Description

Table of contents

• 1) Why are pine trees important?
• 2) Pines are being lost due to pine-wilt disease
• 3) The cause of pine-wilt disease is a tiny nematode
• 4) It is difficult to prevent the spread of B. xylophilus
• 5) RNAi is a powerful weapon to fight against the nematodes
• 6) Delivery of dsRNA to stylet-type nematodes using yeast

1) Why are pine trees important?

Throughout history, the people of Japan have cherished beautiful sightseeing spots such as Matsushima (“ Island of pines ”), Miyajima, and Amanohashidate as “ Japan's three representative scenic spots ” [1].⁠ If you take a look, you will notice that a certain type of tree forms an important background in all these spots: the Japanese pine, or “ Matsu ”.

Pine trees have been widely used as a building material and fuel, and is also important as a raw material for industrial resin. Pine trees are a critical forest component acting as a windbreak to prevent the sea breeze from the sea [2].⁠ Pine trees are resistant to high salt and can even grow in dried soil with poor nutrition, so even though it is no longer a principle building material, planting activities on the coast continue to maintain windbreak forests [3].⁠ However, even though pines form an essential part of our scenery, they are withering on a global scale due to the epidemic called pine-wilt disease [4].

Figure 1 Japan's three representative scenic spots.
From left, Matsushima, Miyajima, and Amanohashidate.
The left photo is offered by Hideaki Tsujii.
The middle photo is from “ http://blue-wind.net/photoimage/224 ”.
The right photo is from “ http://photo53.com/amanohashidate.php ”.

2) Pines are being lost due to pine-wilt disease

In 1905, the first report about pine-wilt disease was issued in Japan [5]⁠. From then, the disease gradually spread, and now this disease is present in all prefectures except Hokkaido [6]⁠. In regions other than Japan, pine-wilt disease has been reported in East Asian countries such as Korea, Taiwan, China, in European countries such as Poland, Spain, Portugal and even in North America including Canada and the USA [7]⁠. As pine trees die from this disease, beautiful landscapes around the world have been greatly damaged. In addition to that, it is estimated that huge economic damage of tens of millions of dollars has been caused [7]⁠. In the forests where pines die, it is known that the forestry phase changes, and evergreen broad-leaved trees take precedence [8].⁠ An increase in evergreen broad-leaved trees reduces the light reaching the forest floor, having harmful effects on herbs and the seedlings responsible for the next generation, which has a serious overall impact on the ecosystem.

Figure 2 The area where pine-wilt disease was confirmed.
Pine-wilt disease is now present in all prefectures except Hokkaido.

3) The cause of pine-wilt disease is a tiny nematode

In 1971, it was clarified that the cause of pine-wilt disease was nematode called Bursaphelenchus xylophilus [9].⁠ B. xylophilus adheres to the spiracles of the Japanese long horn beetle and is delivered to pine branches by it. When the beetle bites a pine branch, B. xylophilus invades the tree trunk through the resin path and reaches the trunk, ingesting pine cells [10]⁠. Through this process, the pine releases defense substances such as volatile terpenes excessively, and bubbles are generated in the tracheid, so that the pine cannot absorb water and this induces death of pines [3].⁠ This is the mechanism of pine-wilt disease.

Figure 3 The life cycle of B. xylophilus.
B. xylophilus are brought from weakened pine to another healthy pine by the long horn beetle. Pine-wilt disease progresses with this cycle.

Figure 4 The flowchart of the mechanism of pine-wilt disease.
Pine cannot absorb water for a while after infected by B. xylophilus.

B. xylophilus feeds by piercing it’s stylet like a straw into the cells of pine, blue stain fungus, or yeast, sucking out their contents. It is known that species with such stylets cover 15% of all nematodes [11]. ⁠Unlike C. elegans, this group of nematodes does not prey on bacteria. It is known that many phytopathogenic nematodes use stylets to feed [11].

Figure 5 The foods of B. xylophilus.
B. xylophilus do not eat E. coli.

4) It is difficult to prevent the spread of B. xylophilus

There are basically three methods commonly used to stop pine-wilt disease. However, all of them have several problems and are not able to achieve their desired results.[6]

• 1. Spraying of insecticide targeting Pinus thunbergii by helicopter
  ---This method targets the beetle, which is a carrier that spreads nematodes. Of course, this method has a potentially harmful effect to human beings and forest creatures, and will inevitably have a huge impact on the ecosystem.
  

  

Figure 6 Spraying insecticide by helicopter.
This method targets the long horn beetle.



• 2. Removal of deadwood to prevent the spread of infection
  ---The longhorn beetle produces eggs in pine trees killed by B. xylophilus. It is known that when longhorn beetles emerge in the following year, nematodes adhere to the young beetles. For this reason, attempts have been made to prevent the spread of infection by disposing of dead pine through incineration. Even in this case, there are places where human beings cannot go, such that not all deadwood can be removed completely, limit the effect of this method for preventing pine-wilt disease.
  

  

Figure 7 Removal of deadwood.
This method also targets the long horn beetle in substance.



• 3. Prevention by trunk injection
  ---In order to combat the nematodes within the pine, a method of injecting pesticide into all the pine trees on a target mountain is performed through the winter. Although this method is effective, it is necessary to perform each year, since if neglected, there are reported cases where the damage actually increases [12]. ⁠Therefore, this method has high economic and physical costs.
  

  

Figure 8 Trunk injection.
This method targets B. xylophilus, but is accompanied by a heavy burden.

5) RNAi is a powerful weapon to fight against the nematodes

In order to develop more effective weapons to fight this nematode we applied feeding RNAi, a strategy which is often used for nematodes such as C. elegans, to B. xylophilus. Using budding yeast as a rich food source, we aimed to create a genetically modified machine that kills nematodes, resulting in a new type of biological pesticide. We first decided to answer the question of whether yeast which kills nematodes can be made, and focused on the practicalities of how to deliver the yeast to nematodes. Regarding our solution to prevent our recombinant yeast from hypothetically leaking into the environment, a powerful solution emerged through our Human Practices activities (Please click here for a link).

RNAi is a gene expression suppression mechanism discovered in 1998 by Fire et al [13]⁠. Within C. elegans incorporating double-stranded RNA (dsRNA), rapid degradation of mRNA having a complementary sequence to that dsRNA is induced. If the target gene is essential for growth, C. elegans dies. C. elegans is a bacterial feeding nematode which can feed on E. coli. Therefore, by preparing E. coli expressing dsRNA as bait, gene expression of C. elegans can be controlled. However, B. xylophilus belongs to the stylet-type of nematode, so we expect that we cannot use E. coli for bait. In order to deliver dsRNA to B. xylophilus it was essential to develop a new dsRNA carrier.

Figure 9 The image of yeast expressing dsRNA.
dsRNA causes RNAi.

6) Delivery of dsRNA to stylet-type nematodes using yeast

B. xylophilus is known to prey on blue stain fungi, so we chose another fungus which can be used experimentally, the budding yeast S. cerevisiae, as a carrier. In addition, although there is no published report, consultation with nematode researchers gave some anecdotal evidence that they might prey on budding yeast. However, when we asked the details, the researcher’s experiences were inconsistent. As of yet, there has been no definitive information on whether or not B. xylophilus could feed on S. cerevisiae.

For the initial step, we set out to prove definitively that B. xylophilus preys on budding yeast. For this purpose, we examined the conditions of B. xylophilus' feeding yeast and established a system of recording live imaging. In this way we succeeded in taking the first video of B. xylophilus' puncturing and sucking out the contents of budding yeast with its stylet. In addition, we introduced a new system for temporarily labeling B. xylophilus that preyed on yeast, and established a method for measuring the phenotype of B. xylophilus incorporating dsRNA.

Figure 10 Delivery of dsRNA.
dsRNA will be took in through their stylets.

Reference

• [1] Nihonsankei, “Nihon-sankei 【 official website 】 The three most scenic spots in Japan.” [Online]. Available: http://nihonsankei.jp/eng/. [Accessed: 21-Oct- 2017].
• [2] Forestry Agency, “Damage of Pine-wood nematodes：Forestry Agency,” 2016. [Online]. Available: http://www.rinya.maff.go.jp/j/hogo/higai/matukui.html. [Accessed: 21-Oct- 2017].
• [3] Kuroda Keiko, “Machanism of pine-wilt disease and characteristics of resistant pine trees,” 2007.
• [4] A. Y. Ryss, O. A. Kulinich, and J. R. Sutherland, “Pine wilt disease: a short review of worldwide research,” For. Stud. China, vol. 13, no. 2, pp. 132–138, Jun. 2011.
• [5] Y. Mamiya, “History of Pine Wilt Disease in Japan 1,” J. Nematol., vol. 20, no. 2, pp. 219–226, 1988.
• [6] Forestry Agency, “The present state of damage of pine-wood nematodes,” 2016. [Online]. Available: http://www.rinya.maff.go.jp/j/hogo/higai/attach/pdf/matukui-1.pdf. [Accessed: 21-Oct-2017].
• [7] D. N. Proença, G. Grass, and P. V Morais, “Understanding pine wilt disease: roles of the pine endophytic bacteria and of the bacteria carried by the disease-causing pinewood nematode.,” Microbiologyopen, vol. 6, no. 2, Apr. 2017.
• [8] Kyoto Association for the Promotion of Traditional Culture of forest, “Danger of Kyoto's three representative mountains,” 2007. [Online]. Available: http://www.kyoto-dentoubunkanomori.jp/topics/img/brochure.pdf. [Accessed: 21-Oct-2017].
• [9] T. Kiyohara and Y. Tokushige, “Inoculation Experiments of a Nematode, Bursaphelenchus sp., onto Pine Trees,” J. JAPANESE For. Soc., 1971.
• [10]C. Vicente, M. Espada, P. Vieira, and M. Mota, “Pine Wilt Disease: a threat to European forestry,” Eur J Plant Pathol, vol. 133, pp. 89–99, 2012.
• [11]Rejendra Singh and Swastik Phulera, “Plant Parasitic Nematodes: The Hidden Enemies of Farmers,” Reserch gate, 2015.
• [12]K. syou Kuroda Keiko, “Lisk of water outage and withering by trunk injection against pine-wilt disease,” 2016.
• [13]A. Fire, S. Xu, M. K. Montgomery, S. A. Kostas, S. E. Driver, and C. C. Mello, “Potent and specific genetic interference by double-stranded RNA in Caenorhabditis elegans,” Nature, vol. 391, no. 6669, pp. 806–811, Feb. 1998.