1. Restoring keystone species through biotechnology
Douglass F. Jacobs
van Eck Professor of Forest Biology
Purdue University
West Lafayette, Indiana, USA
Thank you to the organizers.

2. Eastern U.S. forest tree species that have been or potentially could be decimated by introduced pests or pathogens:
American elm (Dutch elm disease)
American beech (beech bark disease)
Ash species (Emerald ash borer)
Eastern & mountain hemlock (Hemlock woolly adelgid)
Oak spp. (Sudden oak death/gypsy moth)
Balsam fir (Balsam woolly adelgid)
Flowering dogwood (dogwood anthracnose)
Butternut (butternut canker)
Walnut (Thousand canker disease)
All Lauraceae (laurel wilt)
**With global trade, exponential increase in introduction of pests and pathogens.
**Many species threatened in eastern US – globally the list is exhaustive and will only increase.

3. Keystone species: disproportionate impact on ecosystem when compared to its abundance
Foundation species: primary producers that dominates an ecosystem in abundance and influence
Loss of such forest tree species: cascading effects on plant and animal communities, nutrient cycling, and carbon storage
Reintroduction may help to 1) meet FLR targets and 2) restore population community and ecosystem function
**Particularly significant when keystone or foundation species are affected.

4. The historical American chestnut
**American chestnut was arguably both a foundation species (large distribution and abundance) and keystone species (e.g., nut production, carbon storage b/c long lived and decay resistant)

5. Chestnut blight Fungus (Cryphonectria parasitica) Girdling canker
**Functionally extirpated from its range in early 1900s due to accidental import of chestnut blight disease. Loss of > 4 billion trees in 40 years.
**Scientists have been working on methods to overcome blight for ~70 years with 3 main approaches currently being pursued.

6. #1 - Biocontrol with hypovirulence
Virulent
no virus
Hypovirulent
virulent virus
Hypovirulent
weak virus
**Hypovirulence is the reduction in virulence caused by a mycovirus (family Hypoviridae) infection of the blight fungus (Milgroom & Cortesi, 2004).
**The reduced virulence causes sublethal or healing cankers (Griffin, 2000; Milgroom & Cortesi, 2004).
**Hypovirulence research continues. May be important in concert with other options. Can work on individual cankers, but not as effective on whole tree or even populations.
e.g., Milgroom & Cortsei 2004 Ann. Rev. Phytopath.

7. #2 - Genetic engineering
Two different approaches based upon source of genetic information: Transgenic or Cisgenic
1. Any genetic material that will confer blight resistance
OxO-gene construct to degrade oxalic oxidase
**The first application of genetically engineered (GE) forest trees will likely be for reintroduction of species decimated by invasive pathogens or pests (Adams et al., 2002; Merkle et al., 2007).
**Transgenic approaches use genes from non-plant organisms or from plants that are not closely related (i.e., sexually incompatible) to the target plant.
**Chestnut has been transformed with a wheat oxalate oxidase (oxo) gene in an effort to degrade the oxalic acid (OA) secreted by the fungus Cryphonectria parasitica, thus decreasing its virulence.
**Powell Lab in SUNY very active in this approach – A threshold response was observed between the OxO expression level and the C. parasitica lesion length - this is the first report on enhanced pathogen resistance in transgenic American chestnut.
**Cisgenic approaches use genes from closely related or even the same species….this has been demonstrated on poplar and being considered for chestnut. **Cisgenics has been noted for its similarity to interspecies backcross breeding (Figure 6; Schouten & Jacobsen, 2008; Han et al., 2010).
e.g., Zhang et al Transgenic Res.

8. #2 - Genetic engineering
Two different approaches based upon source of genetic information: Transgenic or Cisgenic
American Chinese Japanese European
2. Genes from closely related species
Chinese chestnut, Japanese chestnut
**Cisgenic approaches use genes from closely related or even the same species….this has been demonstrated on poplar and being considered for chestnut. **Cisgenics has been noted for its similarity to interspecies backcross breeding (Figure 6; Schouten & Jacobsen, 2008; Han et al., 2010).
2. Genes from closely related species
Chinese chestnut
Japanese chestnut

9. #3 – Classical tree breeding
BC3 F1
BC1
BC2 P
BC3F2
BC3F3
**Use of backcross breeding method proposed nearly 40 yr ago – hybrids with Chinese chestnut backcrossed to American – BC3F3 is 94% American.

10. collaborative networks
Jacobs et al New Phytologist
government
policy & regulation
cultural & economic
value
restoration
goals
public acceptance
intellectual property
collaborative networks
Society
Technology
Ecology
Restoration
management
field testing
monitoring
breeding
genetic modification
biocontrol
novel replacement
potential ecological barriers
ecological implications
optimal habitats
**While advances have been made through biotechnology….
….maximizing restoration success requires increasing the capacity and integration among social, ecological, and technological spheres.
**Research in understanding ecology of the species in last decade.
***…the critical need for progress in the social sphere has only recently been fully recognized; development in this area is in its infancy and remains perhaps the weakest link toward achieving restoration success.

11. Gustafson et al. 2017 Ecosphere
**We used new knowledge in seed dispersal and ecophysiology of regeneration to model spread of chestnut following reintroduction.
**Passive spread very slow, despite aggressive competitiveness. Aggressive reintroduction more effective but spread still limited after 200 yr.

12. Barriers to restoration
Society: acceptance of GE trees or hybrids as “native species”
Scale of reintroduction of foundation / keystone species under FLR
Production of resistant germplasm
Complex silviculture – many generations
Intensive management
Costs

13. Many forest tree species have been extirpated or are severely threatened by introduced pests and pathogens
Reintroduction offers FLR an added opportunity to help meet targets of Bonn Challenge, while simultaneously restoring species
Enabling insect and disease resistance in forest trees is complicated, but advances in biotechnology have made reintroduction feasible
Societal and ecological barriers to FLR with threatened species remain – most effective is policy that confines the spread of pests and pathogens