Case Study 1: American chestnut American Chestnut Research & Restoration project SUNY College of Environmental Science & Forestry (not-for-profitChestnut, restoration program Blight with TACF since 1990)
Current research team: Andy Newhouse (PhD grad student) Bill Powell (Director) Tyler Desmarais (MS grad student) Chuck Maynard (Co-Director Emeritus) Dakota Matthews (MS grad student) Linda McGuigan (TC lab Manager) Vern Coffey (MS grad student) Allison Oakes (Post doctoral fellow) Yoks Bathula (MS grad student) Kaitlin Breda (Admin assistant) Xueqing Xiong (MS grad student) Vanessa Gravenstone (Greenhouse Man.) Erik Carlson (MS grad student) Andrew Teller (Research Analyst) Hannah Pilkey (MS grad student) Masoumeh Khodaverdi (grad student) Many undergrads, high school students, collaborators, and many volunteers…
The work of well over 100 people over 28 years
1 Public engagement (from beginning) • in 1989, founders of TACFNY, Herb Darling and Stan & Arlene Wirsig, asked us to try GE – Annual meetings, tours, and reports • ~20 public presentations per year with Q&A – Given by team members and myself – Flower shows and Garden clubs, landowner groups, libraries, fairs, schools K- 12, NASA, etc. • Many articles - The Conversation >88K reads – PBS TV and radio, CBC TV and radio, WIOX community radio, The Atlantic, The Wall Street Journal, National Geographic, Scientific American, The Economist, Smithsonian, etc. Public engagement • Interacted on multiple occasions with Haudenosaunee leaders in New York State • Reddit ask me anything (AMA) with 1,240 participants, 89% up-voted – unsolicited Reddit open discussion involved 35,415 participants, with 90% up-voted • engaged non-regulatory governmental agencies – New York State Department of Conservation (DEC) – U.S. Forest Service – Fish and Wildlife Service – National Parks Service Public engagement • Webpage: www.esf.edu/chestnut • Two Facebook pages (~2,800 followers ) – facebook.com/americanchestnutgroup/ – facebook.com/groups/esfchestnut/ • Also interact with chestnut growers on TACF- [email protected] • Workshops • Answer many email questions • And more… Wood products
Agricultural Social/historical
Chestnuts roasting on an open fire, The Christmas Song (by Torme and Wells in 1946) 1915 William Bray (first Dean of college) and American chestnut
“It is claimed that in certain districts the farmers realized more income from the sale of chestnuts than all other farm products.”
Not orchards, but wild trees
Bray, W. L. (1915). The development of the vegetation of New York State (Vol. 16, No. 2). The University. Keystone forest species (environmental benefits) Ecosystem restoration goals different than agricultural goals European chestnut American chestnut (Chinese similar) Other differences: Restoration – tall canopy trees Agriculture – short orchard trees
Restoration – diversity Agriculture - consistency Forest History Society Invasive fungal specie: Cryphonectria parasitca
Chestnut blight in the U.S. ~50 years spread through natural range killing ~4 billion American chestnut trees Chestnut blight on related species: Allegheny Chinkapin, C. pumila var. pumila Ozark Chinquapin, C. pumila var. ozarkensis
1876 Japanese chestnut started to be imported
In 1904, discovery of chestnut blight in the Bronx Zoo (Merkel)
Chestnut blight also survives on oaks After over a century of unsuccessful attempts at combating the blight, what are the choices for restoration? Chestnut hybrids are OK for ornamentals or crops, Not for restoration Japanese chestnut C. crenata
American chestnut Chinese chestnut C. dentata C. mollissima
European chestnut C. sativa Are hybrids suitable for restoration? Unlikely to replace the American chestnut
Lion: Panthera leo Tiger: Panthera tigris
American chestnut Castanea dentata
Chinese chestnut Castanea mollissima Liger: Panthera hybrid Better ways: ~38,000 CC genes + ~38,000 AC genes Unwanted traits TACF Meadowview Farm, VA Dr. Fred Hebard (started 1983) Unwanted traits
Dr. Jared Westbrook (current) Unwanted traits
Goal is for 1/16 What if you Chinese chestnut didn’t have to genome to contain select out any the required 3 to 6 unwanted more blight traits? resistance loci (#genes?) Breeding & Transgenics: (Both viable options & both have advantages & disadvantages) Chestnut has ~ 38,000 gene pairs 1/16 Chinese chestnut genes: vs
CC AC
10 pages or 2,375 words It was very exciting at that season to roam the blight resistant then boundless chestnut Making very small woods of Lincoln, … changes, adding Henry David Thoreau, “Walden: or ” only 2 words Life in the Woods, 1899 100% American chestnut + blight resistance Oxalate oxidase (OxO) from wheat ubiquitous enzyme in many plants & fungi (non-gluten enzyme)
Wheat Rice Goatgrass Barley Stiff brome Sorghum Wild einkorn Banana Perennial ryegrass Oil palm Castor bean Date palm Insulin plant Barrel clover Spiny amaranth Strawberry Beet Azalea Cacao Mosses (6 spp.) Fungi Peanut Peach & Apricot Oxalate oxidase (OxO) from wheat ubiquitous enzyme in many plants & fungi (non-gluten enzyme) Detoxifies oxalate (oxalic acid)
Not a pesticide (more like an antitoxin) Does not kill the fungus, no ‘cidal’ activity. Since the fungus survives, less selective pressure to overcome the oxalate oxidase. On transgenic American chestnut, changes the fungal lifestyle from a pathogen to a saprophyte (coexist). (like on Chinese chestnut & some oaks) Oxalate oxidase assay
Very important to have negative controls without oxalate
Run time: 2 hours for wheat germ 4-12 hours for chestnut
(NRC GE Tree webinar) Examples of OxO leaf disk assays (- OA left, + OA right)
Ellis 1 (NT) Darling 38 (T) Darling 28 (T)
Darling 11 (T) Darling 311 (T) Darling 18 (T)
Zhang et al. 2013 OxO protects plant from oxalic acid (OA) OA leaf disk assay (no fungus)
Green – living tissues Brown – dead tissues
Wild type (Ellis 1) American chestnut
‘Qing’ Chinese chestnut Chinese chestnut has no Oxalate oxidase or Oxalate decarboxylase
So, ? oxalate-CoA ligase Oxalyl-CoA Decarboxylase Formyl-CoA Hydrolase Formate dehydrogenase
(Dakota Matthew’s assays)
Isolation of C. parasitica from small stem assay cankers
Example of Darling 54 cankers Isolation of C. parasitica 52 DPI
Darling 54 Darling 54
Qing
Tree & fungus co-exist. Inheritance of blight tolerance Pollination with transgenic pollen Inheritance of blight tolerance “mother tree” Inheritance of blight tolerance Pollination with transgenic pollen Inheritance of blight tolerance Pollination with transgenic pollen Inheritance of blight tolerance Bags opened in lab Small stem inoculations 21 DPI D58 T1s from tissue culture
Ellis 1
Qing
D58 T1-01 D58 T1-20 Canker height D58T1s Girdling status 21 DPI (0.3– 0.5 mm diameter stems – EP155)
Outdoor shade tent, Note: no-takes were not analyzed not temp controlled (~30% of total, ~40% T and ~10% NT) greenhouse 2.5-year-old Darling T1 seedlings three D58, one D54, OxO minus siblings 18 DPI (still early), EP155
Initial inoculation
Approximate area
W x H 2 Tests for regulators Samples Darling 58 insertion site in chestnut genome
Darling 54 found to be inserted in an intron of a predicted gene Set aside pending further testing. Focusing on Darling 58. Transgene did not affect respiration or photosynthesis of T1 chestnuts in an orchard setting
Light Full light response response curves of curves attached leaves
P-value of Data at low Variable transgene light: leaf (P-value < 0.05 significant difference) effect respiration in Respiration in the dark 0.2 dark and light via Kok Respiration in the light 0.6 method
Light-saturated photosynthetic rate 0.3
Quantum yield 0.7 Rlight
Light compensation point 0.3 Rdark Light saturation point 0.9
Dr. John Drake and Cindy Duong Many typical comparative studies, plus additional experiments for restoration trees
(slide by Andy Newhouse) Tadpole Survival: Cox Proportional Hazard Model
SM – Sugar Maple AB – American beech D4 – Transgenic NT – WT American chestnut HY – Hybrid chestnut CC – Chinese chestnut Increasing Survival Hazard Tadpole Development
American chestnut
Goldspiel, HB, Newhouse, AE, Gibbs, JP, and Powell, WA. 2018. Effects of Transgenic American Chestnut Leaf Litter on Growth and Survival of Wood Frog Larvae. Restoration Ecology (in Press) Bumblebee Interactions with Chestnut Pollen + OxO
Photo: William Powell Bee/pollen with OxO feeding study
Cox hazard ratio for bee survival. Higher hazard ratios indicate more hazardous conditions in terms of increased mortality. “Pollen” indicates OxO concentration; “Source” (A, B, C) indicates source colony (hive).
Newhouse, AE, Allwine, AE, Oakes, AD, and Powell, WA. 2018. Effects of Oxalate Oxidase enzyme on bumblebees feeding on pollen. Manuscript in preparation for submission to Ecotoxicology and Environmental Safety Chestnut is not a weed
• As American chestnuts moved west, not considered a weed • Natural chestnut reproduction rates (Rogstad and Pelikan 2014). : – zero offspring per year from trees aged 0-7 years – 0.05 offspring/year from 8-year-old trees – 0.13 offspring/year from 10-year-old trees – 0.2 offspring/year from 12-year-old trees – 0.5 offspring/year from 17-year-old trees, – 4.9 offspring/year from trees aged 70 to 113 years • Natural spread of American chestnut would likely be very slow, possibly at the rate of a few kilometers per century; though after many decades this could accelerate as the volume of annual seed source increases (Paillet and Rutter 1989). Rogstad, Steven H., and Stephan Pelikan. 2014. “Restoring the American Chestnut: Optimizing Founder Spacing to Promote Population Growth and Genetic Diversity Retention.” Restoration Ecology 22 (5): 668–75
Paillet, Frederick L., and Philip A. Rutter. 1989. “Replacement of Native Oak and Hickory Tree Species by the Introduced American Chestnut (Castanea Dentata) in Southwestern Wisconsin.” Canadian Journal of Botany 67 (12): 3457–69 What about crossing with organically grown chestnut hybrids?
1. Growers already have a system to deal with this in the case of Internal kernel breakdown (IKB) 2. Internal kernel breakdown is caused when Chinese cultivars pollenate European hybrids like Colossal. 3. Recommendation, separate by at least 1000ft.
Same recommendations can be used. A unique opportunity with the Darling lines of blight resistant American chestnut:
Rescuing the surviving genetic diversity.
18 in. DBH American chestnut Manlius, NY Unique feature of the ‘Darling’ American chestnut trees: Rescuing genotypes surviving trees Supplier of pollen Genotypes Regionally adapted ½ mother & ½ father
Parental allelic pollen seed composition Continue to maximize out-crossing
Allows: Transgenic Allelic rescue, American local adaptation, chestnut and increases genetic diversity TACFNY LSC “Mother” Trees Offspring Or surviving wild population 50% OxO & fully blight resistance
Or backcross trees 43 Outcrossing Darling 58 American chestnut Lead Event Progress to Date on USDA Permitted Sites
McCabe R39TA, NY mother tree Darling 58 (D58), Ellis 1 background Fert9T2, NY mother tree
Outcross 1 (OC1) D58+ #16001 T1 D58+ #16020 T1
pollen American Chestnut mother trees in 2018 (12 new AC lines): WB275-27 (southern source) Johnnybrook 40A Lasdon Fert2T2 12 new mother tree Pending H1-12H66 backgrounds from 2 Regulatory Fert3T1 father trees. approval, Outcross 2 (OC2) Brag 8G Lasdon this is what In progress Crumhorn Fert9T3 Pollen from the may be I64-J2 resulting OxO+ distributed for Pond seedlings can make breeding (808+ nuts) AC Seedling McCabe Plot OC3 generation. from SUNY- Fert9T2* (repeat from last year) ESF
Others for experimental use: B3F3 (Fred’s selected) F1 Hybrid (Chinese/American) Allegheny Chinquapin EC Marron du Var (European chestnut)
"We humans are more than consumers, we have gifts of our own to give to the earth." Dr. Kimmerer at the U.N.
Questions?