Biotechnology and Forest Tree Pests and Diseases Kevin J. Hackett, - PowerPoint PPT Presentation

Biotechnology and Forest Tree Pests and Diseases Kevin J. Hackett, Ph.D., USDA-ARS NASEM Study Panel December 1, 2017 American Poplar Chestnut Blight Chinese

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 Maple Ailanthus Oak Ash Lanternfly EAB GM ALB

Ornamentals Oak Maple Ash Gypsy Moth ALB EAB Double strand RNA- RNA interference in the Development of mediated RNA Asian Longhorned RNAi method for interference through Beetle: Identification screening candidate feeding in larval of Key RNAi Genes and genes to control gypsy moth, Reference Genes for emerald ash borer, Lymantria dispar RT-qPCR Agrilus planipennis Thais B. Rodrigues 1, Ramesh Thais B. Rodrigues 1, Lynne K. (Lepidoptera: Kumar Dhandapani1, Jian J. Duan2 Rieske1, Jian J. Duan2, Erebidae) & Subba Reddy Palli1 Kanakachari Mogilicherla1 & SAIKAT KUMAR B. GHOSH and DAWN SCiEntiFiC REPOrTS | 7: 8913 | Subba R. Palli1 E. GUNDERSEN-RINDAL 21 August 2017 Scientific REPOrTS | 7: 7379 | Eur. J. Entomol. 114 : 170 – 178, 2017 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 Citrus Citrus Greening Papaya Ringspot Plum Pox Virus

Orchard Trees Papaya Ringspot Plum Pox Virus 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

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

 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. Crimson Rocket Earliscarlet Sentry peach peach nectarine TruGold peach Bluebyrd plum Sweet-N-UP peach Bounty 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.

FasTrack – The Future of Space Exploration! Most fruits come from woody perennial plants not suited to such growing systems.

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 3 hurricanes 60% reduction 2004 in 9 yrs from post-HLB peak 72% since 03-04 Lowest in 70 yrs Latest estimate for 2017/18 projects another 15% reduction

True species: Very narrow germplasm base Citrus Citrus maxima reticulata Citrus micrantha Citrus medica www.flckr.com

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) Xcc Infiltration results with transgenic plants containing thionin, D4E1 and chimera 10 10 6 10 10 4 10 10 6 10 10 4 10 10 6 10 4 10 10 10 6 10 4 10 10 10 7 10 10 5 10 7 10 10 10 5 10 7 10 10 10 10 7 10 10 10 5 5 Non transformed control Thionin-C12 Chimera-C9 D4E1-C20

New Tissue-specific Promoters • Almost all transgenics in commercial use have “1 st 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 1 st to be highly active • Gene architecture for very high phloem expression and very high root expression (also abscission zone/ fruit specific) D35S constitutive GUS SCAmpP 396SS phloem-specific 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 Ash Maple

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 could be shortened Research and regulatory data accumulation 1990 – 2005 (Over 30 publications from this work) 14 Years Stakeholders input 6 Years Regulatory submissions APHIS 2004, FDA 2006, EPA 2007 Regulatory Approval Next time - submit to 3 agencies simultaneously

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 Open access Compliance with the Convention on Biological Diversity and the Nagoya Protocol on Access and Benefit Sharing (ABS) International EBP working group already established.