ling Xu,Jin-Yi Ji,Martin Heil

doi:10.1371/journal.pone.0038095

Abstract

Pine wilt is a disease of pine (Pinus spp.) caused by the pine wood nematode (PWN), Bursaphelenchus xylophilus. However, the pathogenic mechanism of pine wilt disease (PWD) remains unclear. Although the PWN was thought to be the only pathogenic agent associated with this disease, a potential role for bacterial symbionts in the disease process was recently proposed. Studies have indicated that aseptic PWNs do not cause PWD in aseptic pine trees, while PWNs associated with bacteria cause wilting symptoms. To investigate the pathogenicity of the PWN and its associated bacteria, 3-month-old microcuttings derived from certain clones of Pinus densiflora Siebold & Zucc. produced in vitro were inoculated under aseptic conditions with aseptic PWNs, non-aseptic PWNs and bacteria isolated from the nematodes. Six-month-old aseptic P. densiflora microcuttings and 7-month-old P. massoniana seedlings were also inoculated under aseptic conditions with aseptic PWNs and non-aseptic PWNs. The results showed that the aseptic microcuttings and seedlings inoculated with aseptic PWNs or non-aseptic PWNs wilted, while those inoculated with bacterial isolates did not wilt. Nematodes were recovered from wilted microcuttings and seedlings inoculated with aseptic PWNs and non-aseptic PWNs, and the asepsis of nematodes recovered from aseptic PWN-inoculated microcuttings and seedlings was reconfirmed by culturing them in NB liquid medium at 30°C for more than 7 days. Taken together, the results indicate that the asepsis of PWN did not cause the loss of pathogenicity.

Highlights

Pine wilt is a disease of pine (Pinus spp.) caused by the pine wood nematode (PWN), Bursaphelenchus xylophilus (Steiner & Buhrer) Nickle
Zhao et al discovered that inoculation with bacteria alone did not lead to the development of disease symptoms, but a combination of axenic PWNs and bacteria led to disease, while seedlings exhibited none or weak symptoms when inoculated with axenic PWNs or axenic PWNs combined with the non-pathogenic bacterium [20]
Inoculation experiments performed under sterilized conditions [19,20,23] and in the field [20,32] showed that surface sterilized PWNs lose their pathogenicity, Tamura reported that 3-year-old P. densiflora inoculated with aseptic nematodes wilted [33]

Summary

Introduction

Pine wilt is a disease of pine (Pinus spp.) caused by the pine wood nematode (PWN), Bursaphelenchus xylophilus (Steiner & Buhrer) Nickle. The first occurrence of pine wilt disease (PWD) was reported in 1905 in Japan [1], PWN was not identified as the causal agent of the disease until 1971 [2]. PWN was believed to be the only pathogenic agent causing the disease [10,11,12,13,14]. Han et al reported that inoculating aseptic black pine seedlings with aseptic PWNs or bacteria alone did not lead to browning or wilting, but inoculation with aseptic PWNs combined with the bacteria isolated from PWN resulted in the onset of severe symptoms [19]. The results led the authors to propose a new hypothesis that PWD was a complex disease induced by both, PWNs and their associated pathogenic bacteria

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