Biotechnology and Forest Tree Pests and Diseases

Kevin J. Hackett, Ph.D., USDA-ARS

NASEM Study Panel

December 1, 2017

Poplar Chestnut

Blight

Chinese

American

Areas of Forest and Ag Biotech Overlap

• Application of forest biotech research to control of pests and diseases of:

– Ornamental trees

– Orchard trees

• Woodlands as potential harbors of pests and diseases

• Protecting pollinators from pesticides used in forests

• The regulatory approval process for GM-trees

Ornamentals Ash Maple Oak Ailanthus

ALB EAB Lanternfly GM

Ornamentals Ash Maple Oak

ALB EAB Gypsy Moth

Double strand RNA-

mediated RNA

interference through

feeding in larval

gypsy moth,

Lymantria dispar

(Lepidoptera:

Erebidae) SAIKAT KUMAR B. GHOSH and DAWN E. GUNDERSEN-RINDAL Eur. J. Entomol. 114: 170–178, 2017

RNA interference in the

Asian Longhorned

Beetle: Identification

of Key RNAi Genes and

Reference Genes for

RT-qPCR Thais B. Rodrigues 1, Ramesh

Kumar Dhandapani1, Jian J. Duan2

& Subba Reddy Palli1 SCiEntiFiC REPOrTS | 7: 8913 | 21 August 2017

Development of

RNAi method for

screening candidate

genes to control

emerald ash borer,

Agrilus planipennis Thais B. Rodrigues 1, Lynne K.

Rieske1, Jian J. Duan2,

Kanakachari Mogilicherla1 &

Subba R. Palli1 Scientific REPOrTS | 7: 7379 | 7 August 2017

Questions: Ornamentals

• What are prospects for creating biotech version of these trees, and, if they are created, getting them through regulatory and certification hurdles?

• Do biotech trees affect biocontrol agents?

Orchard Trees

Plum Papaya

Plum Pox Virus

Papaya Ringspot

Citrus

Citrus Greening

Orchard Trees Plum Pox Virus Papaya Ringspot

Stable transformation of papaya via microprojectile bombardment Fitch et al. 1990. Plant Cell Reports 9:189-194. Virus resistant tomato plants derived from tissues bombarded with the coat protein gene of papaya ringspot virus. Fitch et al. Biotechnol. 10 Nov 2002

FasTrack

Traditional breeding of stone fruits is a 3-4+ year cycle. Breeding, carried out in the field, is affected by climate, diseases, and insect pests. Not every year is successful.

Pollination

Field planting seedlings

The Problem

After 3 - 4 years in the field the seedlings will produce their first crop of fruit that can be evaluated.

Each year 3,000 to 6,000 peach and nectarine seedlings are produced, the results of approximately 12,000 to 24,000 hand pollinations.

Less than 1% of these seedlings become advanced selections.

Less than 10% of advanced selections will become named varieties.

Bounty peach

Sentry peach Earliscarlet

nectarine

Bluebyrd plum Sweet-N-UP peach

Crimson Rocket

peach

TruGold peach Orablue plum

Early Flowering Research at the Appalachian Fruit Research Station

Used a flowering gene (PtFT) from poplar

Some of the

transgenic plum plantlets flowered within 2 months of regeneration

FasTrack plums: Designing plants for space exploration.

Most fruits come from woody perennial plants not suited to such growing systems.

FasTrack – The Future of Space Exploration!

Orchard Trees Citrus Greening

An Existential Threat to World Citrus Industries

• Long known in Asia, first identified in Florida in August 2005

• Vectored by the Asian Citrus Psyllid, in FL since 1998, widespread in FL

&TX, spreading in CA, finds in AZ.

• Associated with a phloem limited bacterium, Liberibacter asiaticus, Within a

few years of infection, many citrus trees become weak, have poor quality

fruit, with lots of fruit drop, and trees may die or become useless

• Estimated that ~80% of FL citrus trees are infected, and some groves no

longer productive

• In FL, estimated average crop reduction of 40% compared to healthy trees

(Singerman, 2015) - many folks out of business

Photos Bové, 2006

Trend in Florida Citrus Production

60% reduction

in 9 yrs from

post-HLB peak

72% since 03-04

Lowest

in 70 yrs

3 hurricanes

2004

Latest estimate for 2017/18 projects another 15% reduction

Citrus micrantha

True species: Very narrow germplasm base

www.flckr.com

Citrus

reticulata

Citrus

maxima

Citrus

medica

Xcc Infiltration results with transgenic plants containing thionin, D4E1 and chimera

Non transformed control Thionin-C12

107

106

105

104

107

106

105

104

Chimera-C9

107 105

106 104

D4E1-C20

106 104

107 105

Thionin and chimeral antimicrobial peptides, designed by

Goutam Gupta (Los Alamos National Laboratory)

Chimera of a citrus serine

protease (cyan) joined to the

lytic D4E1 peptide (red) by a

GSTA linker (yellow)

New Tissue-specific Promoters • Almost all transgenics in commercial use have “1st generation”

construct components, like promoters that express the genes

everywhere & all of the time (constitutive promoters like D35S)

• Working with Bill Belknap & Jim Thomson, have “new” genes

FROM CITRUS for new tools

• Tissue specific promoters, perhaps the 1st to be highly active

• Gene architecture for very high phloem expression and very

high root expression (also abscission zone/ fruit specific)

SCAmpP 396SS phloem-specific GUS D35S constitutive GUS

Other Citrus Biotech

• Antibody (ScFv) directed to external CG-pathogen epitopes (Hartung designed)

• Peptides directed at the insect vector gut (Shatters identified)

• De-novo peptides designed based on biophysical models of interaction with bacterial membranes (Gupta)

• Transgenes designed to disrupt CG-pathogen quorum-sensing

• Recombinase mediated cassette exchange (Thomson designed) to remove antibiotic markers and facilitate gene stacking

• RNAi and CRISPR Studies

Grape – Citrus Connection Pierce’s Disease of Grapevine

Other Approaches • Para-transgenesis: Synthesizing Insect-Vectored,

Plant-Colonizing and Cross-Protecting Bacteria

Citrus Greening

• Gene Drives

Questions/Other Ideas

• Where might there be additional areas of productive collaboration to produce useful biotechnologies, or shared knowledge of ecological impacts, for the forest and agricultural communities?

• Please share other connections might you uncover in your study, e.g., tree architecture?

• Will a combined exogenous/endogenous transgene approach be best?

• Is there any work on self-reporting or self-treating trees?

Areas of Forest and Ag Biotech Overlap

• Application of forest biotech research to control of pests and diseases of orchard and ornamental trees.

• Woodlands as potential harbors of pests and diseases.

• Protecting pollinators from pesticides used in forests.

• The regulatory approval process for GM-trees.

Woodlands as Pest and Disease Harbors

Brown Marmorated Stink Bug

Pierce’s Disease of Grapevine Glassy-winged Sharpshooter

Question: Forest-Ag Border Areas

• Are there areas of potential cooperation in shared border areas?

Areas of Forest and Ag Biotech Overlap

• Application of forest biotech research to control of pests and diseases of orchard and ornamental trees.

• Woodlands as potential harbors of pests and diseases.

• Protecting pollinators from pesticides used in forests.

• The regulatory approval process for GM-trees.

Protecting Pollinators

Maple Ash

Questions: Pollinators

• Can biotech-based pest and disease control protect pollinators better than chemical approaches?

• What are examples of such approaches and what are the gaps in our knowledge that, when addressed, will help us better protect pollinators and other species of interest to biodiversity, e.g., the monarch butterfly?

Areas of Forest and Ag Biotech Overlap

• Application of forest biotech research to control of pests and diseases of orchard and ornamental trees.

• Woodlands as potential harbors of pests and diseases.

• Protecting pollinators from pesticides used in forests.

• The regulatory approval process for GM-trees.

The Regulatory Process Development of a GE virus resistant plum variety

Gene discovery, Vector construction, Transformation 1990 - 1992 Plant establishment, Propagation, Greenhouse testing 1992 - 1995 Field testing U.S. (APHIS) 1995 – 2005 Field testing Europe 1996 – 2005 Research and regulatory data accumulation 1990 – 2005 (Over 30 publications from this work) Stakeholders input Regulatory submissions APHIS 2004, FDA 2006, EPA 2007

14 Years

could be shortened

Next time - submit to 3 agencies simultaneously

6 Years

Regulatory Approval

Questions: Regulatory

• What is the status and the need for long-term persistence studies for biotech products?

• How will biotech tree stocks affect assessment of the invasive risk of pests and pathogens?

• Can we use transient expression of Cas9 and sgRNA to reduce regulatory hurdles?

i5K and EBP

GLOBAL NETWORK OF COMMUNITIES

International EBP

working group

already established.

Open access Compliance with the Convention on Biological Diversity and the Nagoya Protocol on Access and Benefit Sharing (ABS)

Questions: Earth BioGenome Project

• What is known about the genomes of forest trees and their pests and fungal pathogens, and how might EBP help resolve critical problems with these pests and pathogens in forests?

• Which forest vegetation and pest and eukaryotic pathogen species are in repositories?

• Has DNA been extracted from these species?

• What are the sequencing priorities for forest trees and their pests and eukaryotic diseases?

• Who are potential partners in this effort?

Biotech Forest ↔ Crop

?↔?

Poplar Orange

Thank you!

How can we apply discoveries in forest biotech to crop biotech, and how might crop biotech speed progress in development and regulatory approval of forest tree biotech?

The Challenge

Tree fruits could significantly improve crew diet as source of antioxidants and high impact, fresh foods

Candidate food crops for space restricted thus far to herbaceous species

FasTrack for Phenolics

• A plum selection with 2x higher phenolic levels than those found in commercial varieties.

• Breeding this trait using the FasTrack system.

0

50

100

150

200

250

300

350

400

Total Phenolics

Grape

$3.5B crop/$162B impact

Research needs

• Disease resistance

• Fruit quality

• Abiotic stress tolerance

From Foe to Friend: Synthesizing Insect-Vectored, Plant-Colonizing and Cross-Protecting Bacteria:

A strategy for rapid development, deployment and dissemination of crop-protecting organisms

USDA ARS, University of Florida, Synthetic Genomics, Inc.

• Produce first synthetic “proto-Liberibacter defensorium” strain

containing a CLas-Lcre hybrid synthetic genome – Outcompete and/or kill the pathogenic CLas – When desired, acquired and transmitted by Asian citrus psyllids only – Culturable – Modular genome for easy manipulation of traits – Trackable and distinguishable from other Liberibacters – Replaceable with upgraded versions – Do everything above in the most acceptable package we can make to gain

permission for release. – For more information, contact Bob Shatters, robert.shatters@ars.usda.gov

EBP Strategy 1: The Phylogenomic Wave

• Domains: 3 (Eubacteria, Archaea, Eukarya)

• Eukaryotic Kingdoms: 5 (animal, plant, fungi, chromista and protozoa)

• Eukaryotic Phyla: 61 (35 animal; 10 plant; 2 fungi; 14 chromists+protozoa)

• Eukaryotic Classes: 266

• Eukaryotic Orders: 1253

• Eukaryotic Families: 9330 (Phase I; reference quality)

• Eukaryotic Genera: 140,000-200,000 (Phase II)

• Eukaryotic Species: ~1.5 million known (Phase III)

EBP Strategy 2: “Google Life”

• Location Sampling (e.g. Ocean Sampling Day Consortium; Genomic Observatories Network; NEON; Critical Zone Observatory; CALeDNA)

• Sequence all organisms in a particular geographical area (e.g., within biodiversity hotspots); soil, land, water and air

• Enables studies of the effect of environmental change on biodiversity (genomic ecology)

• Produce a multidimensional and dynamic view of life on earth