Title: Citrus industry update
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Title: Citrus industry update
Series Title: Citrus industry update
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Language: English
Creator: Institute of Food and Agricultural Sciences, University of Florida
Publisher: University of Florida Institute of Food and Agricultural Sciences
Place of Publication: Gainesville, Fla.
Publication Date: October/November 2008
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Bibliographic ID: UF00086519
Volume ID: VID00008
Source Institution: University of Florida
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I J UNIVERSITY of
UF FLORIDA
IFAS


Citrus Industry Update


Working
To Keep You
Informed


Published by the University of Florida, Institute of Food and Agricultural Sciences, with the mission
of keeping the Florida Citrus Industry informed of current research concerning canker and greening.


Genetic Engineering of Murraya as a Psyllid
Control Tool
Fred Gmitter

Murraya paniculate (orange jasmine) is a citrus
relative and a common ornamental plant
known for its attractiveness. Its utility and
desirability by homeowners/landscapers is well
known. However, it is the alternate host of
Asian citrus psyllids. The rapid proliferation and
spread of psyllids, and subsequently the spread
of HLB in Florida, was aided by the transport
and sale of Murraya plants through the nursery
trade. Accurate, rapid, and robust
methodologies for the detection and removal
of HLB infected trees and better disease
management strategies are required to slow
the spread of HLB.

One strategy to manage psyllid populations
may be by genetic engineering of Murraya with
genes encoding products deadly upon ingestion
by psyllids. Deployment of such plants in the
urban landscape would satisfy homeowner's
desires for the attractive ornamental, while
diminishing the potential urban/suburban
reservoir of vectors and the HLB pathogen.
However, to develop a transformation system
for Murraya, a previously defined tissue culture
system for plant regeneration is a prerequisite.

To standardize the protocol for in vitro
propagation of Murraya, seedling stem
segments were used as explants and cultured
onto various tissue culture media
supplemented with different concentrations of
cytokinins and auxins in combination or alone
to induce regeneration. Nineteen different
tissue culture media were tested of which only
two media gave satisfactory regeneration
efficiencies of around 62% and 75%. Work is
being carried out to standardize the protocol
for the genetic transformation of Murraya
using marker genes. This standardization will


enable us to first develop a reliable protocol for
genetic transformation of Murraya, and then
test genes encoding compounds/products that
may be deadly upon ingestion by psyllids to
produce a potentially toxic trap plant.

For questions and further details, please
contact Dr. Fred G. Gmitter Jr.: 863-956-1151,
fgmitter@ufl.edu

Methods for Killing HLB Infected Trees and
Abandoned Groves
Gene Albrigo

We are still carrying out limited research on
ways to kill HLB infected citrus trees without
removal as we wait for possible additional
funding from the FCPRAC grant process. With
the recent citrus tree census reporting that
over 120,000 acres of abandoned citrus trees
exist in the state, viable procedures to
eliminate these trees and individual affected
trees in commercial groves is perhaps more
important than ever. Of particular concern is
the recent report of active movement of
psyllids from abandoned groves to nearby
commercial groves (Stelinski, CREC). For the
abandoned tree issue, an aerial spray test is
scheduled by the South Florida Water
Management District that will be conducted on
an area scheduled for reversion to natural
habitat. We have nearly completed tests of
trunk application of herbicides to HLB infected
trees in a commercial grove. Spray applications
were made over several shallow cuts into the
wood. Some of the treatments appear
successful, but many herbicides did not have
any noticeable effect compared to direct
canopy sprays. A newer fumigant (methyl iodide)


2008 Citrus Research and Education Center, University of
Florida, Institute of Food and Agricultural Sciences, 700 Experiment
Station Road, Lake Alfred, FL 33850, Phone: 863-956-1151.


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is also being tested, and preliminary results
suggest this may be a viable alternative to
herbicide use. In general, tests are planned for
winter periods to see if chemicals have the
same efficacy during cold temperatures as they
do during warmer periods with active tree
growth.

For more information, contact Dr. L. Gene
Albrigo, albriqo@crec.ifas.ufl.edu

Growth Regulator Use for Controlling Excess
Citrus Tree Flushing
Tim Spann

Because psyllids feed on and require new flush
to reproduce, one horticultural management
strategy for slowing the spread of HLB is to
reduce or eliminate excess flush. This past
summer, greenhouse trials were initiated to
test the efficacy of the plant growth regulator
Apogee (prohexadione calcium, BASF Corp.) to
control citrus tree growth. Apogee is currently
labeled for and routinely used to control the
growth of apple trees in high density plantings.
Apogee is considered to be a growth retardant
and not a growth inhibitor, which means that
treated plants will still produce new flush, but
less of it.

Our initial studies showed that Apogee reduced
the growth of Swingle seedlings under
greenhouse conditions similar to moderate
drought stress. One application at 200 ppm was
effective for approximately 6 weeks. Beginning
in mid-August, three field trials using Apogee
were initiated in a hedging trial in a commercial
block of 'Hamlin' trees in Lake Alfred. Based on
the greenhouse studies, Apogee was again
applied at 200 ppm. The results of these field
studies have been mixed. Apogee effectively
reduced growth on both the north and south
side of trees in one study, had no effect in
another study, and only reduced growth on the
south side of trees in the third study. These
results may be due to the greater vigor of the
field-grown mature trees compared to the
greenhouse seedlings, suggesting that higher
rates may be needed in the field. They may also
point to a difference in sensitivity to Apogee
between Swingle and Hamlin. Additional trials


with Apogee are planned for both the
greenhouse and the field next season.

Additional trials are planned for next season
using other plant growth regulators which are
classified as growth inhibitors, meaning that
they prevent vegetative growth for some
duration of time. One of these products, NAA
(napthaleneacetic acid), has been used in citrus
in the past to prevent trunk sprouts under the
trade name "Tre-Hold." NAA-based products
are still labeled for use in citrus if they prove to
be effective at controlling growth in the
canopy. Other products, such as dikegulac
(Atrimmec, PBI/Gordon Corp.) or paclobutrazol
(Bonzi, Syngenta), are routinely used to control
vegetative growth of landscape trees and
shrubs and greenhouse ornamentals. However,
if such products are effective on citrus,
considerable research will need to be
conducted to test their safety on a food crop
before they can be labeled for use.

For more information, contact Dr. Tim Spann,
spann@ufl.edu

Ongoing Developments on Exploiting Psyllid
Chemical Ecology for Improved HLB
Management
Lukasz Stelinski

Investigations of psyllid behavior and chemical
ecology continue in Dr. Stelinski's laboratory.
Following the discovery that a female-produced
volatile sex attractant pheromone is produced
by the Asian citrus psyllid, work has continued
on its chemical identification. Postdoctoral
researcher, Ebenezer Onagbola, in
Dr. Stelinski's lab is hot on the trial of this
pheromone, having already identified multiple
components that show behavioral activity. The
exact blend and ratio of the components have
not yet been determined. Dr. Onagbola has
also recently identified two components of the
pheromone of the psyllid's main parasitoid
species, Tamarixia radiata. Research is
on-going to confirm whether this is the
complete pheromone. Furthermore, citrus
volatiles have been found to attract both sexes
of Asian citrus psyllid, which has moved
research towards discovery of plant-based


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attractants. A recent breakthrough headed by
Dr. Russ Rouseff, chemist at the CREC, in
collaboration with Dr. Stelinski's lab has been
the discovery of specific sulfur compounds that
likely explain guava's repellent effects against
the psyllid. Dr. Onagbola is finding that guava
volatiles are both repellent and in some cases
toxic to the psyllid. Candidate attractants and
repellents are being tested that may be
developed into practical pest control
applications in the near future. The pheromone
of the parasitic wasp Tamarixia will be used to
monitor the establishment of this natural
enemy in groves throughout Florida and to
perhaps recruit these beneficial insects into
groves to improve biological control.

For more information contact Dr. Lukasz
Stelinski (stelinski@ufl.edu).

The "Hidden" Benefits of Systemically Applied
Imidacloprid
Lukasz Stelinski

Postdoctoral researcher, Raj Boina, is an insect
toxicologist working in Dr. Stelinski's lab.
Dr. Boina has recently uncovered many subtle,
but very important physiological and behavioral
effects of systemically applied imidacloprid at
sublethal concentrations that are not high
enough to kill psyllids. Such concentrations are
commonly found in citrus trees after
application of imidacloprid because of a) the
variation in the spatial and temporal patterns
of imidacloprid uptake and systemic
distribution within trees; b) a time lag between
application and the attainment of lethal
concentrations within trees; and c) metabolic
degradation of the pesticide over time within
trees. However, Dr. Boina has found that the
sublethal concentrations of imidacloprid
negatively affect the survival, developmental
time, longevity, and reproductive capability of
Asian citrus psyllid that likely contributes to
population reductions over time. Also, at the
highest sublethal concentrations, Dr. Boina has
found that imidacloprid substantially reduces
psyllid feeding. These results are very
encouraging and point to other potential
positive impacts of soil applied imidacloprid at
sublethal concentrations in addition to its


direct action of killing psyllids at lethal
concentrations.

For more information contact Dr. Lukasz
Stelinski (stelinski@ufl.edu)

Bridge between Research and Management of
Citrus Greening
Nian Wang

In 2007 and 2008, multiple projects were
conducted in the Wang Lab to understand the
citrus greening pathogen and the potential
implication in controlling citrus greening.
Consequently, five papers were produced from
those studies on aspects related but not limited
to the following areas: early diagnosis, bacterial
diversity associated with greening diseased
trees, distribution of the pathogen in citrus,
and host response to the greening pathogen
infection (One published, one in press, three
submitted). Here the connection between the
research and its potential use in citrus greening
management is discussed.

Reliable and robust diagnosis is necessary. The
current management strategy of greening is to
remove infected citrus trees and reduce psyllid
populations with insecticides. This strategy
requires sensitive and reliable diagnostic
methods for early detection. Symptoms have
been the most convenient means of diagnosis
and are widely used in the field. However,
symptom based diagnosis could be confusing.
A Phytoplasma sp. was reported to cause
greening-like symptoms in citrus in Brazil.
Some greening-like symptoms were also
observed in Polk County without detection of
the potential causal agent: Candidatus
Liberibacter asiaticus. Economically, it makes
sense by confirming the greening disease by
other means including PCR and Quantitative
PCR besides symptoms, rather than just cutting
the suspect which might not be greening at all.
Losing productive trees with some greening-like
symptoms without confirmation is far more
costly than a simple confirmation.

Early diagnosis is necessary and possible. Our
results indicated that a minimum bacterial
concentration was required for greening


Citrus Industry Update
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symptom development in studying the
population of the greening pathogen in
symptomatic and asymptomatic leaves.

A speculation has existed between the
threshold of the greening pathogen population
and successful psyllid transmission. Thus, early
diagnosis will provide means to limit the spread
of the greening pathogen by psyllids by cutting
greening diseased trees before the greening
pathogen reaches certain population in the
phloem. We have optimized detection
sensitivity of the greening pathogen with
different PCR based methods with
primers/probe targeting 16S rDNA, beta-
operon, or 16S rRNA. Preliminary data
indicated that we could detect the greening
pathogen up to 6 months before showing any
symptoms.

What pathogens are associated with citrus
trees showing greening symptoms? Our
results revealed that citrus leaf midribs can
support a diversity of microbes by a
comprehensive study of two citrus groves in
Florida. However, our data clearly indicated
the dominance of the greening pathogen
Ca. Liberibacter asiaticus in symptomatic
leaves. These data strongly implicated
Ca. Liberibacter asiaticus as the pathogen
responsible for greening disease in Florida.

Where are the citrus greening pathogens? We
found that 'Ca. Liberibacter asiaticus' was
distributed in bark tissue, leaf midrib, roots,
and different floral and fruit parts, but not in
endosperm and embryo, of infected citrus
trees. Consequently, we recommend digging
the whole tree out to prevent new flush from
the cut greening diseased trees in the future.

New means to control citrus greening. It is
challenging to control any disease whose
pathogen lives in the phloem. Our previous
data indicated that accumulation of callose and
up-regulation of phloem proteins was
associated with greening disease symptom
development. Currently, we are investigating
the possibility to reduce greening disease by
manipulating callose and phloem proteins.


For questions and further details or paper
requests, please contact Dr. Nian Wang:
863-956-1151, nianwang@ufl.edu

Economics Projects Evaluating Citrus
Greening
-Ron Muraro

Proposals for four projects dealing with the
economic implications of citrus greening have
been submitted to the Florida citrus production
research advisory council for funding. These
are: (1) "Survey to Estimate the Rate of
Greening Infection in Florida Citrus Groves"; (2)
"Long-Run Processed Orange Production and
Price Impacts Associated with Citrus Greening
in Florida and Sao Paolo, Brazil"; (3) "Economic
Evaluation of Alternative Replanting Production
Systems." and (4) "Long-Run Structural
Implications of Citrus Greening in Florida."
Researchers working on one or more of these
projects include Tom Spreen, professor, Food
and Resource Economics Department, UF/IFAS;
Ron Muraro, professor and farm management
economist, CREC; Allen Morris, associate
extension scientist and agribusiness economist,
CREC; Fritz Roka, associate professor of Food
and Resource Economics, SWFREC; Mark
Brown, research economist, and Robert
Norberg, deputy executive director of research
and operations, FDOC; Richard Gaskalla,
director and Mark Estes, environmental
administrator, Division of Plant Industry,
FDACS; Jim Ewing, deputy director, and Candi
Eric, agricultural statistics administrator, Florida
Agricultural Statistics, National Agricultural
Statistics Service.

The first project is a survey of Florida citrus
groves conducted as part of the FDACS Division
of Plant Industry's CHRP plan survey of
greening and canker management practices.
The objective of this (IFAS) part of the project is
to develop reliable estimates of the degree that
greening currently infects Florida citrus groves
(as measured by symptomatic trees), the rate it
is spreading, and the extent that growers are
following management practices to slow its
spread. Data gathered from this joint FDACS-
IFAS survey will be statistically expanded to
provide estimates for each of the five citrus


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growing regions and state totals by the
National Agricultural Statistics Service. Results
from this study will provide the basis for
long-run production and price forecasts for
processed citrus and evaluation of long-run
structural impacts of citrus greening on the
Florida citrus industry. These estimates should
also be useful to other research that seeks to
determine various future impacts of greening
on the Florida citrus industry. Allen Morris is
the principal investigator. Collaborators
include Tom Spreen, Ron Muraro, Richard
Gaskalla, Mark Estes, Jim Ewing, and Candi Eric.

The second project will utilize results from this
greening survey in Florida and estimates of the
incidence of greening in Sao Paulo from surveys
conducted in Brazil to identify plausible
scenarios of production impacts from citrus
greening and incorporate these impacts into a
model of the world orange juice market.
Orange juice supply regions in the model will be
Sao Paulo, Brazil and Florida, and demand
regions will be the United States, Canada,
Europe, and Japan. Scenarios to be developed
will consider alternative tree survival rates,
various demand growth assumptions, and the
possible impact of greening on new tree
planting. Expected output from the study
includes projections of orange production and
processed orange prices in the two supply
regions and consumer impacts in the demand
regions. The principal investigator is Tom
Spreen. Collaborators are Mark Brown, Ron
Muraro, and Allen Morris.

The third project evaluates replanting groves to
higher tree densities as a strategy to offset
increased tree attrition and thus increase the
economic life of future groves infected with
greening. Two alternative high-density planting
production systems will be analyzed: (1) tree
densities of 33% to 100% higher than
traditional tree densities using microsprinkler
irrigation along with traditional management
programs and (2) an open hydroponic system
at more than double traditional tree densities
where nutrients and water are precisely
managed through a drip irrigation system.
Capital requirements, operating costs, fruit
yields and production, additional costs required


for citrus greening management, increased tree
loss and removal due to greening, revenues,
and other variables will be analyzed for each
production system. Break-even prices, time
required to break even, and internal rates of
return on investment will be evaluated and
compared to a traditional lower-density
planting system also experiencing tree attrition
from greening and under a greening
management program. The principal
investigator is Ron Muraro. Collaborators are
Allen Morris and Fritz Roka.

The fourth project will integrate the results
from these other three studies and consider
the consequences of endemic citrus greening
on the infrastructure of the Florida citrus
industry including citrus processing capacity,
fresh packing capacity, associated input supply
industries, and the economic impact on
communities located in citrus producing
regions of Florida. The expected results of this
project address a fundamental issue
confronting the citrus industry: what is the
long-run impact of endemic citrus greening on
the Florida citrus industry and its size and
economic performance in Florida? The
principal investigator is Tom Spreen.
Collaborators are Ron Muraro, Allen Morris,
and Bob Norberg.

If funded, these studies can have wide-ranging
implications for all participants in the industry
including research agencies such as ARS-USDA
and UF/IFAS, organizations such as FDOC and
Citrus Mutual, legislators and policy makers,
input supply companies, lenders, and
processing and packing companies that may be
facing significant decisions regarding product
supply. Estimation of the true economic cost of
citrus greening also has implications with
respect to the allocation of research dollars to
confront it.

For more information, contact Ron Muraro,
rpm@ufl.edu


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Progress towards Incorporation of
Antimicrobial Peptides for the Development of
HLB/Canker Resistance in Commercial Citrus
-Manjul Dutt, Ahmad Omar, Vladimir Orbovic,
Gary Barthe, and Jude Grosser

What are antimicrobial peptides?
Antimicrobial peptides are usually small
proteins, usually 12 and 50 amino acids long.
They form the first line of host defense against
pathogenic infections and are a key component
of the innate immune system. Antimicrobial
peptides are involved in the antimicrobial
defense system among all classes of life.

Why use antimicrobial peptides? Antimicrobial
peptides have broad spectrum antimicrobial
activity. Antimicrobial peptides act by
disrupting the structure or function of microbial
cell membranes. Antimicrobial peptides are
usually bacteriocidal (bacteria killer) instead of
bacteriostatic (bacteria growth inhibitor).


Our research objectives: (1) Design codon
optimized antimicrobial peptide genes for
citrus to combat HLB and Canker; (2) Target
trans-protein in phloem tissue where HLB
resides; (3) Produce a large number of
transgenic lines using Agrobacterium and
protoplast mediated transformation; and
(4) Challenge plants with disease causing HLB
and canker bacteria to evaluate resistance.

Antimicrobial peptides and HLB/canker: Both
diseases are caused by gram negative
bacterium. No natural resistance exists in any
known commercially cultivated sweet orange,
grapefruit, or tangerine. Antimicrobial peptides
have been shown to provide resistance to
bacterial diseases. The LIMA gene has been
shown to control Xylella fastidiosa, the causal
organism for Pierce's disease in grapes (Dennis
Gray, MREC, UF/IFAS). This same gene can
theoretically be used to combat both HLB and
canker. Several other promising constructs are
also being tested.


Transgenic Citrus Plants Produced Containing Antibacterial Constructs (All Are Being Propagated
by Micrografting for HLB and Canker Disease Challenge Assays)


Cultivar


Duncan
Hamlin
Misc Grapefruit
Valencia, Hamlin, OLL-8
Carrizo
Misc Grapefruit
Valencia, Hamlin, OLL-8
Carrizo
Key Lime
Misc Grapefruit
Hamlin
Valencia
Carrizo
Carrizo
Valencia, Hamlin, OLL-8
Key Lime
Misc Grapefruit


Gene


AttacinE
AttacinE
LIMA
LIMA
LIMA
PTA
CEMA
CEMA
CEMA
CEME
CEME
CEAD
CEAD
LIMA under AtSuc2 promoter
LIMA under AtSuc2 promoter
LIMA under AtSuc2 promoter
LIMA under AtSuc2 promoter


No. of plants in soil
27
15
43
56
8
12
20
20
6
14
6
6
12
25
23
17
12


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Use of phloem specific promoters to target
foreign gene expression in the phloem tissue,
where HLB resides: HLB resides in the phloem.
Targeting the trans-protein in the phloem can
minimize issues regarding the subsequent
presence of the protein in the fruit and juice,
thus facilitating FDA approval. Two phloem
specific promoters are currently under
evaluation and working well in citrus:
(1) Arabidopsis (mustard) Sucrose synthase
promoter; and (2) Rice Sucrose synthase
promoter.


GUS expression in citrus leaf phloem tissue
using the Rice Sucrose Synthase promoter -
shows targeted foreign gene expression only
in the phloem.

Transgenic plant challenge via grafting with
HLB infected budwood: Transgenic plants are
being graft challenged with HLB infected sweet
orange budwood. Transgenic plants are
evaluated for HLB symptoms and infection that
is verified by qRT-PCR. The first Duncan
grapefruit plants containing the LIMA construct
were challenged over 14 months ago, and some
are showing no symptoms and remain PCR-
negative, whereas the inoculum budstick
remains PCR-positive (see photo top right).
Some of the same plants are also showing
resistance to citrus canker. Plans are underway
to test these and all the other mentioned
transgenic plants in field tests under heavy HLB
and canker pressure, as necessary to validate
any resistance.


Transgenic Duncan grapefruit containing the
LIMA construct showing putative resistance to
HLB 14 months after inoculation with HLB.


THANKS!

This research is supported by a block grant
from FCPRAC provided to the UF Citrus
Improvement Team (Fred G. Gmitter, Jr., Jude
W. Grosser, William S. Castle, and Gloria
A. Moore) entitled: SURVIVING HLB AND
CANKER: GENETIC STRATEGIES FOR IMPROVED
SCION AND ROOTSTOCK VARIETIES. The
authors also thank Ron Brlansky, Jim Graham,
and Marta Francis for assistance with disease
screening; and the UF Core Citrus
Transformation Facility for producing some of
the transgenic plants.

For more information, contact Dr. Jude Grosser,
jgrosser@ufl.edu

Identification of Metabolite Profile Changes in
'Valencia' Orange Leaves Induced by HLB
Dr. Jose Reyes-De-Corcuera

Over the past 14 months Dr. Reyes-De-
Corcuera's team has worked on the
determination of changes in metabolite profiles
in 'Valencia' orange leaves caused by HLB using
high performance liquid chromatography-mass
spectrometry (HPLC-MS), capillary
electrophoresis (CE), and gas chromatography-
mass spectroscopy (GC-MS). HPLC-MS
untargeted analysis revealed about
190 compounds that differ in concentration
between HLB-infected and healthy orange
leaves. These compounds were grouped by


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mass-to-charge ratio into three major groups.
Two of these groups showed significant
increase in overall concentration while the
other showed significant decrease. The extent
of change was correlated with an arbitrary
scale of intensity of symptoms. CE experiments
under optimized extraction and separation
conditions consistently revealed significant
differences in 6 of 24 compounds. GC-MS
experiments revealed changes in several sugars
and sugar alcohols, amino acids, phenols, and
organic acids. Dr. Reyes-De-Corcuera's team is
now starting the development of an
amperometric enzyme biosensor that targets
one of these metabolites while validating the
correlation between its presence in HLB
infected trees and not produced under other
plant stresses. CE results were recently
submitted for publication to "Electrophoresis."
Most of the research results have been
presented at the 2009 annual meeting of the
Florida State Horticultural Society and at the
59th Citrus Processor's and Subtropical
Technology Conference.

For more information, contact Dr. Jose Reyes-
De-Corcuera, jireyes@ufl.edu


EVENT UPDATE

Florida Ag Expo
November 5, 2008
UF/IFAS Gulf Coast Research and
Education Center, Balm, Florida 33503
For more information and registration,
visit
http://www.floridagrower.net/flgevents/in
dex. htm

International Research Conference on
HLB: Reaching Beyond Boundaries
December 1-5, 2008, Orlando
For more information and registration,
visit
http://www. doacs.state.fl. us/pi/hlb confe
rence/


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