New approaches to control Huanglongbing November 1, 2019; Citrus - - PowerPoint PPT Presentation

New approaches to control Huanglongbing

Ozgur Batuman Assistant Professor, Department of Plant Pathology Southwest Florida Research and Education Center, Immokalee, FL November 1, 2019; Citrus Pathology (PLP5115C) Guest Lecture

In 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
• ne-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)

Zha Zhang et al.

• al. 20

2019 19

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 (a (antibiotics)

• Antibiotics are essential for control of bacterial diseases of

plants, especially fire blight of pear and apple and bacterial spot of peach.

• Streptomycin

is used in several countries; the use

• f
• xytetracycline, oxolinic acid and gentamicin is limited to only a

few countries.

• Springtime antibiotic sprays suppress pathogen growth on

flowers and leaf surfaces before infection; after infection, antibiotics are ineffective.

• Antibiotics are applied when disease risk is high, and

consequently the majority of orchards are not treated annually.

(Stockwell & Duffy, 2012) Erwinia amylovora Xanthomonas campestris pv. pruni

Bactericides (a (antibiotics)

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

ingredient) was applied to orchards, which is 0.12% of the total antibiotics used in animal agriculture.

• Antibiotics are active on plants for less than a

week, and significant residues have not been found on harvested fruit.

• Antibiotics have been indispensable for crop

protection in the United States for more than 50 years without reports of adverse effects on human health or persistent impacts on the environment.

(Stockwell & Duffy, 2012)

Bactericides

(EPA, Section 18c).

Bactericides

• Foliar spray: the epicuticular wax on citrus leaf

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

• Soil drench (not permitted): may result in poor

absorption and translocation of bactericides.

• Antibiotics

are highly photodegradable and biodegradable, and vulnerable to

• ther

environmental conditions .

• Trunk injection (not permitted) is a labor

extensive technique and may cause severe phytotoxicity in citrus.

Bactericides (T (Trunk inje jection)

• Tree injection, also known as trunk or stem

injection, is a method of target precise application

• f pesticides, plant resistance activators, and

fertilizers into the xylem vascular tissue of a tree with the purpose of protecting the tree from pests

• r 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 (T (Trunk inje jection)

• 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 in

citrus was reported around 70’s in S. Africa.

• Li et al. 2019: Min. OTC required for initial inhibition of

CLas growth in planta are ∼0.17 and ∼0.215 µg/g in leaf

• tissues. The highest OTC residue in fruit from the field trial

was 0.038 μg/g fresh tissue (about 9 months after injection).

• United States maximum residue limits of 0.01 μg/g for OTC

in or on citrus fruit (US EPA 2018).

• So, can we apply bactericides via

trunk 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 Slashing Flap-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)

Phyt ytotoxicity aft fter trunk inje jection

Trunk Injection (Rhodamine) Trunk Injection (Water)

Dye movement in citrus vasculature

Trunk Injection (Rhodamine) Soil Drench (Rhodamine)

A micro-computed tomography (m (micro-CT) scan

• f

f the citrus vascular system

(Killiny et al. Unpublished)

Rhodamine and acid fuchsin applied citrus trees

Scared of f needle?

Derma microneedles Tattoo needles

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

2-year-old Valencia 1-year-old macrophylla

Rhodamine 2X Non-Grafted Macrophylla (1 yo) Water Rhodamine 1X Water Rhodamine 1X Grafted Valencia (2 yo) Petiole Epidermis Midrib Root

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

Ba Bark Stem em

1% Rhod

• damin

ine e Dy Dye

24 Hours after application 48 Hours after application 24 Hours after application Control

Le Leaf petio tioles

Vessels Root

• ots

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

Funnel pics IMG_ 1026 – 1034 1073 – 1101 3359 – 3463 9262 Shoot dip IMG_2675- 2707 IMG_9174-9179 App area red 5598-5610

(Killiny et al. 2019)

Oxyt ytetracycline and Streptomycin translocation in in cit itrus

(Killiny et al. 2019)

NATI-application of f bactericides in citrus

OTC and Strep were applied 100 ppm (a low dose?)

Project Title: Development of f an automated delivery ry 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 Members

Stakeholders: 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 Obje jectives

1 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 f our project: Automated Delivery ry System (A (ADS)

NATI-application in citrus (m (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 (m (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)

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

near future!

?

Acknowledgements

• NATI Team
• Advi

visory ry Bo Board Members

• USD

SDA NIF IFA (A (Award No: : 2015-70016-23011)

• USD

SDA NIF IFA (A (Award No: : 2019-70016-29096)

• Cit

Citrus In Initia itiativ ive Grant of f Univ iversit ity of f Flo lori rida

• Bayer

r U.S .S. . LLC Crop Sc Scie ience, Bio iolo logic ics

Ana Redondo, Kellee Britt, Shahrzad Bodaghi, Aditi Satpute, Samantha Gebben, Bo Meyering, Jessica Torres, Manali Motghare, Alec Pica, Keanu Tomas, Nico Tezna