Artificial induction of third-stage dispersal juveniles of Bursaphelenchus xylophilus using newly established inbred lines.

Suguru E Tanaka,Kenji Fukuda,Natsumi Kanzaki,Takuya Aikawa,Yuko Takeuchi-Kaneko,Mette Vestergård

doi:10.1371/journal.pone.0187127

Abstract

The pine wood nematode, Bursaphelenchus xylophilus, is the causal agent of pine wilt disease. This nematode has two developmental forms in its life cycle; i.e., the propagative and dispersal forms. The former is the form that builds up its population inside the host pine. The latter is specialized for transport by the vector. This form is separated into two dispersal stages (third and fourth); the third-stage dispersal juvenile (JIII) is specialized for survival under unfavorable conditions, whereas the fourth-stage juvenile (JIV), which is induced by a chemical signal from the carrier Monochamus beetle, is transported to new host pines and invades them. Because of its importance in the disease cycle, molecular and chemical aspects of the JIV have been investigated, while the mechanism of JIII induction has not been sufficiently investigated. In an effort to clarify the JIII induction process, we established inbred lines of B. xylophilus and compared their biological features. We found that the total number of nematodes (propagation proportion) was negatively correlated with the JIII emergence proportion, likely because nematode development was arrested at JIII; i.e., they could not develop to adults via the reproductive stage. In addition, JIII induction seemed to be regulated by a small number of genes because the JIII induction proportion varied among inbred lines despite the high homozygosity of the parental line. We also demonstrated that JIII can be artificially induced by the nematode’s secreted substances. This is the first report of artificial induction of JIII in B. xylophilus. The dauer (dispersal) juvenile of the model organism Caenorhabditis elegans corresponds functionally to JIII of B. xylophilus, and this stage is known to be induced by a chemical signal referred to as daumone, derived from the nematodes’ secretion. The artificial induction of JIII suggests the presence of daumone-like material in B. xylophilus.

Highlights

Nematodes of the genus Bursaphelenchus have various types of associations with plants [1,2,3] and insects [4,5,6,7]
The total number of nematodes in lines ST4 and ST5 was significantly higher than in the other six lines, while the JIII formation proportion of ST1 and ST2 was significantly higher than in the other lines, and that of ST4 and ST5 was significantly lower than the others
This tendency was supported by the significant negative correlation between the total number of nematodes and JIII formation proportion (r2 = 0.50, P < 0.01; Fig 5)

Summary

Introduction

Nematodes of the genus Bursaphelenchus have various types of associations with plants [1,2,3] and insects [4,5,6,7]. The pine wood nematode (PWN), Bursaphelenchus xylophilus, which causes pine wilt disease, is one of the most important forest pathogens [1, 8,9,10,11,12,13] This mycetophagous/plant-parasitic nematode is native to North America [14,15,16], and is spreading through East Asian and European countries [17]. This nematode invades host pine trees through maturation feeding or oviposition wounds caused by the vector Monochamus longhorn beetle [18, 19], propagates enormously, and kills the host pine [12]. Adult beetles that have loaded nematodes in their tracheal system emerge from the dead pine tree, and spread the disease to healthy pine trees (Fig 1) [20, 21]

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Discussion

Conclusion