Team:NOVA LxPortugal/Description

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Pine Wilt Disease

The Pine Wilt Disease (PWD) was first discovered in the 20th century in Japan1. This disease affects pine trees and it is caused by the pinewood nematode (PWN), Bursaphelenchus xylophilus1. The infection of healthy pine trees by this disease requires a complex biological system: the nematode, the insect vector and a susceptible host1. This disease is characterized by the cavitation in pine sapwood and by the collapse of photosynthesis, which result in the browning of the needles and a reduction of the oleoresin flux in the tree. Consequently, the infected trees start to present wilting and ultimately die within a few weeks to few months1. Currently, this disease is widely spread around the globe affecting coniferous forests in several countries in Asia, America and Europe1,2.

Figure 1- The browning of the needles in Pinus pinaster infected with Pine Wilt Disease. (® Instituto de Conservação da Natureza e Florestas)

In Portugal, the appearance of B. xylophilus was first registered in 19993, with an affected area of 510,000 ha of Pinus pinaster forest in the Setúbal peninsula.4 In the last years, the disease has been spreading over the country and new clusters of infections have been identified in other regions of the Portuguese territory, such as Arganil4, Lousã4 and Madeira.5

Bursaphelenchus xylophilus

B. xylophilus, as previously mentioned, is the nematode responsible for PWD. The transmission of PWN between host trees is mediated by insect vectors, the pine sawyer beetles of the genus Monochamus. Monochamus spp. (M. galloprovincialis in Portugal) carry B. xylophilus inside their tracheal system and on their body surfaces. The insect is attracted by healthy trees and releases nematodes during maturation feeding on young pine shoots (primary transmission), which then spread through the tree’s resin canals and vascular system. Due to the emission of volatile compounds, such as monoterpenes, Monochamus is also attracted by dead or dying pine trees for oviposition (secondary transmission). B. xylophilus feeds on the blue stain fungi that develop as a consequence of the insect penetration. Before the adult insect vector emerges, the nematodes are attracted to the pupal chamber and enter the bug’s tracheal system, restarting the cycle by being transported to the next source of food.3,6,7

”Nematode ”Insect"
Figure 2- Bursaphelenchus xylophilus (®Instituto Nacional de Investigação Agrária e Veterinária)
Figure 3-Monochamus galloprovincialis (®Instituto Nacional de Investigação Agrária e Veterinária)

The importance of Pinus pinaster in Portugal

Maritime pine is estimated to cover around 605 thousand hectares of Portugal 8. The interest in pine wood has been increasing over the last few years 9, totalling a value of 4.5 Mm3 in 2019, which represents 3.1 % of Portuguese exports - around 1,876 million of euros - leading to the creation of 57,843 jobs 10. It also represents around 88 % of the forest industries10.
The major economic importance that the pine industry represents in Portugal goes beyond the several products directly obtained from pine trees, such as resin or wrapping boxes 9. These trees also play a major role in combating coastal erosion by stabilizing dunes3 and constitute the largest carbon reservoir in the national forest, absorbing 90.3 Gg of CO2e10.

Pinus pinaster
Figure 4- Pinus pinaster (® Centro Pinus)

How to control PWD?

After the detection of this pathogen in Portugal for the first time in 1999, the first attempts to control the spread of the disease consisted in using border-belt areas. This strategy was unsuccessful, resulting in a spread of the disease towards the Centre-North of Portugal and to the Madeira Island4,5.
Currently, the most used control methods for PWD include the use of traps to capture the insect vector or spraying trees with insecticides to prevent the insect vector’s maturation feeding. Attacked trees are eliminated during the winter period and when the disease affects large areas, clear cut belts can be created around the affected zones to try minimizing the disease dispersion3. Another solution presented to us by the Business Manager of Syngenta Professional Solutions was an endotherapy application machine with biodegradable valves adapted to the maritime pine, with the cost of around 15 euros per pine tree in 2012. According to Syngenta, this solution is still utilized in Japan and South Korea.
According to Professor Manuel Mota, the director of the nematology laboratory at ICAAM (Instituto de Ciências Agrárias e Ambientais Mediterrânicas), between 2018 and 2020 an estimated 6.8 million euros were spent in trapping and felling the pine trees.

Why did our team choose the combat of PWD as the focus of our iGEM project?

The United Nations (UN) has declared 2020 as the International Year of Plant Health (IYPH), highlighting the importance of plants in sustainable development. Climate change and human activities have altered ecosystems and reduced biodiversity, thus creating new niches where pests such as the PWN can thrive. Globalization has also contributed to the spread of these pests around the world, which are often impossible to eradicate once they are established and managing them is time-consuming and expensive11,12.
We intend to honour this UN landmark by first fighting to preserve a plant species that is endogenous to Portugal. We also want to raise awareness of this threat to our pine tree forests, already endangered by the wildfires every year and neglected by private owners. As previously mentioned, PWD constitutes not only an economic issue in Portugal, but also an environmental threat due to the importance of the maritime pine in the Portuguese ecosystems. As the current solutions are not sufficient to avoid the spread of this fatal disease, our team has developed a totally novel approach by combining systems and synthetic biology to engineer a bacterium that can kill the nematode.
It all started because João, our student leader, lives in Setúbal, a beautiful city in Portugal known for its vast forest, which harbours many species of trees, including Pinus Pinaster. João had noticed that in Setúbal the maritime pines had become more and more affected by the Pine Wilt disease, so when he heard about iGEM he thought it was the perfect opportunity to find a way to cure and prevent the spreading of the disease. It actually came as a surprise when we found out that Setúbal was the place where the nematode responsible for PWD was documented for the first time in Portugal. Ultimately, we thought this project was most promising: not only was it relevant to our society but it was also related to many of the UN’s sustainable development goals.

Using Synthetic Biology against the Pine Wilt Disease

In Figure 5, a schematic representation of the PWD as well as our strategy to combat this disease are illustrated. As already stated, the infection of healthy trees starts with the feeding and egg deposition of Monochamus galloprovincialis in the xylem, introducing Bursaphelenchus xylophilus in the tree. After the pathogen infection, monoterpenes (in particular alpha-pinene) concentrations increase as a tree stress response and can lead to its death 13. Spectinabilin is a compound produced by Streptomyces spectabilis and Streptomyces orinoci that, besides having reported antiviral and antimalarial activities, has been shown to reduce nematode populations by interfering with hatching of B. xylophilus14. In the referred study, B. xylophilus treated with spectinabilin displayed tissue disorganisation and vacuole formation.
The nematode seems to inherit part of its microbiome from the insect vector, with P. putida being one of the most abundant and frequently detected genera in association with B. xylophilus and also present in the insect’s microbiome 15. In sum, we intend to prevent and control the spreading of PWD by introducing in the pine tree’s microbiome a genetically engineered Pseudomonas putida, capable of producing spectinabilin in response to alpha-pinene. In this strategy we will take advantage of the naturally produced compound alpha-pinene by pine trees in response to infection by B. xylophilus, activating the spectinabilin production pathway and resulting in a decrease over time in the nematode population.

Figure 5 - Infection of healthy Pinus pinaster by Bursaphelenchus xylophilus carried by Monochamus galloprovincialis and subsequent spread and tree death versus disease resolution expected when introducing our bioengineered Pseudomonas putida, capable of producing a nematicidal compound, spectinabilin. Bursaphelenchus xylophilus - BX; alpha-pinene - AP; Pseudomonas putida - PP; spectinabilin - SB

Our main goal with this project is to combine synthetic and systems biology not only to prevent the economic damage caused by PWD but also to raise awareness of all society to the threats to our forests. We aim to fight against deforestation and climate change by protecting our pine trees, which are crucial for the portuguese ecosystem's preservation. To achieve this ambitious goal, we developed a Pseudomonas putida model with the spectinabilin production pathway to determine the optimum genotype for our desired phenotype: maximum spectinabilin production while maintaining the maximum growth rate possible. After the in silico validation of the spectinabilin production in P. putida, we proceeded with the wet-lab work by implementing the heterologous production pathway of spectinabilin by expressing the respective genes in P. putida under the control of an alpha-pinene’s inducible promoter. The results obtained from the computational analysis will support the further development of our engineered bacteria by manipulating its metabolism in order to maximize the flux production of spectinabilin.

  1. Vicente, C., Espada, M., Vieira, P. & Mota, M. Pine Wilt Disease: a threat to European forestry. Eur. J. Plant Pathol. 133, 89–99 (2012).
  2. Futai, K. Pine Wood Nematode, Bursaphelenchus xylophilus. Annu. Rev. Phytopathol. 51, 61–83 (2013).
  3. Mota, M. M. & Vieira, P. Pine wilt disease: A worldwide threat to forest ecosystems. Pine Wilt Disease: A Worldwide Threat to Forest Ecosystems (2008). doi:10.1007/978-1-4020-8455-3.
  4. Vicente, C. S. L., Nascimento, F., Espada, M., Mota, M. & Oliveira, S. Bacteria associated with the pinewood nematode Bursaphelenchus xylophilus collected in Portugal. Antonie Van Leeuwenhoek 100, 477–481 (2011).
  5. Fonseca, L. et al. The pinewood nematode, Bursaphelenchus xylophilus, in Madeira Island. Helminthologia 49, 96–103 (2012).
  6. Proença, D. N., Grass, G. & Morais, P. V. Understanding pine wilt disease: roles of the pine endophytic bacteria and of the bacteria carried by the disease-causing pinewood nematode. MicrobiologyOpen (2017) doi:10.1002/mbo3.415.
  7. Kikuchi, T. et al. Genomic Insights into the Origin of Parasitism in the Emerging Plant Pathogen Bursaphelenchus xylophilus. PLoS Pathog. 7, e1002219 (2011).
  8. Centro Pinus. A Fileira do Pinho em 2018: Indicadores da Fileira do Pinho. (2019).
  9. Centro Pinus. Market perspective of pine trees. (2020).
  10. Centro Pinus. A Fileira do Pinho em 2019: Indicadores da Fileira do Pinho.
  11. Food and Agriculture Organization. International Year of Plant Health. (2020).
  12. Instituto da Conservação da Natureza e das Florestas. National Plan for the control of the nematode in pine trees. (2018).
  13. Li, Y. et al. Comparative Transcriptome Analysis of the Pinewood Nematode Bursaphelenchus xylophilus Reveals the Molecular Mechanism Underlying Its Defense Response to Host-Derived α-pinene. Int. J. Mol. Sci. 20, 911 (2019).
  14. Liu, M. et al. Screening, isolation and evaluation of a nematicidal compound from actinomycetes against the pine wood nematode, Bursaphelenchus xylophilus. Pest Manag. Sci. 75, 1585–1593 (2019).
  15. Alves, M. S., Pereira, A., Vicente, C., Mota, M. & Henriques, I. Pseudomonas associated with Bursaphelenchus xylophilus, its insect vector and the host tree: A role in pine wilt disease? For. Pathol. 49, e12564 (2019).