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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
Streptomycin sulfate
Penicillin-G potassium salt
22.40%
100.00%
>85.00%
Adjuvants for addition to antibiotics
fatty acids potassium salts + nutrients
organosilicone wetting agent
Firewall 17 WP Agrosource
Bioreagent 50 g Fisher Scientific
Bioreagent 100 g Fisher Scientific
AgriSolv C-100/-120
Dyne -amic
AgriSolv
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, IFASIRREC.
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
Comparison of Average qPCR Ct Values in leaves from Citrus
Trees Injected with an Antibiotic Solution
35.00
X Ct Values
30.00
25.00
20.00
15.00
10.00
5.00
0.00
Day 0
NT
Day 10
S
Day 19
P
NT=Not Treated; S=Streptomycin; P=Penicillin
Average Las Titer in Leaves of All Treatments
Mature
Young
35
30
Ct Values
25
20
15
10
5
0
Strep
Injection
Pen
Injection
Pen Spray
Strep
DyneAmic
Strep C100
C100
Control
Average Las Titer in Leaves Sprayed with Penicillin G Six Times
35
Ct Values
30
25
20
15
10
5
0
1
2
3
4
5
6
Tree
7
8
9
Control
Mean
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