Carson Poltorack's research on two chemical treatments for Citrus Greening Disease.

1. C A R S O N P O L T O R A C K
D R . G R A C I E L A L O R C A
U N I V E R S I T Y O F F L O R I D A , D E P A R T M E N T O F
M I C R O B I O L O G Y
Plantibiotics:
Phytotoxicity and Detection of ‘Compound A’ and
‘Compound B’ application for treatment of Citrus
Greening Disease in the Valencia Orange

2. Background
 Citrus Greening Disease (CGD)
 Disease phenotype- mottled leaves; unhealthy branches/shoots; premature tree death;
lopsided, low quality, low yield fruit
 Mechanism: callose deposition blocks phloem sieve pores, deprives plant of nutrients
 Tremendous economic threat to Florida
 Citrus is a $9B industry, providing 76,000 jobs
 Between ‘06-’12, $4.5B cost to industry, destroying 8,000 jobs
 Candidatus Liberibacter asiaticus
 Vectored by Asian Citrus Psyllid
 Unculturable
 Phloem-limited: exposed to high degree of osmotic stress
 ‘Compound B’
 ldtP= L,D transpeptidase, peptidoglycan cross-linkage/cell wall remodeling in response to
osmotic stress
 CPD B stabilizes and inactivates ldtR, a transcriptional activator of ldtP
 ‘Compound A’
 Disrupts RNA Polymerase Bridging via CarD

3. Purpose
 Assay toxic doses of drugs to determine max treatment
dose
 Detect drugs in plant tissue to determine how it is
trafficked around plant
 Examine the pharmacokinetic relationship between drug
and plant
 Adapt ‘Compound A’ and/or ‘Compound B’ for
commercial usage as a CGD treatment

4. Methods
 Phytotoxicity Assay
 128 uninfected, immature Valencia Orange trees
 16 treatment groups
 8 groups per drug
 3 concentrations + 1 buffer control (1, 10, 100 uM)
 Buffer used to solubilize each drug contained DMSO an dTris
pH 8
 2 different exposure mechanisms (poured on roots, sprayed on
leaves)
 Dosage administered at a single time
 Octuplicates
 Data analyzed via ANOVA

5. Methods
 Reverse Phase High Pressure Liquid
Chromatography
 Cut out midrib from untreated leaf samples, lyophilize, grind
to powder
 Extract organic phase via ethyl acetate, dry, resuspend residue
in desired fashion
 Inject into HPLC machine
 Mobile phase A: 300:700:5 acetonitrile:water:acetic acid
 Mobile phase B: 800:200:5 acetonitrile:water:acetic acid
 Ran using isocratic gradient; T=0, 80% A, 20% B/T=30 min,
100% B; detection at 313 nm

6. Phytotoxicity Assay Raw Data
Root Treatment Mean % Growth Standard Deviation
Buffer Control Roots 2.90% 5.33
CPD B 100 Roots -1.43% 0.520
CPD B 10 Roots 2.03% 4.79
CPD B 1 Roots 3.33% 5.94
CPD A 100 Roots 0.989% 1.84
CPD A 10 Roots 0.170% 1.69
CPD A 1 Roots 2.40% 3.96
Leaf Treatment Mean % Growth Standard Deviation
Buffer Control Spray 3.77% 6.57
CPD B 100 Spray 1.63% 4.11
CPD B 10 Spray 5.38% 7.48
CPD B 1 Spray 1.35% 2.55
CPD A 100 Spray 8.43% 10.1
CPD A 10 Spray 1.75% 4.33
CPD A 1 Spray 9.57% 9.79

7. Phytotoxicity Assay
 Data in the bar graph are expressed as mean percent
change in growth from time of exposure to time of
measurement. A higher bar signifies lesser toxicity
(more growth) and a lower bar signifies greater
toxicity (less growth).

8. Phytotoxicity Assay Results: Drug Comparison
100 uM B>Control p=.034
10 uM A>Control p=.146
100 uM B>A p=.003
10 uM A>B p=.317

9. Phytotoxicity Assay Results: Drug Comparison

10. Phytotoxicity Discussion
 It was impossible to determine which drug was less
toxic overall, as each exhibited a different
concentration-dependent pattern of toxicity
 Max toxicity
 Compound A: 10 uM
 Compound B: 100 uM
 Taken individually, the data are still valuable
 Given the degree of growth inhibition I saw, an
appropriate and tolerable dosage probably lies
between 10 and 100 uM for CPD B

11. Phytotoxicity Assay Results: Roots vs. Leaves
10 uM Roots>Leaves p=.304
100 uM Roots>Leaves p=.400

12. Phytotoxicity Assay Results: Roots vs. Leaves
10 uM Roots>Leaves p=.354
100 uM Roots>Leaves p=.075

13. Phytotoxicity Discussion
 When drug type and concentration are held
constant, the toxicity can be used as a proxy for
determining drug absorption into the plant.
 I uniformly observed at the 10 and 100 uM
concentrations that root exposure caused greater
toxicity than leaf exposure, meaning root exposure
caused greater uptake of the drug into the plant.

14. HPLC Results: Plant + H2O

15. HPLC Results: Plant + DMSO + Tris pH 8

16. HPLC Results: Plant + H2O +DMSO+Tris pH 8+ 100 uM CPD B

17. HPLC Discussion
 First, I wanted to see what a baseline chromatogram of
just plant material looked like (Plant+H20)
 Then, I wanted to see if either of the solubility buffers
could be detected from a sample of plant material
(Plant+DMSO+Tris pH 8)
 The first two chromatograms are nearly identical, signaling that the
buffers eluted when the plant material eluted out
 Could NOT detect the presence of these buffers (this was a good
thing)
 Finally, I spiked the prior sample with 100 uM CPD B
 Peak at 26 minutes suggests this protocol was successful in detecting
CPD B from a sample of plant material

18. Overall Discussion/Future Direction
 Phytotoxicity
 100 uM toxic, but not possibly not too toxic
 Longer period of observation necessary
 1 uM nontoxic, but possibly would not be effective
 Pair toxicity data with efficacy data to determine dosage
 When comparing treatment mechanisms, toxicity is a proxy for drug
absorption
 Roots more toxic=absorbed better
 HPLC
 Able to detect CPD B in a sample of extracted plant material
 Future: process phytotoxicity plants and detect CPD B without spiking
the samples; later, treat infected plants to determine differences in drug
movement due to pore blockage
 Detection of CPD A could not be completed due to complications with
synthesis of an affinity column

19. References
 Wang N, Trivedi, P. (2013) Citrus Huanglongbing: A Newly Relevant
Disease Presents Unprecedented Challenges. Phytopathology 103(7):652-
665.
 Pagliai FA, Gardner CL, Bojilova L, Sarnegrim A, Tamayo C, et al. (2014)
The Transcriptional Activator LdtR from ‘Candidatus Liberibacter
asiaticus’ Mediates Osmotic Stress Tolerance. PLoS Pathog 10(4):
e1004101.
 Kim JS, Sagaram US, Burns JK, Li JL, Wang N. (2009) Response of Sweet
Orange (Citrus sinensis) to ‘Candidatus Liberibacter asiaticus’ Infection:
Microscopy and Microarray Analyses. Phytopathology 99(1):50-7.
 Hodges AW, Spreen TH. (2012) Economic Impacts of Citrus Greening
(HLB) in Florida, 2006/07–2010/11. Food and Resource Economics
Department, University of Florida EDIS FE903.
 Koh, E. J., Zhou, L., Williams, D. S., Park, J., Ding, N., Duan, Y. P., and
Kang, B. H. 2011. Callose deposition in the phloem plasmodesmata and
inhibition of phloem transport in citrus leaves infected with ‘Candidatus
Liberibacter asiaticus’. Protoplasma 249:687-697.