Progress of an Antimicrobial Treatment for Huanglongbing: The Florida Citrus Arboretum Wayne N. Dixon Richard Gaskalla, Xiaoan Sun, Greg Hodges, Tim Schubert, Mike Kesinger, Ben Rosson, Callie Walker Florida Department of Agriculture and Consumer Services Division of Plant Industry
The Florida Citrus Arboretum
The Florida Citrus Arboretum
The Florida Citrus Arboretum Located in Winter Haven 4.5 acre collection of 212 cultivars of citrus and citrus relatives Established in 1975 to ensure that valuable citrus and citrus related germplasm are always available for study and use in Florida
The Florida Citrus Arboretum Huanglonging (citrus greening) was first detected in the arboretum in 2007 Number of trees infected has steadily increased
The Florida Citrus Arboretum For several years after the initial infection, infected trees were quickly removed after being confirmed by a PCR test By 2012, the infection rate jumped to over 44% from initial 7% Today, most of the trees are infected with citrus greening or are showing visual symptoms of the disease
Antimicrobials Antimicrobials have been utilized for crop protection in United States for over 50 years Several agricultural commodities use either oxytetracycline or streptomycin to control plant pathogens apple, bean, celery, nectarine, peach, pear, pepper, potato, quince and tomato In citrus, streptomycin has been as a treatment for citrus canker
Antimicrobials Aubert and Bove (1980) - Effect of Penicillin or Tetracycline Injections of Citrus trees Affected by Greening Disease. 36 trees were each injected with 2 liters of liquid with an antibiotic Penicillin and streptomycin were evaluated with 18 g/tree being utilized for penicillin and 6 g/tree being utilized for streptomycin Both were effective against citrus greening, but tetracycline can be phytotoxic
o Initiated experiment for potential mitigation of HLB in the arboretum trees Foliar spray applications (6) as monthly applications (May - October 2014) Streptomycin sulfate + adjuvants Penicillin + adjuvant and Adjuvant alone Passive injection 10 trees selected for of Streptomycin sulfate, 10 trees for Penicillin-G 3 untreated trees as controls A rescue operation with a layer of data collection and analysis
Experimental Design Antibiotics Streptomycin sulfate 22.40% Firewall 17 WP Agrosource Streptomycin sulfate 100.00% Bioreagent 50 g Fisher Scientific Penicillin-G potassium salt >85.00% Bioreagent 100 g Fisher Scientific Adjuvants for addition to antibiotics fatty acids potassium salts + nutrients AgriSolv C-100/-120 AgriSolv organosilicone wetting agent Dyne -amic Helena Chemical Co. Treatment Applications Whole tree coverage via 200 gal skid sprayer with 5.5 Hp engine, diaphragm pump, and hand wand Tree injection via external delivery units Pre- and Post Treatment Observations and Samples Pre-treatment observations and samples - Whole tree photograph of all treated trees - Fruit of five varieties collected
Antibiotic treatment of the Florida Citrus Arboretum for Huanglongbing Note on experimental design This is not a controlled study Arboretum contains > 200 cultivars of Citrus and Citrus relatives Tree size varies from resets to large trees with a circumference of 30.25 inches (at 4 inches above soil line)
Tree Injection applications o 10 for streptomycin o 10 for penicillin o Selected trees differ at age (4-23 yrs old), variety, size, HLB expression, and foliage loss (10%-60%). o Injection devices and methods were provided by Dr. Muqin Zhang, IFAS- IRREC. o A 7/32 inch drilling bit and 1,000 ppm antibiotic solution were used for tree injections. The amount of antibiotic for each tree was adjusted to tree size (150-1,200 ml)
Drilling hole for injection jet Foliar Spray with wand Passive injection bag and jets in tree
Testing Las in leaves via qpcr o Injection: A composite sample was taken from 20 citrus trees. 3 untreated citrus served as controls - one leaf from each direction of the canopy - four leaves in total o Trees were sampled before the infusion, then again at 10 and 19 days after the treatment. All composite samples were tested for Las target DNA using the current USDA qpcr HLB testing protocol o New leaves were processed separately from the older ones after the treatment o At day 19, a composite sample (4) was also collected from each of 4 citrus trees that were foliage sprayed with the 4 different antibiotics and adjuvants treatments
X Ct Values Comparison of Average qpcr Ct Values in leaves from Citrus Trees Injected with an Antibiotic Solution 35.00 30.00 25.00 20.00 15.00 10.00 5.00 0.00 Day 0 Day 10 Day 19 NT S P NT=Not Treated; S=Streptomycin; P=Penicillin
Ct Values Average Las Titer in Leaves of All Treatments Mature Young 35 30 25 20 15 10 5 0 Strep Injection Pen Injection Pen Spray Strep DyneAmic Strep C100 C100 Control
Ct Values Average Las Titer in Leaves Sprayed with Penicillin G Six Times 35 30 25 20 15 10 5 0 1 2 3 4 5 6 7 8 9 Control Mean Tree
Penicillin Residue Detection in Fruit 3 Months after Injection About three months after the trunk injection, 25 fruit were collected from 8 arboretum citrus trees that were injected with Penicillin-G in May 10, 2014 2-4 green fruit were collected depending on the availability of fruit on those trees Orange juice was squeezed for the test and 1 gram of peel from some of those fruit were also tested
Table 3. Penicillin G Residue Detection in 25 Fruit via HPLC 80 Days after Treatment HPLC Sample # Tree Location Direction Position (ppb) 1 ARB-07-16 West High ND 2 ARB-07-16 East Low ND 3 ARB-10-07 East Mid ND 4 ARB-10-07 South Low ND 5 ARB-10-07 Interior Mid ND 6 ARB-10-14 East Mid ND 7 ARB-10-14 South Low ND 8 ARB-10-14 West High ND 9 ARB-12-05 West Mid ND 10 ARB-12-05 North Low ND 11 ARB-12-05 East High ND 12 ARB-12-05 Interior Mid ND 13 ARB-13-02 Interior Mid ND 14 ARB-13-02 East High ND 15 ARB-13-04 North Low ND 16 ARB-13-04 Interior High ND 17 ARB-13-04 Interior High 0.33 18 ARB-13-08 North Mid ND 19 ARB-13-08 South Mid ND 20 ARB-13-08 West High ND 21 ARB-13-08 East Low ND 22 ARB-18-09 West High ND 23 ARB-18-09 South Low ND 24 ARB-19-09 South High ND 25 ARB-19-09 East Low ND
Tree Condition Improved Streptomycin Injected Penicillin Injected
Tree Condition Not Improved -Yet
General Observations: Las titer in mature leaves sprayed with penicillin at a rate of 700 ppm did not change much 19 days after first treatment, but decreased in young leaves and in mature leaves 15 days after the fourth foliar spray Las titer significantly decreased 15 days after trees were sprayed with 700 PPM penicillin for the sixth time Las titer in old and new leaves did not change at day 19 on the three citrus trees that were not treated Injected penicillin showed greater effectiveness in reducing Las populations in both mature leaves and young leaves after 19 days Injected streptomycin slightly reduced Las titers in mature leaves after 19 days
General Observations: Fruit from the penicillin-injected trees had a very low residue of penicillin 80 days after the treatment HPLC testing results indicated that those individual fruit contained no penicillin residue except one fruit from which a 0.33 PPB residual penicillin was detected Using a bioassay method, penicillin was detected at concentrations ranging from 0.1 PPM to 5 PPM in leaves and fruit 24 hours after trees were sprayed with penicillin at a rate of 700 PPM and no penicillin was detected in leaves 15 days after treatment. A trace amount of penicillin was detected in some fruit 15 days after the spray
Cost of Tree Injection Ideal target citrus: 7-10 yrs old with 30% foliage loss due to HLB Antibiotic: $1.00 Injection device: $1.50 Labor and other: $2.50 Total: $5 per tree. $650 per acre big caveat about scope of work Effectiveness: 1-2 years or maybe longer
Continuing Work Still a work in progress regarding: Growth response Residue analysis Las Titer in leaves, fruit and roots Spraying 4 applications of Penicillin to foliage at two weeks apart started last week Another phase of work is occurring in Dundee and Ft. Meade Valencias, Hamlins and Grapefruit and replicated
If we are not squashing citrus greening, we are stomping giant African land snails