New approaches to control Huanglongbing

Ozgur Batuman Assistant Professor,

Department of Plant PathologySouthwest Florida Research and Education Center, Immokalee, FL

November 1, 2019; Citrus Pathology (PLP5115C) Guest Lecture

Introduction• Control

• Reduction of the Asian citrus psyllid (ACP) populations• Visual identification and prompt removal of infected trees• Production of propagation material in insect-proof facilities

• HLB disease:• Remove and destroy infected trees• Quarantine program• Chemotherapy and nutrition treatment • Thermotherapy (Heat/steam treatment)• Bactericides, antimicrobials and ‘snake oils’• CRISPR, RNAi and transgenic approaches?

• Psyllid vectors:• Chemical and biological control• Reflective mulch• Protective screens (CUPS and IPC)• Removal of preferred alternative hosts –M. paniculata

Thermotherapy

• Treating planting materials with heat is a one-century-old method of disease control that has proved to be efficient against various pathogenic microorganisms.

• Thermotherapy, simple in principle, consists in heat treatment of plant parts at temperature/time regimes that kill the conserved pathogen and that are only slightly injurious to the host.

• Heat is applied mainly by water, air, or vapor.

Photo Credit: Shirin Ghatrehsamani and Ampatzidis et al.

Thermotherapy

Photo Credit: Shirin Ghatrehsamani and Ampatzidis et al.

Thermotherapy

Photo Credit: Shirin Ghatrehsamani and Ampatzidis et al.

Thermotherapy

Photo Credit: Jaafar Abdulridha and Ampatzidis et al.

(Treatment at 60°C for 30s)

Zhang et al. 2019

Thermotherapy

‘Although the steam heat treatment and additional nutrition did not eliminate or suppress CLas over the long term, these treatments did positively affect tree growth and recovery in the short term.’

‘Heat treatment and defoliation treatments reduced growth, but did not affect systemic delivery of OTC. We conclude that neither heat treatment nor leaf age strongly affect systemic OTC delivery. …The low concentrations of OTC in tissues from the present study appear to be sufficiently below those of the trunk injection studies that they may not represent effective levels of CLas control.’

Thermotherapy -To sum up…

• Using steam as a heat source, thermotherapy can successfully eliminate CLas from the bud sticks at treatments 55°C for 90 and 120 s

• Further optimization of temperature and time combination necessary to achieve high graft survival, with CLas elimination

• Heat injury was observed at treatments 55°C for 30 and 60 s, which corroborated the graft experiment results

• In the field conditions, single application of thermotherapy is not effective

• Seasonality of CLas should be considered for any experiment in controlling CLas population as higher reduction in CLas titer was achieved when the CLas population was greater

• High yield loss was recorded on the first-year post-treatment, however yield recovered mostly at the second-year harvest

• Total yield and quality of the yield improved on third year of harvest, however it might not be due to thermotherapy only

Slide Credit: Naweena Thapa and Megan Dewdney

Bactericides (antibiotics)

• Antibiotics are essential for control of bacterial diseases ofplants, especially fire blight of pear and apple and bacterialspot of peach.

• Streptomycin is used in several countries; the use ofoxytetracycline, oxolinic acid and gentamicin is limited to only afew countries.

• Springtime antibiotic sprays suppress pathogen growth onflowers and leaf surfaces before infection; after infection,antibiotics are ineffective.

• Antibiotics are applied when disease risk is high, andconsequently the majority of orchards are not treated annually.

(Stockwell & Duffy, 2012)

Erwinia amylovora

Xanthomonas campestris pv. pruni

Bactericides (antibiotics)• In 2009 in the United States, 16,465 kg (active

ingredient) was applied to orchards, which is0.12% of the total antibiotics used in animalagriculture.

• Antibiotics are active on plants for less than aweek, and significant residues have not beenfound on harvested fruit.

• Antibiotics have been indispensable for cropprotection in the United States for more than 50years without reports of adverse effects onhuman health or persistent impacts on theenvironment.

(Stockwell & Duffy, 2012)

Bactericides(EPA, Section 18c).

Bactericides• Foliar spray: the epicuticular wax on citrus leaf

surface and structural degradation under UV orvisible light might affect bactericide uptake.

• Soil drench (not permitted): may result in poorabsorption and translocation of bactericides.

• Antibiotics are highly photodegradable andbiodegradable, and vulnerable to otherenvironmental conditions .

• Trunk injection (not permitted) is a laborextensive technique and may cause severephytotoxicity in citrus.

Bactericides (Trunk injection) • Tree injection, also known as trunk or stem

injection, is a method of target precise applicationof pesticides, plant resistance activators, andfertilizers into the xylem vascular tissue of a treewith the purpose of protecting the tree from pestsor nutrition for correction of nutrient deficiencies.

• Usually used for pest control in trees of forest,urban and palm (i.e., Lethal Yellowing of Palm).

‘drill-plug-inject’ method

Bactericides (Trunk injection) • This is how we do it in our experiments:

‘drill-plug-inject’ method

Bactericides

Bactericides

Bactericides

(ca. 1973)

• Bactericide application via Trunk Injection to control HLB incitrus was reported around 70’s in S. Africa.

• Li et al. 2019: Min. OTC required for initial inhibition ofCLas growth in planta are ∼0.17 and ∼0.215 µg/g in leaftissues. The highest OTC residue in fruit from the field trialwas 0.038 μg/g fresh tissue (about 9 months afterinjection).

• United States maximum residue limits of 0.01 μg/g for OTCin or on citrus fruit (US EPA 2018).

• So, can we apply bactericides viatrunk injection in citrus?

• Yes?• No?

Needle assisted trunk infusion (NATI) of therapeutic material for controlling HLB and

its psyllid vector

Agrochemical application methods

Soil Drench

Trunk Injection

Foliar Spray

Stem SlashingFlap-inoculation

Microneedle Injection

Particle Bombardment

Virus inoculation methods

Dye application method, and movement in trees

Bernholt 1941; Sano et al. 2005

Safranin- and acid fuchsin-stained xylem of Populus sieboldii

Dye application method, and movement in trees

Etxeberria et al. 2015

NBDG tracer-applied citrus ‘Valencia’ tree

Dye movement in citrus vasculature

Trunk Injection (Rhodamine) Soil Drench (Rhodamine)

Phytotoxicity after trunk injection

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Dye movement in citrus vasculature

Trunk Injection (Rhodamine) Soil Drench (Rhodamine)

A micro-computed tomography (micro-CT) scan of the citrus vascular system

(Killiny et al. Unpublished)

Rhodamine and acid fuchsin applied citrus trees

Scared of needle?

Derma microneedles

Tattoo needles

A Novel Method: Needle-Assisted Trunk Infusion (NATI)2

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Non-Grafted Macrophylla (1 yo)

Water Rhodamine 1X Water Rhodamine 1X

Grafted Valencia (2 yo)P

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A Novel Method: Needle-Assisted Trunk Infusion (NATI)

Bark

Stem

1% Rhodamine Dye

24 Hours after application

48 Hours after application

24 Hours after application

Control

Leaf petioles

Vessels

Roots

A Novel Method: Needle-Assisted Trunk Infusion (NATI)

Funnel pics IMG_1026 – 10341073 – 11013359 – 34639262 Shoot dip

IMG_2675- 2707IMG_9174-9179

App area red 5598-5610

(Killiny et al. 2019)

Oxytetracycline and Streptomycin translocation in citrus

(Killiny et al. 2019)

NATI-application of bactericides in citrus

OTC and Strep were applied 100 ppm

(a low dose?)

Project Title: Development of an automated delivery system for therapeutic materials to treat

HLB infected citrus

Period of Performance: 4 years (Jan 2019 through Dec 2022)

USDA NIFA Award Number: 2019-70016-29096

Emergency Citrus Disease Research and Extension Competitive Grants Program (CDRE)

Role Name Title Institution City, State

PD Ozgur Batuman Assist. Prof. Univ. of Florida Immokalee, FL

Co-PD Yiannis Ampatzidis Assist. Prof. Univ. of Florida Immokalee, FL

Co-PD Ute Albrecht Assist. Prof. Univ. of Florida Immokalee, FL

Co-PI Fernando Alferez Assist. Prof. Univ. of Florida Immokalee, FL

Co-PI Tara Wade Assist. Prof. Univ. of Florida Immokalee, FL

Co-PI Nabil Killiny Assoc. Prof. Univ. of Florida Lake Alfred, FL

Co-PI Amit Levy Assist. Prof. Univ. of Florida Lake Alfred, FL

Co-PI Veronica Ancona Assist. Prof. Texas A&M University Kingsville Weslaco, TX

Co-PI Louise Ferguson Prof., Extension

Specialist

Univ. of California Davis Davis, CA

Project Leaders

Stakeholder Advisory Board MembersStakeholders:

Michael Monroe: General Manager, Sun Ag LLC, Fellsmere, FL

Michael Irey: Dir. of Res. and Business Develop., Southern Gardens Citrus, Clewiston, FL

Ron Mahan: Chief Financial Officer, Tamiami Citrus LLC, Fort Myers, FL

Cody Lastinger: Manager, Horticulture Services, Consolidated Citrus, Venus, FL

Joby Sherrod: Director, Grove Operations, Duda Inc., Felda, FL

Forrest Taylor: Sr. Manager, Barron Collier Inc., Naples, FL

Charles Mellinger: Plant Pathologist; Owner, Glades Crop Care, Jupiter, FL,

Raina King, Technical Sales Representative, Biosafe Systems, TX

Eric Bream, Central Valley citrus grower, CA

Scientists:

William J. Lucas, Distinguished Prof. Emeri., Dept. Plant Biology, UC Davis, CA

Bryce Falk, Distinguished Prof., Dept. Plant Pathology, UC Davis, CA

Bill Dawson, Eminent Scholar, Dept. Plant Pathology, UF, CREC; Lake Alfred, FL

Ed Etxeberria: Prof., Dept. Horticultural Sciences, UF, CREC; Lake Alfred, FL

Project Objectives1 Design and development of an automated and economically feasible system to efficiently

deliver HLB-therapeutic materials (including, but not limited to, bactericides) to citrus

trees.

2 Deciphering the path of citrus vascular transport for uptake, movement, and distribution of

therapeutic materials throughout the plant.

3 Optimization of therapeutic application through delineation of daily and seasonal vascular

transport dynamics of citrus trees.

4 Evaluation of the automated delivery system (ADS) in field-grown citrus trees.

5 Evaluation of the economic feasibility of adopting the ADS and comparison of its benefits

with currently used disease management strategies in Florida, Texas and California.

6 Development and implementation of an energetic outreach and extension program.

B

A

C

Goal of our project: Automated Delivery System (ADS)

NATI-application in citrus (many questions!)

• When, what kind of, and how much therapeutics can be

applied by NATI?

• In what frequency?

• What type of citrus plants (cultivar; young vs. old; infected vs.

healthy etc.) can be treated by NATI?

• How and when to assess a change in CLas titer after

applications?

• Is DNA-based detection method giving us any idea about

titer change?

• Do we have RNA- or protein-based detection method

available?

HLB control in citrus (many questions!)

• What treatment is working and what is not?

• Nutrition (spoon-feeding)

• Thermotherapy

• Bactericides (spray application)

• ACP control

• In what frequency to apply treatments?

• What application method to use; how and when?

• Is there a ‘silver bullet’ to HLB? What is it?

…best control would be relaying on integrated pest management (IPM)

of ACP and HLB and having resistant (or tolerant) citrus cultivars in

near future!

?

Acknowledgements• NATI Team • Advisory Board Members• USDA NIFA (Award No: 2015-70016-23011)• USDA NIFA (Award No: 2019-70016-29096)• Citrus Initiative Grant of University of Florida • Bayer U.S. LLC Crop Science, Biologics

Ana Redondo, Kellee Britt, Shahrzad Bodaghi, Aditi Satpute,

Samantha Gebben, Bo Meyering, Jessica Torres, Manali

Motghare, Alec Pica, Keanu Tomas, Nico Tezna