The Microbiome of Endophytic, Wood Colonizing Bacteria from Pine Trees as Affected by Pine Wilt Disease

Diogo Neves Proença,Gisle Vestergaard,Romeu Francisco,Michael Schloter,Susanne Kublik,Paula V Morais,Anne Schöler

doi:10.1038/s41598-017-04141-6

Diogo Neves Proença, Gisle Vestergaard + Show 5 more

Open Access

https://doi.org/10.1038/s41598-017-04141-6

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Abstract

Pine wilt disease (PWD) is a devastating forest disease present worldwide. In this study we analyzed the effects of the invasion of the pinewood nematode Bursaphelenchus xylophilus, the major pathogen causing PWD, on the endophytic microbiome of adult P. pinaster trees. Wood samples from trees with different degrees of PWD disease were collected at two sites (A and M) in Portugal. Endophytic bacteria were characterized based on directly extracted DNA by fingerprinting and barcoding using the 16S rRNA gene as marker. Furthermore, cultivation-based approaches were used to obtain isolates of the major taxa to study their ecophysiology. The endophytic microbiome from P. pinaster trees differed significantly between the two sampling sites. Main bacterial OTUs belonged to the Proteobacteria (39% (site M) - 97% (site A)), and Firmicutes (0.70% (site A) - 44% (site M)). However, consequences of the invasion with the pathogen were comparable. Interestingly diversity of wood endophytic bacteria increased with the severity of the diseases, with highest diversity levels observed in in the most affected trees. Our results suggest that in the first stages of the disease, the defence mechanisms of plants are repressed by the pathogen, resulting in a colonization of the wood interior by soil microorganisms.

Highlights

A relationship of mutuality has been suggested between bacteria and B. xylophilus[7] in the development of the disease
Taking in consideration that the pinewood nematode produces effector proteins, as cellulases and peroxiredoxin[4], that modulate the plant response and might reduce the plant defenses, we postulated that the infection with B. xylophilus of pine trees will change the structure of bacterial endophytes
The overall diversity of wood colonizing bacterial endophytes was assessed from 43 P. pinaster trees, with differing severity of Pine wilt disease (PWD) as well as unaffected healthy trees from two regions in Portugal (Table S1)

Summary

Pine Trees as Affected by Pine Wilt

Diogo Neves Proença 1, Romeu Francisco[1], Susanne Kublik[2], Anne Schöler[2], Gisle Vestergaard[2], Michael Schloter2 & Paula V. In this study we analyzed the effects of the invasion of the pinewood nematode Bursaphelenchus xylophilus, the major pathogen causing PWD, on the endophytic microbiome of adult P. pinaster trees. Taking in consideration that the pinewood nematode produces effector proteins, as cellulases and peroxiredoxin[4], that modulate the plant response and might reduce the plant defenses, we postulated that the infection with B. xylophilus of pine trees will change the structure of bacterial endophytes. We hypothesize that the diversity of endophytes will increase mainly in heavily infected trees, due to reduced plant defense mechanisms against entering the plant interior This might overall accelerate the course of the disease. We used molecular fingerprinting and barcoding methods to describe bacterial diversity based on extracted community DNA and amplified 16S rRNA genes, as well as isolation based approaches to assess the phenotypes of major (cultivable) wood colonizing endophytes

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