New approaches to control Huanglongbing November 1, 2019; Citrus Pathology (PLP5115C) Guest Lecture Ozgur Batuman Assistant Professor, Department of Plant Pathology Southwest Florida Research and Education Center, Immokalee, FL
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 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 (Treatment at 60°C for 30s) Photo Credit: Jaafar Abdulridha and Ampatzidis et al.
Zha Zhang et al. al. 20 2019 19
Thermotherapy ‘Although the steam heat treatment and additional nutrition ‘Heat treatment and defoliation treatments reduced did not eliminate or suppress CLas over the long term, these growth, but did not affect systemic delivery of OTC. We treatments did positively affect tree growth and recovery in conclude that neither heat treatment nor leaf age strongly the short term.’ 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) Erwinia amylovora • 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 of oxytetracycline , oxolinic acid and gentamicin is limited to only a Xanthomonas campestris pv . pruni 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)
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 other 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 of pesticides, plant resistance activators, and fertilizers into the xylem vascular tissue of a tree with the purpose of protecting the tree from pests or 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 • 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? (ca. 1973)
Needle assisted trunk infusion (NATI) of therapeutic material for controlling HLB and its psyllid vector
Agrochemical application methods Microneedle Injection Foliar Spray Stem Slashing Flap-inoculation Soil Drench Particle Bombardment Virus inoculation methods Trunk Injection
Dye application method, and movement in trees Safranin- and acid fuchsin-stained xylem of Populus sieboldii Bernholt 1941; Sano et al. 2005
Dye application method, and movement in trees NBDG tracer- applied citrus ‘Valencia’ tree Etxeberria et al. 2015
Dye movement in citrus vasculature Trunk Injection (Rhodamine) Soil Drench (Rhodamine)
Phyt ytotoxicity aft fter trunk inje Trunk Injection (Rhodamine) jection Trunk Injection (Water)
Dye movement in citrus vasculature Trunk Injection (Rhodamine) Soil Drench (Rhodamine)
A micro-computed tomography (m (micro-CT) scan of f the citrus vascular system Rhodamine and acid fuchsin applied citrus trees (Killiny et al. Unpublished)
Scared of f needle? Tattoo needles Derma microneedles
A Novel l Method: Needle-Assisted Trunk In Infusion (NATI) 1-year-old macrophylla 2-year-old Valencia
Non-Grafted Macrophylla (1 yo) Grafted Valencia (2 yo) Water Rhodamine 1X Water Rhodamine 1X Rhodamine 2X Midrib Epidermis Petiole Root
A Novel l Method: Needle-Assisted Trunk In Infusion (NATI) Stem em Ba Bark
Leaf petio Le tioles 24 Hours after application 24 Hours after application 48 Hours after application 1% Rhod odamin ine e Dy Dye Control
Root oots Vessels
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?)
Emergency Citrus Disease Research and Extension Competitive Grants Program (CDRE) Project Title: Development of f an automated delivery ry system for therapeutic materials to treat HLB infected citrus USDA NIFA Award Number: 2019-70016-29096 Period of Performance: 4 years (Jan 2019 through Dec 2022)
Project Leaders 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 Univ. of California Davis Davis, CA Specialist
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