Citrograph March / April 2010

P R S R T S T D
U . S . P O S T A G E P A I D
P O N T I A C , I L 6 1 7 6 4
P E R M I T 1 2 5
C I T R U S R E S E A R C H B O A R D , P . O . B o x 2 3 0 , V i s a l i a , C A 9 3 2 7 9
A d d r e s s S e r v i c e R e q u e s t e d
March/April 2010
Citrograph
Citrograph
Lindcoves
50th
Anniversary
March/April 2010 Citrograph 3
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An Ofcial Publication of the Citrus Research Board MARCH/APRIL 2010 VOLUME 1 NUMBER 2
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Canadian & Foreign:
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EDITORIAL BOARD
4 Editorial
6 Industry Views
8 University of California Lindcove Research
and Extension Center celebrates its
50th Anniversary
12 CRB 2009Annual Report
16 G. Harold Powell and the defeat of
citrus decay, 1904-1912
20 Descriptions of new varieties recently
distributed from the Citrus Clonal
Protection Program
27 Parasitoid preference for citricola scale in
southern California citrus versus San
Joaquin Valley citrus
30 Novel immunocapture technology for eld
deployable nucleic acid-based detection
of plant pathogens
32 Identication of Spiroplasma citri secreted
proteins as detection markers for citrus
stubborn disease
33 Probiotic diet for SIT Medy
4 Citrograph March/April 2010
EDITORIAL
Industry adopts vehicle to battle pests/diseases
A
s we begin a new decade, our industry once again shows its age-old ability to work together
for the common good. Witnessing the disaster in Florida associated with the Asian citrus
psyllid and Huanglongbing, Californias citrus industry leaders, growers and organization
directors all saw the need for a vehicle to avoid the catastrophe now occurring in Florida.
Temporarily, that need and vehicle has been our Citrus Research Board (CRB). But the program
and effort now being designed and implemented by federal and state government in partnership with
industry will become controversial. The program will require additional spraying. It will require the
loss of organic production. It will require constant intrusion into residential homes, and it may well
cause the elimination of backyard trees and landscape material.
This controversy cannot be shouldered by our Research Board because inevitably it would then
become incapacitated to perform its core function research! Whether it be research to enable our
industry to create revenue streams via climate change legislation, research for benecial bugs, research
to help thwart ACP and HLB, or research for new varieties, CRB cannot become embroiled in constant
controversy and legal attacks.
It was recognized that another vehicle was needed to specically address this ACP/HLB threat
and similar threats. It was also recognized that our State government was in a scal meltdown, and
many questioned whether it would be there to help when and if another invasive threatened our ability
to produce, harvest and market fresh citrus. Florida didnt have that vehicle, and they are paying a
price. Texas doesnt have that vehicle, and they are behind the eight ball. Mexico, Brazil and other
production areas did not have the industry tool to address challenges such as the ACP/HLB threat.
Thus was borne the legislation to facilitate an industry tool to address pests and diseases specic
to the California citrus industry. Dubbed the 281 committee, which is the legislation number, the
California Citrus Pest and Disease Prevention Committee is now in its early stages. Fourteen growers
within our industry have stepped forward to help give birth to this effort.
Those 14 asked a Fresno County producer, Nick Hill, to be their rst Chair. They asked a
Coachella Valley grower, Craig Armstrong; and a Tulare County producer, Richard Bennett, to also
serve on the executive committee. The balance of the 14 represent a cross section of our industry from
small to large producer; from PCA experience to the handling of fruit. They are Dan Dreyer, Bob
Felts, John Gless, Jim Gorden, Gus Gunderson, Link Leavens, Mark McBroom, George McEwen,
James McFarlane, Dr. Etienne Rabe, and Kevin Severns. There are two oating alternates who are Earl
Rutz and Don Barioni Jr.
They come from all the production areas of this state, and they come with one goal to prevent
the introduction of Huanglongbing and its spread throughout the citrus industry. Their meetings are
public, and their agenda is ambitious. In the next few months they will be discussing an action plan
or protocol should HLB be found in this state. They will take the scientic recommendations, add a
touch of practicality, and subsequently make recommendations to our Secretary of Agriculture. Theyll
determine the best use of industry funds to ll voids in the existing effort. Theyll determine what
components of the existing CRB program will transfer to the AB 281 committee. Actually there is a
whole lot more, but one can see how ambitious their agenda will become.
The Committee will be staffed by the California Department of Food and Agriculture and it will
have the authority to advise the Secretary on specic pest and disease issues. It will have the power
to create the funding necessary to protect our valuable industry. The closest vehicle to this tool is the
glassy-winged sharpshooter program and advisory group, the Pierces Disease Control Program. This
new citrus program is far more encompassing and, again, a bit more precedent-setting.
But thats what we should expect from an industry that provides leadership to California
agriculture and the nations specialty crop industry. So while it may be a new decade, our industry
leadership is once again coming back to the future and creating a mechanism that is sure to be copied.
Next to our citrus fruit, that is the commodity we are best known for!
California Citrus Mutual (CCM) is a nonprot, grower-based trade association formed in 1977 by citrus
growers, for citrus growers. The organization is headquartered in Exeter, California.
BY JOEL NELSEN, President, California Citrus Mutual
They will take
the scientic
recommendations,
add a touch of
practicality, and
subsequently
make recom-
mendations to
our Secretary of
Agriculture.
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District 2 Southern California Coastal
Member Alternate
Earl Rutz, Pauma Valley Alan Washburn, Riverside
William Pidduck, Santa Paula James Finch, Santa Paula
Joe Barcinas, Riverside Ken Kelley, Hemet

District 1 Northern California
Member Alternate
Allan Lombardi, Exeter Kevin Severns, Orange Cove
Donald Roark, Lindsay Dan Dreyer, Exeter
Jim Gorden, Exeter Dan Galbraith, Porterville
Joe Stewart, Bakerseld Franco Bernardi, Visalia
Etienne Rabe, Bakerseld Richard Bennett, Visalia
John Richardson, Porterville Jeff Steen, Strathmore
Kevin Olsen, Pinedale David Dir, Visalia
CITRUS RESEARCH BOARD MEMBER LIST BY DISTRICT 2009-2010
Citrus Research Board
323 W Oak, Visalia, CA 93291
PO Box 230, Visalia, CA 93279
(559) 738-0246
FAX (559) 738-0607
E-Mail Info@citrusresearch.org
District 3 California Desert
Member Alternate
William Stein, Oasis John Turco, Indio

Public Member
Member Alternate
Seymour Van Gundy, Riverside Steve Garnsey, Fallbrook
The Mission of the Citrus Research Board:
Develop knowledge and build systems for grower vitality.
Focus on quality assurance, clonal protection, production research,
variety development, and grower/public education.
INDUSTRY VIEWS
What is the hottest topic you are
facing at this time?
asks: Citrograph
I
would be surprised if any California citrus nursery owner answered anything other than ACP/
HLB to this question. We are business as usual on the outside, but inside we are all about the
upcoming changes in our industry. We have begun the physical transformation of our nursery,
from eld-grown trees to container trees grown in hot houses, protected from the Asian citrus
psyllid. We continue to do research on matters of containers, soil mixes, cold frame structure vs.
climate-controlled houses and many other questions related to the impending changes. We are
evaluating all the options so that we will create the highest quality tree at a reasonable price for
our customers. So far the ACP/HLB threat has been a huge challenge that is keeping our entire
staff on its toes; however, we at Willits & Newcomb are excited about developing new methods of
producing quality citrus trees. Jackie Maxwell, President, Willits & Newcomb Citrus Nursery
T
he Asian citrus psyllid/Huanglongbing threat is our greatest focus in our citrus nursery
program. At Duarte Nursery our efforts are focused on delivering growers disease-free
citrus trees while operating under conditions of, hopefully distant, future ACP infestations
throughout California. We have introduced over 50 commercial varieties of citrus scion and
rootstock into in vitro culture for both production and germplasm banking and will continue with
another 40 50 this year. We have begun screening of our greenhouses. This fall we will begin
building our specialized clean citrus propagation facility where laboratory-produced plantlets
of both scion and rootstock will be grown into rootstock liners, scion blocks and nished trees
under fully protected screened greenhouses near Modesto, CA. It is thought that major inter-
regional spread of the HLB in Florida and other southern states was through infected nursery
stock plants. We believe that our preparations to maintain an in vitro germplasm bank and to
produce HLB-free trees under eventual ACP pressure are very important to the long-term
viability of the California citrus industry. In either case, whether genetic cures arise for HLB,
California growers will need to protect their current generations of plantings while cycling in
newly developed, and currently nonexistent, commercially acceptable resistant varieties. We
at Duarte Nursery are preparing for a future when disease-free nursery stock is paramount.
John Duarte, President, Duarte Nursery, Inc.
C
itrus nurseries in California have enjoyed growing in the outdoors in the last 100 years in
an environment with few diseases that were vectored by insects. Although stubborn dis-
ease and Citrus tristeza virus (CTV) have been endemic in citrus growing areas for years, the
incidence in citrus nurseries has been a rare occurrence. The threat of HLB disease vectored by
the Asian citrus psyllid (ACP) coupled with the ongoing concern of CTV infection has caused
citrus nurserymen to rethink their business plans and evaluate the costs of moving into an insect
excluding environment. Greenhouses or screenhouses built to exclude insects have been in use
in Brazil for the past 10 years and became mandatory in Florida four years ago. The concept is
simple keep out the insect vector and you keep out the disease. Entry doors are equipped with
air curtains that blow on you as you enter, and when an outside door is open, an inside door
is closed, which is called a double entry door system. All air intakes are covered with screen
boxes, and any opening to outside air is either screened or sealed off. In Brazil, some nurseries
even require workers to change clothing before entering the facilities to prevent bad bugs from
hitchhiking on clothing. The success of these measures proves out in Florida where no ACP have
been found in a citrus nursery since they all moved indoors. The cost of these measures is high,
from $10-$15 per square foot, so nurseries will soon be measured in square feet not acres. Citrus
trees grown in protected environments will have to be smaller but can be as good as the old
reliable eld-grown ball & burlap tree if 21st century technology in soil media, pot design and
environment control are used. Growers can count on quality trees grown in nurseries that are
protected from disease, but they will have to adapt to the new look of these trees and the higher
cost that will inevitably be required to fund these expensive measures to insure a strong future
for the California citrus industry. Roger Smith, General Manager, TreeSource Citrus Nursery.
8 Citrograph March/April 2010 8 Citrograph March/April 2010
T
he University of California
Lindcove Research and Exten-
sion Center (Lindcove LREC)
celebrated its 50th Anniversary on
December 11, 2009. The celebration in-
cluded a dedication of the new research
laboratory (funded by the University
of California) with a ribbon cutting by
Associate Vice President of Programs
Barbara Allen-Diaz and Lindcove REC
Director Beth Grafton-Cardwell. A new
screenhouse (funded by the Citrus Re-
search Board-CRB) that protects Citrus
Clonal Protection Program (CCPP)
budwood source trees from pests and
diseases was dedicated with a ribbon
cutting by Chairman of the CRB Jim
Gorden, CRB Vice President of Science
& Technology MaryLou Polek, Vice
President of UC Agriculture and Natu-
ral Resources Dan Dooley, and Director
of the CCPP Georgios Vidalakis.
Additional events included the annu-
al Fruit Display and Tasting in the Con-
ference room, sensory taste testing of
citrus fruit by UC Riverside Subtropical
University of California Lindcove
Research and Extension Center
celebrates its 50th Anniversary
Beth Grafton-Cardwell, Georgios Vidalakis, and Ray Copeland
Director Beth Grafton-Cardwell with Barbara Allen-Diaz, Bill
Frost, Shawn Tibor and Luzanne Martin dedicate the new
laboratory at Lindcove REC.
Jim Gorden, Dan Dooley, Georgios Vidalakis and MaryLou
Polek (left to right) cut the ribbon for the new CCPP
screenhouse, funded by the Citrus Research Board.
Horticulture Extension Specialist Mary
Lu Arpaia and USDA-ARS researcher
Dave Obenland, and a walking tour of
the citrus trees in the Lindcove REC
demonstration block by UC Riverside
Senior Museum Scientist Tracy Kahn.
The history of the Lindcove REC
(until 1992 known as Lindcove Field
Station) is closely linked to global events
such as the end of World War II and sev-
eral California citrus industry decisions,
such as the petition to the University
of California for the development of
variety foundation plantings which
resulted in the establishment of the
Citrus Variety Improvement Program
(CVIP) in 1957 (today known as the
CCPP) and the creation of the CRB in
1968 (Calavan et al., 1978).
Following World War II, southern
California became the aviation manu-
facturing center for the USA as well as
much of the world. The need for skilled
labor in the factories plus the ideal
subtropical climate acted as a magnet
in drawing many of the returning GIs
to the area. The huge inux of people
created a need for housing, turning
productive farm land into housing tracts.
One of the prime areas for new citrus
groves in the 1950s was near the foothills
in Kern, Tulare and Fresno counties of
the San Joaquin Valley, and many south-
ern California citrus growers made the
migration north.
As the citrus industry grew in the San
Joaquin Valley, the need for information
concerning citrus production increased.
Most of the citrus research in California
had been done through the University
of California (UC) Citrus Experiment
Station located in Riverside, California
under conditions quite different than
those in Tulare County. In the mid-1950s
with leadership from UCCE Tulare
County Farm Advisor Karl Opitz and
UC Cooperative Extension Director
Sheldon Jackson, an advisory committee
of citrus growers was formed to nd a
site in Tulare County that would be suit-
able for the establishment of research
plots by UC Riverside researchers. A
March/April 2010 Citrograph 9 March/April 2010 Citrograph 9
Dedication ceremony of the new CCPP screenhouse.
number of sites were reviewed with the
nal selection of the Lindcove site which
provided researchers an opportunity to
look at problems and conduct research
in the San Joaquin Valley citrus grow-
ing areas.
Initial property for the station was
donated by Adna Neil, a citrus nurs-
eryman and grower. By the spring of
1959, the grower committee, chaired
by citrus grower Roy McLean, turned
over the keys to Dan Aldrich, Dean of
Agriculture Sciences for the University
of California Riverside in a dedication
ceremony on the hillside overlooking
the current shop and greenhouse areas
(Citrograph 1959). Willard Bitters of the
Department of Botany and Plant Sci-
ences, UC Riverside, was the rst acting
director of the station. In the rst year,
an ofce was constructed, land was lev-
eled and irrigations systems installed in
preparation for the planting of the rst
orchards in 1960.
The funding of this off-campus
research facility was difficult for the
Botany and Plant Sciences Department,
and so in 1964 UC Riverside turned over
the operation of the Lindcove REC to
the statewide UC Agricultural Field Sta-
tions. From 1965 to 1987, UCCE Tulare
County Farm Advisor Ray Copeland
was the Superintendent of the station,
and this period was characterized by
development of the land and facilities
(Citrograph 1985). Land was donated
or purchased to bring the station up to
its current 175 acres, and research proj-
ects expanded to include variety trials,
rootstock trials, and pest management
studies.
Land purchased in 1959 was largely
dedicated to the establishment of the
primary variety foundation planting
(in addition to three secondary founda-
tion plantings in Ventura and Orange
counties) (Reuther et al., 1972). In
1961, ve years after the establishment
of the CCPP (then named CVIP), the
rst disease-free budwood source trees
were moved out of the Riverside CCPP
quarantine facilities and in to the Lind-
cove REC for planting in what is known
today as the CCPPs Foundation and
Evaluation Blocks. Since then, 807
different citrus varieties from around
the world have completed the rigor-
ous Variety Introduction-VI disease
testing and therapy program under
quarantine at the CCPP, and more than
4,000 budwood source trees have been
propagated and safely moved for plant-
ing at Lindcove REC.
For decades now, the Lindcove REC
has served the very important function
of maintaining the CCPP budwood
tree sources as well as assisting with
the trueness-to-type evaluations and
budwood distribution to nurseries,
growers and researchers in California,
Arizona, Texas, Alabama, and several
countries around the world. CCPPs
budwood distribution records indicate
that 750,000 buds have been distributed
from the Lindcove REC-housed CCPP
budwood source trees since 1982. Speak-
ing conservatively, a citrus nursery can
produce 200 eld trees from each CCPP
bud within a year. Thus, in the last 28
years the Lindcove REC has been the
source of at least 150 million citrus trees
worldwide.
Outbreaks of diseases have always
been driving forces for major changes
in the California citrus industry and
research. The widespread occurrence of
psorosis in the 1930s and the epidemic
of tristeza in the 1940s to the 1960s, in
combination with the rapid spread of the
exocortis and stubborn disease in the
1950s, formed many of todays citrus cul-
tural practices as well as the current reg-
istration and certication program for
citrus budwood. In a similar manner, the
increased threat of the tristeza disease
around the Lindcove REC prompted
the CRB to fund the construction of a
screenhouse for the establishment of
the rst CCPP Protected Foundation
Block in 1998.
Today, the Protected Foundation
Block covers 65,000 square feet which
contain approximately 1,000 trees rep-
resenting 400 different citrus varieties.
In 2007, after almost 50 years, the Pro-
tected Foundation Block in Lindcove
REC replaced the open eld trees of
the Foundation and Evaluation Blocks
as the primary source for budwood
distribution. Over the past three years,
the Protected Foundation Block at
Lindcove REC has fullled 400 orders
for 280 different varieties that total ap-
proximately 100,000 buds.
Since its establishment in 1968, the
CRB has provided grant funding for
the CCPP program and the majority of
the research projects that take place at
Lindcove REC. In the 1990-2000s, when
Walter Stutzman and Louis Whitendale
were superintendents, the CRB became
an especially important partner in the
rapid expansion of the facilities of
Lindcove REC.
In 1995, the Citrus Research Board,
California Citrus Quality Council
(CCQC) and a number of citrus indus-
try equipment manufacturers provided
the funding and equipment to build a
packing facility to conduct postharvest
fruit quality evaluations. Prior to the
construction of this facility, researchers
manually counted, sized, and rated the
damage of fruit from research trees
on tables in the orchard. Manual labor
limited the amount of data that could
be collected and the number of proj-
ects that could be supported each year.
With the building of the packline, which
includes a high pressure washer, waxer
and dryer, sorters, and an electronic
eye that can count, size, and grade fruit,
researchers are able to rapidly collect
data from individual trees in their plots.
This equipment provides a much greater
depth of knowledge of the effects of
scions and rootstocks on fruit size, yield
and quality.
The Citrus Research Board also pro-
vided funding to build a Fruit Quality
Laboratory. The laboratory has equip-
ment and staff that allows researchers to
measure parameters such as fruit length
and width, color, rind thickness, fruit and
juice weight, % juice, total soluble solids,
% sugar/acid ratio, and % crease and
puff -- again, expanding the parameters
that researchers can study.
In 2004, the University constructed
a conference building at Lindcove REC.
The conference building hosts meetings
of University and citrus industry organi-
zations and hosts educational programs
on citrus varieties, pest management,
and horticultural practices for citrus
growers, PCAs and nurserymen as well
as Master Gardeners and the general
public. Each December, the conference
building hosts a two-day event to display
more than 100 varieties of citrus from
the Foundation Block for tasting by the
growers and the general public. For the
growers, this provides them an opportu-
nity to evaluate new citrus varieties and
helps them make decisions on planting
and topworking. For the homeowners,
the fruit display educates the public
about the large number of varieties of
citrus available and the timing of their
maturation.
Lindcove REC has always had an
impressive collaboration between UC
Riverside and UC Davis researchers and
extension specialists, UC Cooperative
Extension Farm Advisors, and USDA
researchers. Lindcove REC currently
supports an average of 30 research
projects per year covering topics such
as freeze damage measurements, volatile
organic compound (VOC), measure-
ments, pruning, irrigation, variety de-
velopment, pesticide treatments for
nematodes, insects, mites, and posthar-
vest diseases, and economic thresholds
for pests. One of the early collaborative
research contributions was the develop-
ment of techniques for detecting and
removing graft transmissible pathogens
such as viruses and viroids from citrus
trees in order to support the CCPP
budwood distribution program.
Because of its ability to conduct
long-term studies under controlled
circumstances, Lindcove REC is able
to provide the citrus industry and
regulatory agencies with information
about fruit production and fruit quality
on a wide array of rootstock and scion
combinations as well as study problems
that develop with time. For example, the
California Department of Food Agri-
culture (CDFA) has been using either
directly or indirectly data generated
by Lindcove REC studies performed
by David Gumpf and Joseph Semancik
(Dept. of Plant Pathology, UC River-
side) for the determination of maturity
and quality standards for harvest and
packing of individual varieties as well
as the commercial use of two selections
of small transmissible RNAs which
have been shown to modify the growth
of citrus varieties growing on trifoliate
orange rootstock. Lindcove REC has
played a signicant role in the expan-
sion of the number of navel varieties
grown in the San Joaquin Valley from
a few navel varieties (Washington, T.I.
and Atwood) to many additional vari-
eties (Powell, Fukumoto, Lane Lates,
Chislet, Barneld, and Bonanza). More
recently, researchers have been study-
ing the many varieties of the wonderful
tasting and easy to peel mandarins at
Lindcove REC.
Examples of several current re-
search projects at Lindcove include the
breeding program of Mikeal Roose,
the fruit quality evaluation program of
Tracy Kahn, and the pesticide evalua-
Director Beth Grafton-Cardwell with
retired nurseryman Raul Gonzales and
superintendents Walt Stutsman and Ray
Copeland (left to right).
Tracy Kahn leads a walking tour of the citrus trees in
the demonstration block at Lindcove REC.
Chet Roistacher planting
the rst citrus tree in the
Foundation Block at LREC.
Photo by E.C. Calavan
tion program of Beth Grafton-Cardwell.
Mikeal Roose of the UC Riverside
Dept. of Botany and Plant Sciences
plants trees from his scion breeding
program at Lindcove REC to determine
their growth characteristics on various
rootstocks under San Joaquin Valley
conditions. In recent years, Dr. Roose
has been utilizing irradiation of citrus
tissue to create trees that are evalu-
ated at Lindcove for qualities such as
seedlessness. From this process, he has
created several seedless varieties of
mandarins, most notably the Tango, a
seedless form of the W. Murcott Afourer.
Tracy Kahn, the curator of the
Riverside Citrus Variety Collection,
began planting trees for evaluation and
demonstration of differences in variet-
ies at Lindcove REC during 1992. This
demonstration block provides San Joa-
quin Valley fruit quality and maturation
data, which is reported on the website
http://www.citrusvariety.ucr.edu/. The
demonstration block also provides an
excellent teaching tool for the eld day
events conducted by Dr. Kahn and the
tour groups that visit Lindcove.
Beth Grafton-Cardwell, Dept. of En-
tomology UC Riverside, has conducted
more than 80 insecticide trials during the
past 20 years at Lindcove REC. In these
studies, she has evaluated the efcacy
and residuality of various rates and tim-
ings of insecticides against pests such as
California red scale, citricola scale, citrus
red mite, katydids, citrus cutworm, citrus
peelminer, citrus leafminer, and ants
and their selectivity favoring natural
enemies. This work could not be done
elsewhere because it often includes un-
registered insecticides that require crop
destruction. Her research is the basis for
the UC IPM pest management guide-
lines for citrus and for recommendations
presented at numerous eld days, slide
shows and workshops.
The research projects and exten-
sion programs at Lindcove REC have
educated thousands of citrus growers
and nurserymen about the best methods
for producing the many varieties of cit-
rus grown in the San Joaquin Valley of
California. The success of this program is
due to the excellent collaboration of the
University and USDA project leaders
and their staff with the citrus industry.
Lindcove REC is toured by visitors
from all over the world who marvel at
the quality of the trees and the research
programs and view it as a jewel of the
California citrus industry.
Beth Grafton-Cardwell is Extension
Specialist and Research Entomologist,
UC Riverside and Director of Lindcove
Research and Extension Center. Georgios
Vidalakis is Extension Specialist and
Plant Pathologist, UC Riverside, and
Director of the Citrus Clonal Protection
Program. Ray Copeland, UCCE-retired,
served as Superintendent of Lindcove
Field Station from 1965-1987.
1959. Lindcove Field Station Dedi-
cated. Citrograph 44 (8): 262.
Calavan C.E., Mather S.M., and
McEachern E.H. 1978. Registration, cer-
tication, and indexing of citrus trees. In
Reuther W., Calavan C. E., and Carman
G. E. (eds.). The citrus industry Vol. IV.
Crop protection. Chapter 3, pages 185-
222. University of California, Division of
Agricultural Sciences.
Reuther W, Calavan C.E., Naurer
E.M., and Roistacher C.N. 1972. The Cali-
fornia Citrus Variety Improvement Pro-
gram after twelve years. p 271-278 in: The
5th Proc Int Org Citrus Virologists. Univ
of Florida Press, Gainesville, Florida USA.
1985. Lindcove at 25: Planning for the
Future. Citrograph 70 (6):120-121.
EXPENSES
RESEARCH PROGRAM
Plant Management
DMS VOC Sensor for Citrus ............................ 189,641
Measuring Ozone Removal............................. 176,958
Subtotal Plant Management ......................... 366,599
T
his past scal year (2008-2009) has been a season
of change for the Citrus Research Board. With the
discovery of the Asian citrus psyllid in 2008, the
Program took on a completely new adventure, that of de-
veloping an Early Detection-Rapid Response operational
program. In late 2008, the industry approved a change to
the authorities for the CRB Program to include an opera-
tions function for pest and disease detection and identica-
tion. This has provided the legal
foundation for the addition of
an Operations Department.
The audited budget gives
the amount of funding to the
Operations Department along
with the initial functions of the
Program. As you can see, con-
siderable attention has been
given to the new program as
a measure of protecting the
California citrus industry. The
2008-2009 funding went to the
formation of the eld teams, the establishment of the data
management system, and the opening of the laboratory in
Riverside. By the end of the scal period in October, all
three systems were up and operating. The current scal year
has seen an expansion of the eld teams to accommodate
expanded trapping programs throughout the state.
The research program for the 2008-2009 scal year
expanded the focus on citrus disease diagnosis and un-
derstanding. The California industry is in a unique posi-
tion in that the pest is just arrived and the bacterium that
causes HLB has not been detected in the state. Therefore,
investments in improved detection systems for both the
pest and the bacterium have been prudent. The Program
continues to create systems that will attract the ACP to the
trapping systems through research in the role of volatile
organic compounds (VOCs) to be attractive to the pest.
This process should lead to a trap that will have a wider
range of detection and increase the probability of nding
CRB 2009 Annual Report
breeding populations early in their development.
Additional areas of research are the continuation of the
pest programs including efcacy trials of new compounds,
work on biological control programs, and resistance man-
agement. IPM continues to be the main driving force in
future research. Understanding resistance management
will help keep the existing crop protection tools working
for the growers as new technologies are developed.
The postharvest investments are aimed at providing
tools for maintaining our markets with fruit that is of the
highest quality as it reaches the consumer. Additionally the
Program includes developing new tools to meet the inter-
national marketing requirements and tools for increased
efciency in harvesting.
The Board welcomes your comments and observations
to the Citrus Research Program. Please feel free to contact
us with your input at any time.
Ted Batkin, President
INCOME
2008 Fund Balance (Carryover) ...................... 921,897
2008-2009 FY Assessment Income ............. 5,049,591
Prior Season Income ...................................... 359,283
Outside Income .................................................. 6,075
Grower Seminar Registration Income ................. 8,058
Investment Dividend Income ............................ 27,690
TOTAL FUNDS AVAILABLE ........................... 6,372,594
CITRUS RESEARCH BOARD
November 1, 2008 through October 31, 2009
Plant Improvement
Citrus Rootstock Evaluation .............................. 80,000
Variety Evaluation for Trueness ........................ 60,000
New Citrus Breeding ...................................... 116,830
Evaluation of Strain Trials ................................. 29,595
Evaluation of Desert Lemons .............................. 9,802
Subtotal Plant Improvement ...................... 296,227
Biotechnology
Improving Peel Quality in Citrus ....................... 10,000
Sweet Orange Physical Map ............................ 28,850
Subtotal Biotechnology ................................. 38,850
Plant Pathology
Septoria Spot ................................................... 39,500
HLB Management Systems .............................. 45,000
Small RNA for HLB ........................................... 89,006
Small RNA for Stubborn ................................... 26,739
Lateral Flow Microarray ................................. 180,000
Fingerprinting of HLB ....................................... 35,413
Cultivation and Sequencing of HLB .................. 58,300
Evaluation of Field Treatments ........................... 6,400
Investigation of Seedling Yellow ....................... 30,000
Identication of Spiroplasma ............................ 57,500
Subtotal Plant Pathology ............................ 567,858
Entomology
Pest Management Infrastructure .................... 195,000
Chemical Control of Thrips ............................... 75,000
Armored Scale Research .................................. 27,040
Rearing Peelminer Parasitoids .......................... 59,540
Parasitoid of Citricola Scale .............................. 45,789
Nematodes Against Diaprepes ......................... 32,959
Dev. Of Attractants/Repellents for ACP ............ 173,625
Probiotic Diet ..................................................... 6,335
Assessment of Systemic Neonicotinoid .......... 156,449
Subtotal Entomology ................................... 771,737
Post Harvest
Treatment Evaluation ....................................... 46,500
New Methods for P.H. Decay ............................ 46,000
Robotic Picker ............................................... 100,000
Ethyl Formate Studies for Bean Thrips .............. 35,900
Subtotal Post Harvest ................................. 228,400
TOTAL RESEARCH PROGRAM .................... 2,269,671
COMUNICATIONS PROGRAM
Core Grower Education Program ...................... 31,439
HLB Task Force .............................................. 124,398
HLB Task Force Activities ...................................... 863
Conferences .................................................... 12,070
HLB/ACP Grower Training ................................. 10,565
TOTAL COMMUNICATIONS PROGRAM .......... 179,335
CITRUS CLONAL PROTECTION PROGRAM
Core Citrus Clonal Protection Program ........... 363,255
Facilities Improvement ................................... 109,741
Screenhouse Construction ............................. 159,544
TOTAL CITRUS CLONAL
PROTECTION PROGRAM ............................... 632,540
OPERATIONS PROGRAM
Salaries Management ................................... 88,589
Benets Management ................................... 14,819
Payroll Taxes Management ............................ 6,777
Travel & Mileage Management ...................... 17,295
Salaries ACP Field Stafng ............................ 45,899
Benets ACP Field Stafng .............................. 4,115
Payroll Taxes ACP Field Stafng ...................... 3,511
Travel & Mileage ACP Field Stafng ............... 11,731
Salaries Laboratory Stafng .......................... 55,208
Benets Laboratory Stafng .......................... 10,451
Payroll Taxes Laboratory Stafng .................... 4,223
Travel & Mileage Laboratory Stafng .............. 4,632
Salaries Data Management ........................... 40,625
Benets Data Management ........................... 12,665
Payroll Taxes Data Management ..................... 3,108
Travel & Mileage Data Management ............... 2,132
Equipment Purchase/Supplies
Data Management .................................... 17,811
Equipment Repair & Maintenance
Data Management ......................................... 780
Ofce Equipment Data Management ............... 2,832
Information Services Data Management ....... 20,372
Supplies Data Management ............................ 2,464
Telephone Data Management ......................... 3,008
Rent Data Management .................................. 7,500
Equipment/Supplies Lab Operations ............. 17,273
Equipment Repairs & Maintenance
Lab Operations ........................................... 7,616
Insurance Bonds Lab Operations ........................ 160
Ofce Equipment Lab Operations .................... 1,163
Supplies Lab Operations ............................... 33,553
Building Re-Model Lab Operations ................ 43,093
Sandy Creighton, Sales Manager
Phone: 559-433-9343
E-mail: screighton@farmprogress.com
W
hether you're selling tractors or other farm
equipment,pickup trucks, irrigation equip-
ment, fertilizer or pesticides...consider the value
of your ad dollar in the pages of Citrograph.
Each issue reaches every commercial citrus grower
in the states of California
and Arizona, plus associ-
ated business members
afliated with the citrus
industry...the people
in charge of purchas-
ing. Your advertising
message is directed
to farm leaders who
use vast amounts of
goods and services.
Circulation reach-
es over 5,000 key
decision makers among California and
Arizona fresh citrus growers, landowners and
industry-involved companies. In the near future,
Citrograph will reach the entire United States.
Dont miss the next issue!
Reach
Commercial
California &
Arizona
Citrus Growers
Contact us today to be
included in future issues
of Citrograph
Utilities Lab Operations ................................... 4,761
Telephone Lab Operations ............................... 8,970
Rent Lab Operations ..................................... 21,413
Equipment/Supplies Field Operations ................. 647
Supplies Field Operations ............................. 41,567
Telephone Field Operations ................................ 1,751
Equipment Purchases Transportation .......... 145,153
Equipment Repair & Maintenance
Transportation ................................................ 151
Insurance & DMV Fees Transportation ............. 2,887
Miscellaneous Expenses ....................................... 446
Printing ................................................................. 178
Ofce Supplies ................................................ 12,808
Other Projects .................................................... 1,089
TOTAL OPERATIONS PROGRAM .................... 725,226
CALIFORNIA CITRUS QUALITY
COUNCIL (CCQC)
CCQC Administration ...................................... 371,600
TOTAL CALIFORNIA CITRUS
QUALITY COUNCIL (CCQC) ........................... 371,600
GENERAL AND ADMINISTRATIVE
Audit Fee ........................................................... 7,498
Equipment/Supplies Purchased .......................... 6,798
Employee Benets ........................................... 87,078
Equipment Repair & Maintenance .................... 80,824
Equipment Rental .............................................. 5,630
Information Services ........................................ 20,193
Insurance & Bonds ........................................... 10,095
Ofce Supplies ................................................ 26,174
Postage ............................................................. 7,842
Printing ............................................................ 18,627
Rent & Storage ................................................ 36,967
Research Consultant .......................................... 5,286
Salaries ......................................................... 345,989
Meeting Costs .................................................. 31,243
Payroll Taxes .................................................... 39,283
Telephone ........................................................ 10,612
Travel & Mileage Consultant .......................... 5,733
Travel & Mileage Members ........................... 54,063
Travel & Mileage Staff ................................... 54,910
CDFA Bureau of Marketing ............................ 55,289
CDFA Handler Audit ......................................... 6,750
TOTAL GENERAL & ADMINISTRATIVE .......... 916,884
SUMMARY
TOTAL EXPENSES ...................................... 5,095,256
RESERVE AT END OF YEAR ........................ 1,277,338
TOTAL ALL ................................................................. 6,372,594
OPERATIONS PROGRAM...continued
CRB launches
special ACP/HLB
survey website
T
he Citrus Research Board
has begun working to detect
the Asian citrus psyllid and
Huanglongbing in commercial cit-
rus groves across the state. CRB
has a crew of nine trappers working
in eight counties and has already
established over 6,700 ACP trap lo-
cations with more being added daily.
The CRB diagnostic laboratory in
Riverside is testing both insect and
plant tissue samples for the pres-
ence of the HLB bacterium.
With the help of the University of
California, CRB has completed the
rst phase of constructing a special
citrus invasive pest website, and we want you to see it. This will be the principal
vehicle for extending the results of the ACP/HLB detection program in com-
mercial citrus. In the future, it will be expanded to include additional exotic
citrus pests and diseases and become a valued resource for growers, packers,
collaborating researchers, regulators, and area-wide pest managers. Despite the
preliminary nature of this launch, the site already has many features and func-
tions with more to come.
Access to the site will be limited to legitimate stakeholders in the California
citrus industry including growers, handlers, researchers, and regulators. Citro-
graph readers, feel free to request your own new account at any time.
Access the site at: https://crbcitrussurvey.uckac.edu/viewer
Through the end of May, use Username: DemoGrower
Password: 123EasyDemo*
CRB invites your comments, questions and suggestions.
Contact Richard Dunn, CRBs data, information & management director, by email
to rick@citrusresearch.org or by phone at (559) 738-0246
H. Vincent Moses
G. Harold Powell and the
defeat of citrus decay,
1904-1912
The directors of this Foundation
are elated and honored in having
this opportunity to showcase
our work through the Citrograph
magazine. Our Mission is to el-
evate the awareness of California
citrus heritage through publications,
education, and artistic work. We
are pleased with the response of the
three current university displays: the
University Library-Special Collec-
tions at Cal Poly Pomona, Pomer-
antz Library at Western University,
and our largest display in the John
M. Pfau Library at CSU San Ber-
nardino. We are especially happy to
report that the Foundations latest
book has just arrived, titled Citrus
Powered the Economy of Orange
County for Over a Half Century In-
duced by A Romance. Please visit
our website www.citrusroots.com.
Citrus Roots
Preserving Citrus Heritage Foundation
B
y 1900, something was very rot-
ten indeed but not in Denmark.
Every year since the emergence
of Californias modern citrus enterprise,
devastating rot had manifested itself in
thousands of refrigerator rail cars laden
with California citrus. These cars carried
the expanding, extremely lucrative and
intensely sought-after orange and lemon
crops of thousands of growers.
Growers complained to the USDA
and the University of California that
they were losing as much as a million
dollars a year to decay while their fruit
was in transit to New York, New Orleans,
Chicago, London (Ontario, Canada) and
other distant market destinations. The
losses were hurting their bottom line
and threatening the future of the entire
California citrus enterprise.
Already practitioners of Professor
Liberty Hyde Baileys scientic agricul-
ture and modern business organization,
these growers wanted answers, particu-
larly to the problem of citrus decay in
transit. Many growers thought this
situation inherent in the keeping quality
of oranges and lemons. They assumed
it had to be considered part of doing
business. Other growers blamed the rail-
roads for mishandling their shipments
and sued them constantly in Commerce
Court or led complaints with the I.C.C.
Certain scientifically astute, busi-
ness-minded growers in Riverside
refused to accept either the inherent
weakness argument or the railroad
bugaboo theory, believing that the
industry could not sustain such losses
without long-term impact on the busi-
ness. Their persistent demands on the
We are proud of our accomplishments
as a volunteer organization, which
means each donated dollar works for
you at 100% [for we have no salaries,
wages, rent, etc.]. All donations are tax
deductible for income tax purposes to
the full extent allowed by law.
Citrus Roots Preserving Citrus
Heritage Foundation
P.O. Box 4038, Balboa, CA 92661
USA
501(c)(3) EIN 43-2102497
USDA eventually prevailed. In short
order, this triumph sealed the future of
the industry, ultimately guaranteeing
its existence as what Harvard business
historian Alfred D. Chandler, Jr. has
described as a modern industrial enter-
prise, on par with the big meat packer
trusts Swift and Armour.
In 1904, orange grower pressure
brought youthful, talented and brash G.
Harold Powell roaring into the region.
One of Uncle Sams missionaries,
fresh from the USDAs Bureau of Plant
Industry, Powell expected to educate
growers in scientic practices. Would
he be surprised! Born February 8, 1872
as part of the post-Civil War generation
that created the Progressive Movement,
Powell grew up on the comfortable and
successful commercial apple orchards of
his inuential father, George T. Powell, a
notable Quaker, at Ghent, New York. G.
Harold Powell worked his way through
Cornell University and took his training
in horticulture under the agricultural
rock star at Cornell, Professor Lib-
erty Hyde Bailey, who helped him gain
appointment as the Graduate Fellow in
Horticulture.
Powell took his Master of Science
in Horticulture and soon landed a job
with the Bureau of Plant Industry of
the United States Department of Agri-
culture, Washington, D.C. It was 1901,
and Powell reveled in his new work as
assistant pomologist assigned to study
the problems of apples in cold storage.
Within two years the Department pub-
lished two of his bulletins, The Apple
in Cold Storage and Cold Storage
with Special Reference to the Pear and
G. Harold Powell (1872-1922), the tallest man in the photograph, standing between B. A. Woodford, President of the California Fruit
Growers Exchange (CFGE) and La Verne grower, and Walter Barnwell, Assistant Freight Agent for the Atchison, Topeka & Santa Fe
Railroad, inspecting one of Powells USDA citrus pre-cooling and refrigeration experiments sponsored by the CFGE, c1907. Photo
courtesy of the Huntington Library.
They launched a
revolution in
the handling of
fresh produce.
Peach. They launched a full-blown rev-
olution in the handling of fresh produce.
His study of barrel spoilage in apples
launched all other such investigations of
the shelf life of perishable agricultural
products in the United States.
As historian Richard Lillard ar-
gued, prior to Powells apple spoilage
experiments, both the USDA and farm-
ers, even the commercial ones like his
father, had concentrated almost solely
on production problems. Then, as a
complement to the apple study, Powell
demonstrated conclusively why Georgia
peaches decayed in transit aboard trains.
Consequently, Powell rose rapidly up
the ladder of his discipline within the
USDAs Bureau of Plant Industry. Just
prior to his rst trip to California in 1904,
B. T. Galloway, Chief of the Bureau,
placed him in charge of the Fruit Storage
and Transportation Division.
As Pomologist-in-Charge, Powell
and his subordinates pursued studies
in ways to obtain more efcient cooling
and air ow in refrigerated rail cars. The
USDA Yearbook of Agriculture pub-
lished the results of his teams research
as The Handling of Fruit for Transpor-
tation in 1905. By this time, however,
the direction and nature of Powells
professional life made a radical shift to
the great West, placing him in position to
ultimately transform the very structure
of agriculture in the entire country.
Here is a reason why. The prominent
leaders among California orange and
lemon growers were previously captains
of industry or doctors,
lawyers, accountants,
bankers, and refugees
from other success-
ful urban professions.
Wi thout prompti ng
from Powell or any oth-
er USDA missionary,
orange growers under-
stood the need to organize for their own
interests along modern business lines.
While some were skeptical of a pure
scientist, Powell soon won them over
with his persuasive manner and practi-
cal knowledge of horticultural practices.
The rise of sanitary engineering,
brought on by Progressive Movement
reformers and muck-raking journalists,
had stimulated leading growers in River-
side, California to seek government help
in nding a solution. In dramatic fash-
ion, Powell and his team quickly identi-
ed both the source of the decay, blue
mold (Penicillium italicum), and the
reasons it took hold in citrus shipments.
Then they submitted proposals for
industrial solutions to the problem, i.e.
careful handling from eld to shipping
crate. Those proposals were adopted
wholesale by growers
and packers
As early as April 7,
1905, Powells team of
agricultural research
scientists had gathered
enough evidence to
provide a searing argu-
ment that decay could
be prevented, and that growers brought
on most of it through their own faulty
practices. April 7th, the date of Powells
well publicized appearance at a Farm-
ers Citrus Institute in Riverside, had
been eagerly awaited and touted by
the regular and agricultural press for
several weeks. Editorials predicted that
Powells work might lead to a revolution
in picking and packing methods.
His Institute presentation, in fact,
Washington navel oranges showing blue mold decay, frontispiece, USDA Bureau of
Plant Industry Bulletin No. 123, Powell et al, The Decay of Oranges While in Transit
from California.
produced radical results. It took on piv-
otal signicance for citriculture practices
in California . These practices would
never be quite the same afterward.
Powell reported to his superior, B. T.
Galloway. The institute which closed
yesterday, he recalled, is said by the
best men here to have been the best one
held in southern California. Set up as a
forum for Powells preliminary ndings,
the two-day meeting drew scores of
growers, packers, and a large contingent
of transportation representatives, both
from the railroads and the refrigerator
car lines such as Armour.
In one fell swoop, he deftly linked
the industry with the scientic prow-
ess of the United States Department
of Agriculture. From that time on, the
industry was his to shape. In 1910, grow-
ers summoned Powell back to California
to head the Citrus Protective League, a
trade association representing 90% of
the industry, and the rst such entity in
agriculture. As manager of the League,
he emerged as a potent spokesman for
the industry, promulgating his ideology
of cooperation wherever and whenever
he could.
By means of his established position
with President Benjamin Ide Wheeler of
the University of California (previously
his wifes Greek professor at Cornell)
and among Washington bureaucrats and
congressmen, he furthered ties between
the state and federal governments and
the growers of California. Lastly, serving
as General Manager of the California
Fruit Growers Exchange, 1912-1922,
Powell insured the future of the industry,
and thus the region, by consolidating the
Exchange into a full-edged managerial
corporation.
Future chapters will put Powell
squarely into the story of California
orange growers, their famous business
organization, the California Fruit Grow-
ers Exchange, and their establishment
of the first professional managerial
corporation in American agriculture.
These additional articles will tell the
story of the arrival of the Revolution
of Corporate Capitalism to California,
and to agriculture. They will describe the
introduction of Country Life ideology
into Southern California via G. Harold
Powell. The story will focus on that
pivotal period when the corporate and
managerial revolution growers led con-
solidated their industry into a modern
business enterprise, with the ability to
shape a regional culture.
Dr. Vince Moses, Owner, VinCate &
Associates Museum and Preservation
Consultants, and member of the Advi-
sory Board, Citrus Roots, Preserving
Citrus Heritage Foundation.
22144 BOSTON AVENUE
EXETER, CALIFORNIA 93221
Tango
Miho Wase
Gold Nugget
Contract Growing
for 2011 Delivery
on
Carrizo Rootstock
559-592-3367 Bus
559-592-4158 Fax
george@mcewen.com
Growing Quality Containerized Trees
at Competitive Prices
C I T R U S A V O C A D O S O L I V E S
Citrus Roots
Preserving Citrus Heritage Foundation
Keeping citrus heritage alive in the minds of those living in California through publications, educational exhibits and artistic works
Citrus Roots...Our Legacy - Volume I
Selling the Gold - History of Sunkist
and Pure Gold
Citrus Roots...Our Legacy - Volume II
Citriculture to Citrus Culture
Citrus Roots...Our Legacy - Volume III
Our Legacy...Baldy View Entrepreneurs
- 25 men & women who left a legacy
All donations are tax deductible for income tax
purposes to the full extent allowed by law.
Citrus Roots Series...
NEWEST RELEASE!!
Citrus Roots...Our Legacy - Volume IV
Citrus Powered the Economy of Orange County
for over a half century Induced by a Romance

By: Rahno Mabel MacCurdy, V.A. Lockabey and others...
compiled and edited by R.H. Barker
GOLD
History of
Sunkist
and Pure Gold
CITRUS ROOTS . . . OUR LEGACY Volume I of III

Selling the
$1500
American Business Cycles from 1810 to 1978
vs. the Life Span of Twenty-Five Entrepreneurs
by Marie A. Boyd and Richard H. Barker
Including a fold out
time line chart of
Our Legacy:
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ENTREPRENEURS
CITRUS ROOTS ... OUR LEGACY Volume III of III CITRUS ROOTS ... OUR LEGACY Volume III of III
For ordering information
visit our website
www.citrusroots.com
(Fed. Tax ID # 43-2102497)
KINGS
ORANGE
IMPERIAL
SAN DIEGO
San Nicolas Island
MADERA
SAN MATEO
MERCED
FRESNO
SANTA
CLARA
INYO
SANTACRUZ
SAN BENITO
MONTEREY
TULARE
San Clemente Island
SAN BERNARDINO
KERN SAN LUIS OBISPO
SANTABARBARA
VENTURA
LOS ANGELES
RIVERSIDE
Santa Cruz Island
San Miguel Island
Santa Rosa Island
UT
NV
AZ
Sonora
Baja California
United States
Department of Agriculture
Animal and Plant
Health Inspection Service
Date Created: April 5, 2010
Data Source:
CADept of Food & Agric.
AZ Dept of Agriculture
USDA, APHIS, IS
TeleAtlas Dynamap
USDA, APHIS, PPQ
Western Region GIS Specialist
650 Capitol Mall, Suite 6-400
Sacramento, CA95814
The U.S. Department of Agriculture's Animal and Plant Health Inspection Service
collected the data displayed for internal agency purposes only. These data may
be used by others; however, they must be used for their original intended purpose.
0 20 40 60 80 100 10
Miles
Asian Citrus Psyllid Cooperative Project
California, Arizona, and Baja California
Coordinate System:
CATeale Albers, NAD83
Legend
Asian Citrus Psyllid, CA_2010 thru 3-29-10
Asian Citrus Psyllid, CA_2009
Asian Citrus Psyllid, CA_2008
Asian Citrus Pysllid, AZ_2010 thru 3-31-10
Asian Citrus Psyllid, AZ_2009
Asian Citrus Psyllid, Mexico_2010 thru 3-25-10
Asian Citrus Psyllid, Mexico_2009
Asian Citrus Psyllid, Mexico_2008
ACP_Traps_Citrus Research Board_3-25-10
Quarantine for Asian Citrus Psyllid, CA_11-17-09
Quarantine for Asian Citrus Psyllid, AZ_12-7-09
Parks in CA
National
State
Local
Map of Asian citrus psyllid detections prior to 3/29/2010 in California and neighboring portions of Arizona and Mexico. This map also
indicates the locations of ACP traps installed by the Citrus Research Board prior to 3/25/2010. Traps installed and maintained by other
agencies are not displayed.
Descriptions of new varieties
recently distributed from the
Citrus Clonal Protection Program
Toni Siebert, Robert Krueger, Tracy Kahn, John Bash and Georgios Vidalakis
C54-4-4 mandarin: Citrus Variety Collection, Riverside,
California, 16 Feb 2010. Photo by T. Siebert
Protected Foundation Block Budwood is budwood
provided from CDFA registered CCPP citrus trees from the
LREC screenhouses and is available from the University of
California in accordance with the CDFA regulations for cit-
rus registration and certication. Protected Foundation Block
Budwood is produced by trees grown in pots and in ground
under protective screen and is intended for individual nurseries
or growers to produce their own registered budwood source
trees or for the production of nursery increase blocks from
which additional budwood may be harvested in accordance
with CDFA (or other appropriate) regulations and used for
the production of certied nursery stock. A signed Waiver
and Release form must accompany all orders for Protected
Foundation Block Budwood. The Waiver and Release form
is available on the CCPP website (http://www.ccpp.ucr.edu).
SRA 337 or C54-4-4 Mandarin (VI 672): First distribution
of buds from the CCPP: September 2009
C54-4-4 was selected for introduction to California in
1997 by members of the California Citrus Nurserymen Society
(CCNS) during a tour of the INRA-CIRAD Station de Re-
cherches Agronomiques in San Giuliano, Corsica, associated
with the Congress of the International Citrus Nurserymens
Society. C54-4-4 is actually a product of California, being
a cross of Clementine X Murcott. The cross was actually
made at the USDA Horticultural Research Laboratory in
T
he Citrus Clonal Protection Program (CCPP)
is operated through the Department of Plant
Pathology and Microbiology at University of
California (UC) Riverside and is funded in large part
by The California Citrus Research Board (CRB). The
CCPP processes citrus propagative material in two
phases. First, during the quarantine phase, citrus bud-
wood of potentially important commercial varieties is
introduced from any citricultural area, germplasm or
breeding program of the world under the authority of
a permit which is issued to CCPP by the United States
Department of Agriculture (USDA) Animal and Plant
Health Inspection Service in cooperation with the Cali-
fornia Department of Food and Agriculture (CDFA).
While in quarantine at the Rubidoux Facility in River-
side (approximately 2.5-3 years), newly imported vari-
eties are tested extensively and any detected pathogens
(such as viruses and bacteria that cause the tristeza,
exocortis, stubborn, or Huanglongbing (HLB) disease
of citrus) are eliminated via therapy. The second phase
includes the production of budwood source trees which
are moved out of quarantine in Riverside and to the
UC Lindcove Research and Extension Center (LREC)
in Exeter, California, where the CCPP Protected Foun-
dation (screenhouse) and Evaluation Blocks (eld) are
housed. Trees established in the Evaluation Blocks are
evaluated for trueness to type by scientists, growers,
and nurserymen and are accessible to the public during
eld and fruit testing days (aka walkthroughs). Trees
established in the Protected Foundation Blocks are off
limits to the public, they are regularly tested for a vari-
ety of pathogens and are registered with the CDFA as
budwood source trees.
Over the past several years, many varieties have
been through the rigorous Variety Introduction-VI
disease testing and therapy program under quarantine
at the CCPP. Varieties that successfully complete the VI
process receive a unique VI identication number that
permanently accompanies the budwood that is made
available to growers, nurseries, researchers, and others.
Little information about many of the CCPP VI variet-
ies is accessible to the public, or may take a great deal
of effort to nd. As a result, the UCR-Citrus Variety
Collection (CVC), USDA-National Clonal Germplasm
Repository for Citrus and Dates (NCGRCD), and CCPP
have compiled information on the 18 most recently dis-
tributed varieties.
Protected Foundation Block Budwood
Orlando, Florida, by Dr Phil Reece but the seeds were sent
to Dr Joe Furr at the USDA Date and Citrus Station in
Indio for testing and development. In 1963, budwood was
sent from California to Texas for evaluation by Dr Heinz
Wutscher, USDA, Weslaco. The characteristics of C54-4-4
under Texas conditions is presented in a paper in the Journal
of the American Society for Horticultural Science, 103:124-127
(1978). Those results indicate that C54-4-4 is a late maturing
mandarin with uniform exterior color with 20 % granulation.
The fruit was large with an average of 22 seeds per fruit. Brix
was intermediate in the varieties reported upon, but acid was
among the lowest reported. Therefore, the brix/acid ratio was
the second highest among the varieties evaluated. Yields were
intermediate. This data was based upon harvest in January or
February for the years 1969 1974. Dr Wutscher introduced
this selection into Florida when he transferred to Orlando
in 1975, and evaluated it in Florida conditions. In Florida, it
proved susceptible to scab (a disease that we do not have in
California). Dr Wutscher later made a scab-resistant selec-
tion of C54-4-4 that has recently been released by Florida
Division of Plant Industry (DPI) as Furr mandarin. Note
that this is not the Furr trifoliate hybrid rootstock recently
released by UC Riverside. In the 1980s, prior to making the
scab-resistant selection, Dr Wutscher sent budwood of C54-
4-4 to Corsica. This is the source of the recent introduction
into California and hence does not represent the scab-resistant
Florida selection Furr.
Imperial Mandarin (VI 684): First distribution of buds
from the CCPP: January 2008
Imperial is reported to have originated in Emu Plains,
near Sydney, Australia, as a chance hybrid of Mediterranean
or Willowleaf and another mandarin, possibly Emperor in
approximately 1890. Imperial is one of Australias most im-
portant and long-established mandarin selections. It is widely
planted throughout existing mandarin-growing regions, with
about 361,000 bearing and 26,000 non-bearing trees in 1999,
mostly in Queensland. Imperial is an extremely early matur-
ing mandarin, equivalent in this regard to Owari satsuma.
Brix:acid ratio reaches 7:1 around mid-March in the earliest
regions of Australia, with later regions reaching this ratio in
early June. Imperial can be harvested up to July or August
in Australia, depending upon the region. Fruit quality is con-
sidered good with a good balance of sugar and acid and good
internal color. The skin is thin and soft, and although adherent,
it peels easily. Juice levels are at least 35 % and fruit are rm
when peeled. Granulation is sometimes a problem because
of the low juice content. There are usually 4 or less seeds per
fruit in both solid and mixed plantings. The fruit is medium
sized (or small in heavy crop years). The external color is
yellow orange, but is more intense in regions where fall tem-
peratures are cool. Fruit generally must be clipped from the
tree rather than plucked. De-greening is necessary when fruit
are harvested early and the post-harvest life is short at 2 - 4
weeks. Imperial responds poorly to heat and cold treatments
for pest disinfestation, which makes it unsuitable for export
(from Australia). Imperial performs adequately on a variety
of rootstocks, although incompatibilities have sometimes
been noted on Carrizo and Troyer. Cleopatra produces
high quality fruit. Cleopatra and Troyer are the most com-
monly used rootstocks in Queensland, whereas Carrizo is
more popular in southern areas. There is a tendency towards
alternate bearing, which sometimes is managed by thinning.
The trees habit is vigorous and upright. Cross-pollinators may
help with set, yield, and size. This variety is currently being
evaluated by Toni Siebert and Tracy Kahn. This information
was summarized from The Citrus Industry, 1967, I:516; and
Saunt, 2000, Citrus Varieties of the World, pp 65-66. A down-
loadable factsheet developed by Australian Citrus Limited
is available at http://www.australiancitrusgrowers.com.au/
aspdev/resources/documents/ImperialB.pdf
Hickson Mandarin (VI 685): First distribution of buds
from the CCPP: June 2008
Hickson is reported to have originated near the town of
Roma, Queensland, Australia, as a sporting limb on Ellendale
tangor. Hickson is similar in many respects to its Ellendale
progenitor. In Australia, it is a mid-season variety, maturing
starting in late June, about 2 weeks earlier than Ellendale.
Hickson is considered to hang better than Ellendale, with
the harvest date extending through August. In August, it is
usually slightly puffed but the juice content is satisfactory.
The general appearance of the tree is similar to Ellendale,
although there are some differences in leaf shape. The fruit
is similar in appearance to Ellendale but the rind is not as
smooth and it peels more easily. The fruit averages 6 cm X 5
cm, has a slight neck, and a smooth, yellowish-orange rind with
some gloss. The orange-eshed fruit is juicy with good avor
and has 12-15 seeds. Hickson is resistant to brown spot and
was initially considered a promising alternative to Ellendale.
However, Hickson declines when propagated on rough lem-
on rootstock probably due to genetic or physiological reasons.
It is also susceptible to a crotch rot associated with Phomopsis
and in addition does not perform well on trifoliate. For these
reasons, it is no longer as popular in Australia. This variety
is currently being evaluated by Toni Siebert and Tracy Kahn.
Information summarized from: Jorgenson, 1972, Queensland
Citrus Bulletin, 1972:23-24; Cox, 1975, NSW Dept of Agri-
culture, Bulletin H2.2.6; Broadbent et al, Proceedings, ISC,
Hickson mandarin: Citrus Variety Collection, Riverside,
California, 16 Feb 2010. Photo by T. Siebert
1978:207-208; Forsyth et al, 1985, NSW Dept of Agriculture,
Agfact H2.1.4.). A factsheet developed by Australian Citrus
unlimited is available at http://www.australiancitrusgrowers.
com.au/aspdev/resources/documents/HicksonB.pdf.
Sudachi Ichandarin (VI 693): First distribution of buds
Thought to be a hybrid of a papeda and a mandarin, Su-
dachi arose as a chance seedling in the Tokushima Prefecture
of Japan, on Shikoku island, where it has traditionally been
grown. When harvested young, Sudachi is considered to
have a distinctive fragrance that is different from Yuzu. The
young fruits are used for cooking while still green, often being
incorporated into vinegars or avoring many different entrees,
especially sh. The avor is now also used in soft drinks and
alcoholic beverages. Fruit of Sudachi was formally evaluated
by Ottillia Bier, Toni Siebert and Tracy Kahn in September
and October of years 2003 to 2007 at Riverside, California.
Signicantly smaller than Yuzu, the average fruit size has a
mean width of 3.8 cm and a height of 3.4 cm. The fruits have an
oblate (spherical and attened at both poles) shape, although
some fruits can be round. Sudachi has a mean weight per fruit
of 27.2 grams. Color break was reached between the rst and
third week of September. The rind texture is slightly pebbly
with a mean thickness of 1.9 mm. The number of seeds per
fruit averages 9. The mean juice weight is 9.6 grams and the
average juice content is 34.4%, which is tting as Sudachi is
primarily used for juice. The juice weight and juice content
increased during the sampling dates. The internal esh color
of Sudachi in the green stage is light green to green-yellow.
Sudachi is slightly more acidic than Yuzu with an average of
5% citric acid. Sudachi trees tend to have a spreading habit
of moderate vigor, but can be considered a small to medium
Australian Finger Lime (VI 697): First distribution of buds
The Australian nger lime, a citrus relative also known
as Microcitrus australasica, is one of six different species of
citrus considered to be native to Australia. This VI is one of 8
different accessions of Microcitrus australasica in the Citrus
Variety Collection, and was imported from Sydney, Australia,
in 1965. Depending on the type of rootstock used (The CVC
has used several: Schaub rough lemon, Cleopatra mandarin,
sized tree, as 26-years-old trees on Carrizo and C-35 citrange
rootstocks are only approximately 8 feet tall, with no indica-
tions of rootstock-scion incompatibility. Thorns up to 5 mm
in length are present in each leaf axil. Leaves are elliptical
in shape, with a small winged petiole. The tree canopy has
dense branching. (Kawada, K., and Kitagawa, H. Storage of
Sudachi (Citrus sudachi Hort. ex Shirai) in Japan. Pro. Int.
Soc. Citriculture, 1084-1085. 1992. Kawada, K., and Kitagawa,
H. Citriculture, Marketing are Different in Japan. Fresh
Citrus Fruits. Avi Publishing Company: Connecticut. 1986.)
Sudachi ichandarin: Citrus Variety Collection, Riverside,
California, 15 Nov 2002. Photo by D. Karp.
Australian Finger Lime: Citrus Variety Collection, Riverside,
California, 5 Dec 2008. Photo by D. Karp and T. Siebert.
C-35 citrange, Carrizo citrange, Calamondin, Citrus macro-
phylla), the Australian nger lime can be a very small (about
5 feet on Schaub rough lemon) to large-sized tree. The leaves
are tiny at approximately one-half inch long and the branches
become very dense and spiny with about 1 thorn set in every
leaf axil. New growth is purple in color and the one-quarter
inch wide owers are white and pink during the main owering
season of February to April. The nger limes are about three
inches long and roughly the size of an average persons index
nger, but fruit from juvenile trees can be less than one inch
long. The skin of the nger lime is usually a greenish black to
very dark purple and thin, but durable. Once the fruit is cut
open the tiny round juice vesicles will slowly seep out of the
fruit without squeezing, and resemble what we like to call
citrus caviar. The round vesicles are usually a clear-green,
but can be very light pink. The juice is very tart, much like a
Mexican lime. Although the tree produces fruit year round,
the main fruiting season in California is November-December
when the fruit falls off in your hand. Australian nger lime is
reported to fetch approximately 40-50 dollars per pound. The
fruits are technically edible, but this is not commonly done.
Its most common use is as a garnish or avor component in
culinary creations.
Persian Lime SPB-7 (VI 708): First distribution of buds
This selection of Persian-type lime is said to be free of the
genetic disorder called wood pocket, which is found in many
of the large-fruited acid limes. Wood pocket was formerly very
common in Florida and caused extensive losses. The industry
requested help from researchers and after screening more
than 100,000 trees, 10 trees were identied that were appar-
ently free of wood pocket, based upon their survival. The
current selection (SPB-7) was entered into the program in
1954 as Li-38-1-1-X. This selection was apparently erroneously
identied as being wood pocket positive and was dropped but
later reinstated and never showed wood pocket symptoms. The
current selection was entered into the Florida DPI foundation
program about 1961 and was imported into California in the
year 2000. The current screenhouse bud source descended
from a tree planted in the Haines City Foundation Grove.
Because of this budsource, the Florida Persian lime industry
became virtually free of wood pocket by the early 1970s. (This
information redacted from an email from Mike Kesinger,
06/25/2006) Trees of this selection have been planted at various
places in California for evaluation as to the development of
wood pocket. One of these locations is the Coachella Valley
Agricultural Research Station in Thermal, CA. Since wood
pocket develops more rapidly at high temperatures, the trees
in Thermal should be the most susceptible to development of
wood pocket. More information on these observations will be
provided as it becomes available.
Lemonade (VI 734): First distribution of buds from the
CCPP: September 2008
Lemonade is reported to be a sweet lemon hybrid of un-
known parentage with a very pleasant taste, and can be readily
but not easily peeled. The fruit is small-medium, and not very
seedy. The trees are semi-dwarfed (on trifoliate rootstock), but
quite productive. The main crop matures in early spring in New
Zealand, with much smaller summer crops also occurring. Un-
fortunately there is no commercial production in New Zealand,
although it is a popular home garden tree. It is susceptible to
citrus scab disease; however, in a drier climate this should be
less of a problem. (This information redacted from an email
from Andrew Harty via Peter Chaires, 12/07/2005) Although
the budsource trees are derived from trees at the CCPP that
tested negative for all known graft-transmissible diseases,
trees of Lemonade propagated in Riverside have shown a
tendency to develop small brown to black lesions on the bark.
The reason for these lesions is currently unknown. Lemonade
trees propagated at Riverside are not particularly vigorous but
the relationships of this to the observed lesions is not known.
Early Release Budwood
Early Release Budwood is budwood provided from se-
lected cultivars that have been recently out of quarantine and
are maintained by the CCPP at the LREC Protected Founda-
tion Blocks for the Early Release program. These cultivars
are grown in pots under protective screen producing limited
amounts of budwood. Therefore, supply of Early Release
Budwood will be limited. A signed Waiver and Release form
must accompany all orders for Early Release Budwood. The
Waiver and Release form is available on the CCPP website
(http://www.ccpp.ucr.edu).
Valentine Pummelo Hybrid (VI 597): First distribution of
buds from the CCPP: September 2009
Valentine is the most promising of the seedy pigmented
low-acid pummelo hybrids selected by Drs. Soost and Cam-
eron in 1986 from a cross of Siamese Sweet pummelo x
Persian lime: Citrus Variety Collection, Riverside, California,
3 Nov 2009. Photo by D. Karp and T. Siebert.
Lemonade: Citrus Variety Collection, Riverside, 3 Nov 2009.
Photo by D. Karp and T. Siebert.
Valentine pummelo hybrid: Citrus Variety Collection
laboratory, Riverside, California. Photo by O. J. Bier.
(Ruby blood orange x Dancy mandarin). It received its
name from former Staff Research Associate for the Citrus
Variety Collection Ottillia Toots Bier, who nicknamed it
Valentine not only because the fruit matures in mid-February
near the Valentines Day holiday, but also because often when
the fruit is cut lengthwise and turned upside down, the esh
of the fruit resembles a vibrant red heart. Valentine fruits
are round to somewhat pyriform (pear-shaped). The average
fruit size is large with a mean width of 10.8 cm (4.25 inches)
and a height (including the neck) of 11.0 cm (4.33 inches).
The mean weight per fruit is 531.1 grams (18.7 ounces). Rind
color is medium to dark yellow. The rind texture is moder-
ately smooth with a mean thickness of 8.8 mm (0.35 inches).
Fruit samples from Lindcove generally have a thicker rind
than samples from Riverside. The number of seeds per fruit
averages 27.6. However, the mean number of seeds per fruit
among 36 different 10-fruit samples ranged from 2.6 seeds
per fruit to 51 seeds per fruit. The mean juice weight is 201.8
grams (7.1 ounces) and the average juice content is 38.6%.
The red esh color of Valentine can be somewhat variable
in its distribution and intensity inside the fruit. Color forma-
tion rst appears in mid-January and becomes more intense
in early to mid February when the solids to acid ratio is an
average of 16:1. Please see Valentine, A Recently Released
Anthocyanin-pigmented Pummelo Hybrid Developed at UC
Riverside from Topics in Subtropics, 2009, 7(3): 2-4, for a
more detailed description of this variety.
Xie Shan Satsuma (VI 621): First distribution of buds from
the CCPP: June 2007
Xie Shan was originally imported from the Institute of
Subtropical Crops of Zheijiang Academy in China in 1992.
Dr. Fred Gmitter, University of Florida, who was responsible
for the collection of this variety, reported Xie Shan to be
extremely early ripening in comparison to other Chinese
satsumas. In a California trial, Dr. Thomas Chao, UC Riv-
erside, reported that Xie Shan developed high brix levels
somewhat earlier than other early Satsuma cultivars tested
(Armstrong, Miyagawa, and Chinese S-9). However, high
acid levels kept the sugar/acid ratio within about the same
range as the other cultivars. Xie Shan and Miyagawa were
considered the earliest cultivars in this trial. Additionally, it
was reported that Xie Shan was completely seedless, easy
peeling, and had a unique taste and avor. Projected harvest
in the San Joaquin Valley was mid-September. (Information
from Topics in Subtropics, 2005, 3(2): 3-5). This variety is cur-
rently being evaluated by Toni Siebert and Tracy Kahn.
China S-9 Satsuma (VI 636): First distribution of buds
China S-9 was part of the rst of two different sets of
several satsumas collected in the Hubei Province of China,
by Dr. H. Huang, Auburn University, in 1995 and given to the
CCPP for introduction and quarantine. Each selection in the
rst set was given the S signier and a number, and was
described in a letter from Bill Dozier, Auburn University, to
the late Dave Gumpf on August 28, 1995. They were selected
from elds in Hubei and Sichuan that had been devastated
after a very bad freeze. Each selection was from branches
on satsuma trees that had survived the freeze, and the three
selection characteristics they looked for were cold hardiness,
fruit quality and tree vigor. Huang rated the tree vigor and
fruit quality of China S-9 as acceptable, and cold hardiness
as good. The second set of satsumas were brought back by
Dozier in either 1996 or 1998 after he visited these same areas
of China where the freeze occurred. Preliminary observations
by Thomas Chao at Santa Paula (see citation in previous de-
scription) suggested that China S-9 should mature early in
the San Joaquin Valley (about the same time as Miyagawa
and Xie Shan). In addition, preliminary observations at
LREC showed that China S-9 has a smoother peel than most
other satsumas. This variety is currently being evaluated by
Toni Siebert and Tracy Kahn.
Pehrson #3 Valencia Orange and Pehrson #4 Valencia
Orange (VI 749 and VI 750): First distribution of buds from
the CCPP: September 2008
These VIs are two of a group of 8 Valencia clones se-
lected by University of California Cooperative Extension
Specialist Emeritus John Pehrson. These were included in a
Valencia Strain Trial established at LREC. In a letter sent by
John Pehrson on April 13, 2009, to the CVC, he described the
Xie Shan satsuma: Citrus Variety Collection, Riverside,
California, 27 Nov 2007. Photo by T. Siebert.
China S-9 satsuma: Citrus Variety Collection, Riverside,
California, 27 Nov 2007. Photo by T. Siebert.
complex history of the Valencia clones. The objective was to
salvage propagative material from high production Valencia
trees of good quality fruit because urban growth was shrinking
Valencia acreage in Orange County, CA. Some of the strains
were reported to be from the Smith Family Ranch and the
Wagner Family Ranch in Placentia, CA, and the Gilman Grove,
Fullerton, CA. Seedlings were grown from collected fruits and
budded onto Troyer citrange. These were planted in coopera-
tion with a grower in a Brea area oil lease that would not last
another 15 years. The time frame was from the late 1950s to the
mid 1960s for collection, growing and planting. Shortly after
planting in Brea, Pehrson was moved from Orange County to
Tulare County, so efforts were made to save these selections.
With propagating material from the plot Pehrson had estab-
lished with the grower in Brea, CA, Ed Nauer cleaned up 8 of
the clones in Riverside, CA, and moved them into a Valencia
strain trial at LREC in 1986 where they were compared with
Campbell, Cutter, Frost, Olinda, and Chapman Valencias. The
8 Valencia clones were put through the VI index as VIs 747 to
754 with budwood taken directly from the nucellar block in
November 2006. Pehrson Valencia #3 and Pehrson Valencia
#4 were selected as being the most promising out of the 8
clones. Pehrson Valencia #3 was selected for having good peel
color, minimal regreening, having a good yield, and is later
maturing. Pehrson Valencia #4 was selected as having the
highest cumulative yield and weight, and a good distribution
of fruit size (information from M.L. Arpaia).
FairchildLS Mandarin (VI 762): First distribution of buds
from the CCPP: January 2010
FairchildLS is an irradiated selection of Fairchild
mandarin developed at UC Riverside. The distinctive trait
of FairchildLS is that it is considered to be low seeded (2.4
seeds per fruit) despite any cross-pollination. FairchildLS
fruit are deeply oblate in shape with no neck. The fruit is
medium sized for a mandarin (classed as Large by State of
California standards) averaging 64 mm (2.5 in.) in diameter
and 56 mm (2.2 in.) in height with a very smooth, deep orange
rind color. The rind is relatively thin and at maturity is consid-
ered moderately easy to peel. The fruit interior has ne esh
texture with 10-11 segments. The fruit are juicy averaging 47%
juice with an average weight of 110g. FairchildLS matures in
winter (late January) and holds its fruit quality characteris-
tics through late March. Fruit from trees on Carrizo citrange
rootstocks average 12.1-13.0% soluble solids and 0.85-1.17%
acid in January increasing to 13.2-14.5% soluble solids with
decreasing acid of 0.76-0.93% by mid-March at four trial
locations. Fruit from trees on C-35 citrange rootstocks aver-
age 11.2-12.8% soluble solids and 0.94-1.23% acid in January
increasing to 13.1-14.0% soluble solids with decreasing acid
of 0.78-1.01% by mid-March. FairchildLS averages 2.4 + 0.6
seeds per fruit in the presence of cross-pollination at all trial
locations throughout California compared to 15-25 seeds per
fruit for regular Fairchild. Pollen of FairchildLS has very
low viability, therefore it has a very low likelihood of causing
seeds in other citrus, particularly mandarins, when planted
nearby. Fruit production for FairchildLS begins in the third
year after planting. Four-year-old trees averaged 62-88 lb,
and ve year old trees averaged 92-108 lbs. Alternate bearing
can be a problem by years seven and eight (information from
M.L Roose and T.E. Williams). Patent and/or propagation
rights for Fairchild LS Mandarin are held by the Regents of
the University of California. Budwood is available only to
nurserymen who have a License Agreement for these cultivars.

Tango Mandarin (VI 765): First distribution of buds from
the CCPP: June 2007
Tango is a patented (Plant Patent #17863) irradiated selec-
tion of W. Murcott mandarin developed at UC Riverside. Fruit
of Tango are similar to W. Murcott in all appearance, quality
and production characteristics with the exception of seed
numbers. Tango fruit are deeply oblate in shape with no neck.
The fruit is medium sized for a mandarin (classed as Large by
State of California standards and size 28 by industry packing
standards) averaging 59 mm (2.32 in.) in diameter and 48 mm
(1.89 in.) in height with a very smooth, deep orange rind color.
The rind is relatively thin and at maturity is easy to peel. The
fruit interior has ne esh texture with 9-10 segments and a
semi-hollow axis of medium size at maturity. The fruit are juicy
averaging slightly over 50% juice with an average weight of
90.6 g (3.2 oz.). Tango matures in winter (late January) and
holds its fruit quality characteristics through April into May.
Production is excellent averaging 800-900 cartons/acre when
planted at densities of 250-300 trees/acre. Fruit from trees
on Carrizo and C-35 citrange rootstocks average 11.1-13.1%
soluble solids and 0.97-1.19% acid in January increasing to
13.5-15.4% soluble solids with decreasing acid of 0.54-0.82%
FairchildLS mandarin: Photo by M. Roose.
Tango mandarin: Citrus Variety Collection, Riverside,
California, 21 Feb 2007. Photo by D. Karp.
in April. Tango averages 0.04 + 0.2 seeds per fruit in the pres-
ence of cross-pollination at seven trial locations throughout
California compared to 11.6-22.6 seeds per fruit for W. Murcott.
Pollen of Tango has very low viability consequently it has a
very low likelihood of causing seeds in other citrus, particularly
mandarins, when planted nearby. Like W. Murcott, trees of
Tango have a tendency to overbear and therefore need to be
regularly pruned to maintain good, but not excessive produc-
tion and to maintain fruit size and prevent alternate bearing.
Crop yields should be limited to about 150-170 lbs/tree (6-7
mandarin boxes) through a combination of pruning and, if
needed, fruit thinning and should be harvested on time, not left
on the trees as this can lead to alternate bearing (information
from M.L Roose and T.E. Williams). Patent and/or propaga-
tion rights for Tango Mandarin are held by the Regents of
the University of California. Budwood is available only to
nurserymen who have a License Agreement for these cultivars.
Primosole Mandarin (VI 777): First distribution of buds
from the CCPP: September 2009
Primosole mandarin is a hybrid of Carvalhais mandarin
and Miho wase Satsuma produced at the University of Cata-
nia, Sicily, in 1980. It is described as being seedless in isolation
and matures very early (at the beginning of October in south-
ern Italy or early April in Australia). Coastal Fruitgrowers
Newsletter (Australia, August 2005) reported, upon release of
Primosole from quarantine, that it reached maturity about
10 days before Okitsu satsuma. Fruits are oblate in shape
and average 150 grams in weight in southern Italy. The trees
are said to be vigorous and productive with an open growth
habit. They do not exhibit alternate bearing tendencies. The
branches do not have spines, and the lanceolate (long, wider
in the middle or lance-shaped) leaves tend to fold as if under
water stress. According to an article published in Plant Disease,
December 2001, pg. 1291, called Extreme Susceptibility of Pri-
mosole Mandarin to Alternaria Fruit Rot in Italy, Primosole
is extremely susceptible to Alternaria fruit rot due to growth
cracks at the stylar end (blossom end of fruit) and sensitivity
to sunburn. Citrogold reports that this variety has orange rind
color, a good to large fruit size, and a good to very good crop
load. The peel separates easily. The avor is said to be between
satsuma and mandarin. The variety handles degreening well,
stores well, handles cold sterilization well, and has a rm rind
in comparison to satsumas. It tends to have a ricyness in the
core of the fruit that lessens with tree age and characteristic
leaf wilting. Primosole is a self-incompatible variety, but will
pollinize compatible varieties such as clementines. Cross pol-
lination is managed by a buffer of 10 rows. It does not require
a plant growth regulator to increase fruit set or size. It is very
sensitive to strong winds and can defoliate under windy con-
ditions. Swingle is reported to not be suitable as a rootstock
for Primosole mandarin, but it does well on C-35, Carrizo,
and Troyer citranges. (Aznar and Fayos, Citricos. Variedades y
tecnicas de cultivo. Mundi-Prensa Libros. 2006. pg 128)
Bitters C-22 Citrange (VI 792), Carpenter C-54 Citrange
(VI 793), and Furr C-57 Citrange (VI 794): First distribution
of buds from the CCPP: September 2009
According to the report provided by Claire Federici,
Ricarda Kupper, and Mikeal Roose, Bitters, Carpenter
and Furr trifoliate hybrids: Three New Citrus Rootstocks
Bitters C-22, Carpenter C-54, and Furr C-57 citrange
rootstocks: Photo by M. Roose.
(information can be found at http://plantbiology.ucr.edu/
faculty/new%20citrus%20rootstocks%202009.pdf), all three
are hybrids of Citrus sunki and Swingle trifoliate orange.
John Carpenter and Joe Furr made all three hybrids as part
of the USDA breeding program at Indio, CA. Professor W.P.
Bitters tested the hybrids for Citrus Tristeza Virus tolerance
in a trial established at the South Coast Research and Exten-
sion Center in Irvine, CA, in 1966 and 1968. All three hybrids
showed good tolerance to the virus. Details about subsequent
eld trials, soils, management, and results compared to other
varieties can be found at http://plantbiology.ucr.edu/faculty/
Summary-of-Active-Rootstock-Trials-5-09v5.pdf
Bitters produces a small tree, with high yield per canopy
volume. Young trees on this rootstock showed good tolerance
to freezing. Fruit quality of late navels was good and granula-
tion was no worse than fruit on Carrizo or C-35. It is toler-
ant to CTV, moderately tolerant of Phytophthora parasitica,
not very tolerant of citrus nematode, and very tolerant of
calcareous soil. It was reported that this rootstock is the best
candidate in Texas to replace sour orange due to its tolerance
of calcareous soil conditions.
Carpenter produces medium to large trees, with good
yield. Young trees on this rootstock showed moderate toler-
ance to freezing. Fruit quality of late navels was good and
granulation was no worse than fruit on Carrizo but was
slightly worse than on C-35. It is tolerant to CTV, moderately
tolerant to P. parasitica, very tolerant of citrus nematode, and
moderately tolerant of calcereous soil.
Furr produces medium to large trees, with good yield.
Young trees on this rootstock showed good tolerance to freez-
ing. Fruit quality of late navels was good and granulation was
no worse than fruit on Carrizo or C-35. It is tolerant to CTV,
very tolerant to P. parasitica, very tolerant of citrus nematode,
and moderately tolerant of calcareous soil.
Please visit the Citrus Clonal Protection Program website
at http://www.ccpp.ucr.edu for more information about how
to obtain budwood of these varieties. Registered users of the
online budwood ordering system may visit http://ccpp.ucr.
edu/budwood/budwood.php. If you are not a registered user
you can e-mail ccpp@ucr.edu with your name, address, e-mail,
and phone number or call (951) 684 8580 and the CCPP will
generate a username and password for you. After becoming a
registered user of the budwood ordering system you will also
receive announcements about future budwood distributions
for other citrus varieties.
C
itricola scale was a key pest in
both southern and central Cali-
fornia citrus during the rst half
of the 20th century. However, it became
scarce in southern California citrus dur-
ing the late 1930s coincident to the in-
troduction of the exotic parasitoids for
black scale. Furthermore, southern Cal-
ifornias complex of soft scales includes
brown soft scale, which provides year
round hosts that support a diverse com-
plex of parasitoids. This complex has
kept citricola scale to near extinction.
By contrast, citricola scale remains a
key pest in the San Joaquin Valley (SJV)
where brown soft scale populations
are absent. Moreover, citricola scale is
developing resistance to Lorsban and
some of the newer pesticides. This is of
real concern. Oil is the most selective
pesticide for control of this scale but it
only reduces its density when it is ap-
plied 1-2 times annually, and there are
concerns that oils will affect yield.
Research Project Final Report
Parasitoid preference for citricola scale in southern
California citrus versus San Joaquin Valley citrus:
which one or several species should we produce and release?
Robert F. Luck, Joseph G. Morse, and Lisa Forster
Fig. 1. A) Citricola scale from SJV foliage moving onto potted citrus at UC Riverside. B) Successful scale transfer to potted citrus.
C) Cages used to protect scale from natural enemies during scale growth and development prior to conducting experiments.
CRB Funded Research Reports 2009
The primary justification for this
project is based on the hypothesis that
the parasitoids present in southern Cali-
fornia, coupled with ant control and skirt
pruning, have driven citricola scale pop-
ulations nearly extinct. However, these
parasitoid species are lacking in the
SJV. We have identied the parasitoid
complex present in southern California
and have determined which parasitoid
species prefers or utilizes citricola scale.
We are now using this information to
develop a sustainable, biologically-based
pest management program for citricola
scale in the SJV.
To determine the species that para-
sitize citricola scale, we brought citrus
foliage with citricola scale from the SJV
to southern California and inoculated
them on potted citrus plants. These
scales were then used in laboratory
and eld experiments to identify which
southern California parasitoid species
attack citricola scale.
Initially we used large potted plants
(15 gal.), but we had great difficulty
keeping the plants and citricola scales
protected from other pests or con-
tamination of predators and parasitoids.
When we switched to small, 1-gallon pot-
ted citrus, we achieved good inoculations
with citricola scale, and we were able to
keep the plants and scales clean of other
pests or predators.
We made cages to t over a modied
tomato trellis and used closed cell foam
around the lower quarter of the pot to
prevent parasitoid and predator access
(Fig. 1). The plants were maintained in
shade and watered as needed. Every
week, ca. 2 to 6 plants were cleaned
and inspected for contaminates (soft
scale parasitoids, mealybug, mites, etc.),
and they were resealed in their cages.
All pots were set in basins of water to
preserve moisture and reduce the likeli-
hood of ants invading through the drain
holes in the pots. Using this method to
A B
C
Fig. 2. Complex of parasitoids from brown soft scale in southern California
(A = past survey, B = current survey {with citricola scale}).
Fig. 3 Brown soft scale on yucca in the eld and closeup of scale.
grow citricola scale on living plants elim-
inated the mealybug and Coccophagus
contamination that we had experienced
with the larger potted citrus.
The design of our field study in-
volved both brown soft scale and citri-
cola scale (=out-plants). Through past
research, we have identied the complex
of parasitoids in southern California
that attack brown soft scale (Fig. 2a).
Moreover, with this particular project,
for every citricola scale- infested plant
we put out, we also put out brown soft
scale as an index to determine what
species attacks brown scale but not
citricola scale.
Citricola scale plants were placed in
the eld in basins with water beneath the
tree canopy edge. This avoided excessive
scale mortality due to high temperatures.
Detached yucca leaves infested with
brown soft scale were put three to four
feet from the citricola scale (Fig. 3). The
citricola scale out-plants (and brown
soft scale out-plants) were placed in
the eld and uncaged (to expose the
scale) only when the sizes of both scale
species were large enough to support
parasitoid offspring. We did not evaluate
eld predation and host feeding of small
scale in this study.
Although there seems to be some
size variation associated with where the
scale settles on the potted tree (e.g., leaf
vein, leaf margin, etc.), citricola scale
has a single generation annually, thus
scale size is fairly uniform at any given
time of the year. All sets of out-plants
(brown soft scale & citricola scale) were
left in the eld for 14 to 18 days before
they were re-caged and returned to the
laboratory for subsequent evaluation.
We used the same sites for this eld
experiment that we used for our original
parasitoid survey.
The same citricola scale-infested
plants were used to study parasitoid
behavior in the laboratory. All the be-
havior studies were conducted on leaves
and stems of plants before the plants
were used in our eld trials. We evalu-
ated four species of Metaphycus (M.
luteolus, M. angustifrons, M. nr. avus
and M. stanleyi) for their preference to
attack citricola scale. Metaphycus spe-
cies are the main parasitoid complex
that controls the soft scale pests.
We conducted 35+ replications for
each parasitoid species using the scales
on leaves and twigs that we removed
from the plant just prior to their use in
an experiment. All females were mated
and fed honey and had not been previ-
ously exposed to live scale prior to the
experiment. Each female was given ve
minutes to oviposit (lay eggs), host feed,
or reject the scale.
Our initial expectation was that
most if not all of the species in this
diverse complex of parasitoids that had
been identied in southern California
on brown soft scale would also attack
citricola scale. This was especially true
of Metaphycus angustifrons, as it was
the most abundant species that we
recovered from brown soft scale in
our southern California survey. We
recovered very similar proportions of
M. angustifrons and M. luteolus from
the brown soft scale out-plants that ac-
companied each citricola scale infested
out-plant. This duplicated our original
survey and veried our results in this
smaller, second survey (Fig. 2a & b).
The behavioral trials clearly showed
that M. angustifrons is not well suited to
citricola scale and has great difculty
drilling through the epidermis of the
scale. The parasitoid primarily host fed
on small scale and killed them (Fig. 4).
The average number of eggs laid by
M. angustifrons was only 0.2 eggs per
scale, and no offspring emerged from
these scale. This contrasts with the other
three species which ranged from 1.47 to
1.8 eggs per scale and yielded female
offspring that had a sex ratio ranging
A B
Fig. 6. Parasitoid
preference in
the eld when
given the choice
of citricola scale
versus brown
soft scale.
Fig. 5. Average number of eggs laid in citricola scale, and percent of female
offspring produced.
Fig. 4. Laboratory experiments of behavior activity: four different Metaphycus
species on citricola scale.
from 50% to 74% females (Fig. 5).
The eld study conrmed what we
observed in our laboratory behavioral
experiments. Although we did not re-
cover M. nr. avus or M. stanleyi from
our out-plants, M. luteolus dominated
the parasitoid species emerging from
our citricola scale out-plants, even when
M. angustifrons was clearly present and
attacking the brown soft scale just 3 feet
from the citricola scale (Fig. 6). Evident
from this eld experiment is the impor-
tance of using the correct parasitoid to
release against a target pest. Moreover,
although Metaphycus species look very
similar taxonomically, they each behave
quite differently when they parasitize
a scale.
In our previous field studies, we
detected substantial Coccophagus
lycimnia parasitism on the very young
scale, scale too small for Metaphycus
spp. This suggests that Coccophagus
may compliment Metaphycus in sup-
pressing citricola scale. This observation
in our earlier trials is conrmed in this
study. Coccophagus representation in
the parasitoid complex emerging from
citricola scale is only 3%. In brown soft
scale it remains below 30%. Moreover,
the concern that male Coccophagus
hyperparasitoids may interfere with
Metaphycus species control of citricola
scales seems unlikely.
Dr. Paul Rugman-Jones (UCR -
Dr. Richard Stouthamer Laboratory)
developed a species-specic identi-
cation tool for both Metaphycus spp.
and Coccophagus sp. which is based
on differences between each species
DNA sequences (PCR). He has con-
rmed the identity of all of our eld
specimens. This was critical for this
system, as the taxonomy of these para-
sitoids (Metaphycus and Coccophagus)
historically have been plagued with
misidentications. After reviewing the
specimens from our last survey, all of
the species identications have been
conrmed genetically and are now be-
ing identied taxonomically by a spe-
cialist in Metaphycus. Only specimens
identied as M. helvolus failed to match
the DNA sequence; thus, it is a differ-
ent species. Our original concern with
the brown soft scale survey was that M.
helvolus would not attack brown soft
scale, which seems to be the case. We
identied one additional Metaphycus
species using Dr. Rugman-Jones pro-
tocols, and we are getting it identied
(e.g., named). We have also identied
all four species of Coccophagus (C.
lycimnia, C. cowperi, C. rusti, and C.
scutellaris) using these molecular tech-
niques. The other benet arising from
Dr. Rugman-Jones technique is that it
gives very rapid results.
Results from this project funded by
the Citrus Research Board have allowed
us to better understand the parasitoid
species complexes attacking soft scale
pests in citrus. Clearly it is a very com-
plicated system, but the information
that we have gained will now allow us
to exploit these parasitoids with refer-
ence to their preferences for particular
scale species. We are currently seeking
USDA funding for a eld project to test
the proof-of-concept for a sustainable
management program for citricola scale
in SJVcitrus that we have developed as
a result of this CRB project.
Project Leaders Robert F. Luck,
Ph.D., and Joseph G. Morse, Ph.D., are
Professors of Entomology, University
of California Riverside. Lisa Forster is
Staff Research Associate, Department
of Entomology, UC Riverside.
Fig. 1. A-B) Laser ablation of nitrocellulose laminated to a polycarbonate substrate can be used to generate uid ow
modulating structures in a planar format. C) Buffer exchange mediating geometries cut in absorbent materials are
incorporated into a low-cost plastic housing to provide an easily used sample preparation system.
R
esearch and development at Mesa Tech Internation-
al, supported in part by the Citrus Research Board
(CRB), is aimed at the simplication of sensitive and
reliable testing for citrus diseases.
Currently, molecular tests for devastating citrus pathogens
require sophisticated laboratories and highly trained person-
nel resulting in high costs and long turnaround times. Mesa
Techs approach, however, will provide the same reliability
and sensitivity as existing laboratory-based tests in completely
self-contained disposable cartridges similar in complexity to
the test strips long employed for home pregnancy tests.
Unlike existing test strip approaches based upon antibody
binding, known as immunoassays, Mesas technology employs
far more sensitive and specic nucleic acid-based molecular
assays. The ease of use and low cost of Mesa Techs approach
to nucleic acid-based assays will empower growers with the
technology they need to accurately monitor their orchards for
such diseases as Huanglongbing (HLB). By placing rapid and
reliable tests directly in the hands of growers and regulatory
agencies, rapid identication of new and emerging diseases,
as well as tracking their movement through ports of entry,
will be greatly facilitated.
Although nucleic acid-based assays for pathogen detection
and identication offer sensitivity, specicity and resolution,
they continue to be relatively elaborate and often costly, limit-
ing their utility for deployment under eld conditions where
Novel immunocapture technology for eld deployable
nucleic acid-based detection of plant pathogens
R. Bruce Cary
a supporting laboratory infrastructure is limited or absent.
One of the most challenging hurdles to moving nucleic
acid testing from the laboratory to the eld has been the
extraction of sufciently pure nucleic acid molecules from
complex samples such as leaf and other plant tissues. The
biochemical processes upon which DNA and RNA testing
hinges are adversely impacted by the remnants of tissue and
other contaminants present in crude samples, typically neces-
sitating lengthy laboratory processing to obtain highly puried
DNA or RNA suitable for test procedures.
With CRB support, Mesa Tech has developed and con-
ducted pilot eld tests of an exciting new approach to nucleic
acid purication. Unlike laboratory methods, Mesa Techs
approach eliminates the need for instrumentation and user
expertise, allowing anyone to isolate readily testable nucleic
acids from eld-collected plant tissue samples. This break-
through technology is now being incorporated into an inex-
pensive and disposable handheld unit that integrates nucleic
acid purication, amplication and detection within the device
without additional user manipulation.
To develop simple nucleic acid sample preparation tech-
nologies, three initial project objectives were established.
These objectives can be summarized as: 1). Develop buffer
solutions capable of disrupting bacterial pathogen cells or
virus particles while simultaneously stabilizing their DNA
and RNA for isolation and later amplication and detec-
Fig. 2. A lateral ow microarray (LFM) was used to detect
amplied CTV RNA from citrus leaf nucleic acids isolated
using Mesa Techs sample preparation system. Field
collected leaf samples were processed at the ranch, and RNA
targets indicative of CTV infection were detected on site.
Panel (A) is a schematic legend of the LFM layout. Positive
controls conrm proper test performance and provide
positional markers (green). Negative controls conrm assay
specicity (black). The location of diagnostic probes for
HLB (Candidatus Liberibacter spp.) and CTV are indicated in
beige and red respectively. (B) One of 20 trees examined was
negative for CTV by both the Mesa Tech sample preparation
method and Qiagen RNeasy, a laboratory-based approach.
(C) A representative CTV positive LFM reveals strongly
positive spots (blue) at corresponding CTV probe locations
on the strip.
tion; 2). Design and build an inexpensive and easily used
disposable device to accomplish nucleic acid binding from
solutions of disrupted plant tissue; 3). Devise methods and
wash solutions to efciently wash bound nucleic acids to free
them of deleterious cell and sample debris; and, 4). Demon-
strate that isolated nucleic acids are suitable for subsequent
amplication and detection.
To accomplish these goals, Mesa Tech developed a com-
pact disposable device and associated system of optimized
buffer solutions to isolate nucleic acids from citrus leaf tis-
sue. Fig. 1 A-C depicts the fabrication of a key component
of the prototype device that controls the sequence and rate
of solution movement (patent applied for). This technology
allows sample and wash buffers to be added to the device at
the same time, yet exhibit optimized ow for sample prepara-
tion without user intervention, pumps, valves or other moving
parts. This component is incorporated into a plastic housing,
shown in Fig. 1C. The user simply adds the sample and two
wash buffer solutions to the device; approximately 15 min-
utes later, puried nucleic acids are ready for amplication
and detection.
To test the ability of the device to yield useful RNA for the
detection of Citrus tristeza virus (CTV), Mesa Tech conducted
a pilot eld study at the citrus ranch of Earl Rutz in Pauma
Valley, California. These studies tested 20 randomly selected
trees for CTV using both Mesa Techs sample preparation
system and a widely accepted laboratory-based method
(RNeasy, Qiagen, Inc.). Comparisons of Mesa Techs methods
with traditional laboratory sample preparation approaches
resulted in perfect agreement between these two disparate
methods.
To accomplish RNA amplication, this study employed a
technique that amplies RNA molecules through a biochemi-
cal reaction that can be conducted in a single tube at a single
temperature. The single temperature nature of this approach
allows target RNA sequences, in this case sequences indicative
of the presence of CTV, to be exponentially amplied without
the complex instrumentation typically associated with such
procedures as polymerase chain reaction (PCR).
Detection of amplied CTV RNA sequences was ac-
complished using lateral ow microarrays, a simple yet rapid
and sensitive method developed by one of Mesa Techs co-
founders (the scientic publication describing this method
is available for download at http://www.ncbi.nlm.nih.gov/
pmc/articles/PMC1904290/ ). Fig. 2 illustrates the lateral ow
microarray detection strips used in this study.
Despite the simplicity and excellent performance char-
acteristics of Mesa Techs sample processing, amplication
and detection methods, these technologies currently exist as
independent steps of an analytical procedure. Although our
prior work has resulted in adaptations of each requisite pro-
cess that are amenable to eld deployment, the manipulations
required, though simple, remain reliant upon user interactions
during the test procedure.
A truly easy to use eld assay for nucleic acids will require
a level of subsystem integration not currently available. How-
ever, the methods we have developed were devised with the
longer-term vision of integration into a low-cost disposable
device suitable for use under eld conditions with little or no
end user training. By developing simplied methods for each
stage of assay performance, Mesa Tech has greatly facilitated
the ultimate integration of existing component technologies
into an easily used sample-to-result diagnostic tool. Indeed,
Mesa Tech is now engaged in a signicant follow-on effort,
supported by the CRB and the USDA, to develop a low-cost
and easily used disposable device that integrates all steps
of molecular diagnostic testing from sample to result. This
system will enable users with no molecular testing expertise
to obtain sensitive and specic results without the time and
costs typically associated with the use of external laboratory
testing.
Project Leader R. Bruce Cary, Ph.D., is Co-founder and
Vice President, Mesa Tech International Inc., Santa Fe, New
Mexico.
Note from: MaryLou Polek, CRB Vice President
of Science & Technology
The Citrograph will now serve to communicate progress
on Citrus Research Board funded projects. This is in lieu
of a separate CRB Annual Report as published in previ-
ous years. Each issue of the magazine will feature one or
two project completion reports and several progress re-
ports. I hope you nd this format useful and informative.
Project introduction and signicance
The phloem-limited and insect
vector-transmitted citrus stubborn
disease, caused by the bacterial patho-
gen Spiroplasma citri, has resulted in
constant/consistent yield losses and
quality reductions to the citrus industry.
Depending on the varieties, the reduc-
tion on the yields from this disease can
be up to 100%.
The currently available diagnostic
methods are either labor-intensive or
inaccurate due to the uneven distribu-
tion, seasonal uctuation, and the low
titer of S. citri in the infected trees. So
far, there are no robust, economic and
reliable detection tools for citrus stub-
born disease, especially for certica-
tion programs. This gap in the current
diagnostic capacity for citrus stubborn
disease greatly impacts the fundamen-
tal control method for the disease, i.e.,
the use of disease-tested propagative
material.
The main goal of this project is to
develop a novel serological diagnostic
method for citrus stubborn disease
by detecting S. citri proteins that are
secreted from the pathogen into citrus
phloem. Suitable protein markers will
be identied from S. citri and then used
to generate antibodies for detecting the
stubborn disease in citrus trees.
Since secreted proteins are not
restricted to the infection sites and can
be relatively abundant, they can be
used as detection markers which will
enable rapid, reliable and cost-efcient
detection of citrus stubborn disease.
The development of such a novel
immunoassay-based diagnostic tool is
particularly important for large-scale
testing in certication programs, such
as the Citrus Clonal Protection Program
(CCPP), which provides clean citrus
propagative materials to growers, breed-
ers, and researchers worldwide. There-
fore, this project holds the potential to
Fig. 1. Citrus Stubborn Disease-Leaf and
Fruit Symptoms. A) Leaf mottling, B)
Lopsided fruit with stylar end greening
or color inversion of maturation and,
C) Lopsided fruit longitudinal and cross
section with aborted seeds.
Progress Report
Identication of Spiroplasma citri secreted proteins
as detection markers for citrus stubborn disease
Wenbo Ma and Georgios Vidalakis
signicantly benet the citrus industry
not only in California, but also in other
citrus-growing areas worldwide.
Thus far, no secreted proteins have
been identified from S. citri or any
other spiroplasmas. In order to identify
suitable secreted proteins as detection
markers, we will rst carry out a systemic
analysis on the secretome (complete
repertoire of the secreted proteins) of S.
citri. Secreted proteins play an essential
role in pathogen infection and disease
development.
In addition to directly providing a
robust detection tool for citrus stub-
born disease, this project, as the rst
systemic analysis on the secretome of
spiroplasmas, will also ll a gap in our
basic understanding on the molecular
basis of bacterial pathogenicity. Phloem-
limited and insect-transmitted bacte-
rial diseases, including citrus stubborn
disease and Huanglongbing (HLB) are
among the most devastating citrus dis-
eases nowadays. The knowledge on how
phloem-limited and insect-transmitted
bacterial pathogens cause widespread
and severe diseases in citrus is urgently
required in order to develop sustainable
disease control strategies.
The extensive similarity on the
host responses, disease symptoms and
pathogen life styles between citrus
stubborn disease and HLB lead to the
hypothesis that similar virulence fac-
tors or strategies might be used by the
bacterial pathogens to cause disease
in citrus. Therefore, novel knowledge
obtained from this project, including the
secreted proteins, protein markers, ge-
nome sequences and detection tools will
provide guidance to the development
of detection strategies for HLB. This is
especially important because the causal
agent of HLB is very difcult to work
with at this time. Moreover, since HLB
research is not possible at this point in
California due to its exotic status, this
project is Californias best option to
study the genes that might be involved
in the pathogenicity of the causal agent
of HLB or other ecologically related
pathogens such as phytoplasmas.
Progress during November 2008
October 2009
This project has been funded by the
Citrus Research Board since November
2008. During the year of November 2008
through October 2009, we have made
signicant progress on the identication
and systemic analysis of the secretome
of S. citri using modern high throughput
approaches. Two distinct but comple-
mentary approaches are employed:
1) a comparative proteomic approach
using mass spectrometry, from which
the secreted proteins in the cell-free
supernatant of S. citri cell culture are
identied; 2) a genomic approach using
the next generation genome sequencing
A.
B.
C.
and bioinformatic analyses.
Proteins secreted from S. citri cells
in liquid culture grown in the articial
media have been identied using mass
spectrometry. We have established and
optimized the bacterial culturing proto-
cols and the induction conditions using
phloem extracts from citrus. We have
tried several liquid media and different
induction conditions, and have decided
to induce the S. citri cells in C3G medium
at room temperature for 24 hours. This
short induction time allows us to iden-
tify early-induced proteins, which are
useful for early disease detection. After
24 hours of induction at room tempera-
ture, the bacterial cells were removed
from the cell culture by centrifugation.
Total proteins were extracted from the
cell-free supernatant and then ana-
lyzed using mass spectrometry these
proteins should be secreted from the
S. citri cells. By comparing the secreted
protein complements of S. citri cell cul-
tures with and without the induction
of phloem extracts, we have identied
proteins that are highly induced by the
phloem extract. Among these proteins,
two or three candidates with relatively
high abundance are targeted for fur-
ther examination as detection markers.
The secreted protein proles were also
compared to that of the phloem extract
to make sure that we are not looking at
any plant proteins.
The protein candidates identified
from mass spectrometry are also sub-
jected to bioinformatics analysis to
predict the presence of the N-terminal
secretion signals. S. citri belongs to the
gram-positive bacteria, in which the ma-
jor protein secretion system is called the
Sec system. Proteins secreted through
the Sec system possess a sequence motif
at the N-termini which lead the secretion
of these proteins. In other words, the
presence of the N-terminal secretion
signal is a strong hint that the protein
is secreted.
Thus far, we have repeated the
experiments several times and came
up with a short list of secreted protein
candidates for further analysis. Some of
these candidates are listed in Table 1.
These proteins are all highly induced in
the presence of citrus phloem extracts.
Moreover, all of them are predicted to
have a potential N-terminal secretion
signal. Therefore, these proteins could
be used as detection markers.
Another signicant point of progress
we have made during the rst year of
the grant is that we have successfully
established the mechanical transmission
of S. citri from pure culture into pine-
apple sweet orange (Citrus sinensis var
Pineapple) seedlings. Four weeks post
inoculation we could detect the systemic
infection using PCR. Once we conrm
the secretion of some of the candidate
proteins, antibodies will be generated to
examine the presence of the proteins in
citrus. The mechanical transmission of S.
citri in citrus is very important to conrm
the secretion and evaluate the candidate
detection markers.
Project Leader Wenbo Ma, Ph,D.,
is Assistant Professor, Department of
Plant Pathology and Microbiology,
University of California Riverside.
Co-Project Leader Georgios Vidalakis,
Ph.D., is UC Cooperative Extension
Specialist and Plant Pathologist, UC
Riverside, and Director of the Citrus
Clonal Protection Program (CCPP).
CAK99227 chromosome 0.998 5 1
Table 1. Candidate secreted proteins of Spiroplasma citri
identied from mass spectrometry.
# of peptide hits using
Mass Spectrometry
Trial 1 Trial 2 Gene Name Location
Probability of N-terminal
secretion signal
C
alifornia and Florida are at con-
siderable risk for the establish-
ment of the Mediterranean fruit
y (MFF) due to their favorable climate
and host availability. Any Medy inva-
sion in these or neighboring states trig-
gers a sobering reality. Colonization of
MFF would result in devastating losses
of crops; personal, state, and national
income; jobs; and global trade.
We can never afford a Medy in-
vasion, and during times like these
Probiotic diet for SIT Medy
Carol R. Lauzon
management of an infestation could
prove ruinous. In light of this, we should
be hyper-critical of our existing pest
management strategies to safeguard
our high-valued products and economy
and continue to work toward improv-
ing existing management programs and
strategies.
Radiation is used to produce adult
males that are reproductively sterile.
These irradiated males are subsequent-
ly introduced into susceptible regions,
Photo by Jack Kelly Clark, University of Cali-
fornia, Agriculture and Natural Resources.
Intact DNA (smear)
from non-irradiated
male Medies seen in
Lanes 1 and 3. Lane 4
shows a DNA ladder.
Lanes 5 and 7 contain
fragmented DNA
(bands) from irradiated
male Medies.
DNA fragmentation
is characteristic
of apoptosis, or
programmed cell death.
DNA from irradiated males that fed on a standard
diet (white arrow). Notice the bands, or fragments
of DNA. DNA from males that fed on agellin
(black arrow). Equal amounts of DNA were used.
Thus, there appears to be less fragmentation of
DNA for males that consumed agellar protein.
N
Transmission electron
micrograph of a sterile
male that fed on
agellar protein. Notice
the mitochondria in
the act of dividing
(arrow) and a nucleus
(N) dense with nuclear
material. There is no
visual indication of
apoptosis.
i.e. the Los Angeles Basin, with the in-
tent to interrupt the reproductive cycle
of any female Medy that may have
invaded the area. This strategy, know as
Sterile Insect Technique (SIT) is part of
a Preventative Release Program (PRP),
and while both SIT and the PRP have
proven benecial for control of Medy
infestations, there are areas within each
that can be improved and/or optimized,
such as SIT male tness.
In recent years, we have shown
that radiation used in SIT damages
the gut of sterile males. This damage
likely compromises the performance of
sterile males because improvements in
sterile male mating performance have
been observed after these males con-
sumed benecial gut bacteria (Niyazi
et al. 2004).
The bacteria used in those diets,
Pantoea agglomerans (formerly, En-
terobacter agglomerans) and Klebsi-
ella pneumoniae, have been shown
to be vertically transmitted, that is,
passed from female to her eggs and to
emerging adults (Lauzon et al. 2009).
Information regarding the relation-
ships between pest fruit ies and these
bacteria is dovetailing (i.e. Lauzon
2004). There really is no question now
that these bacteria exert positive inu-
ences on Medy physiology, but there
are several questions regarding how
these bacteria do what they do.
Our research was directed toward a
more complete understanding of how
bacteria can be used to improve the
tness of SIT Medies. It utilized new
ways of assessing the degree of nontar-
get radiation damage (tness costs) in
SIT Medies. The aim of the work is
to nd a practical way to facilitate gut
repair and/or protect the Medy gut
from radiation damage by enlisting
the bacteria themselves or using some
microbial product to improve the t-
ness of SIT males. The goal of the work
is to strengthen the Preventative Re-
lease Program and protect California
more soundly from a Medy invasion.
The intent of this short term one-year
project was also to determine if suf-
cient evidence could be garnered to
support a larger research plan.
In earlier work, our laboratory
found that cells within the midgut of
SIT males were apoptotic. Apoptosis,
also known as programmed cell death,
is a common cellular phenomenon that
occurs, within insects for example, to
delete unwanted cells, whether they
are cells irreversibly damaged, tumor
cells, or superfluous (Dorstyn et al.
2002). This process is highly controlled
and self-inicted and in the case of
SIT males is due to radiation expo-
sure. Visual signs of apoptosis include
cell organelles, such as mitochondria,
that appear aberrant in shape. Other
evidence includes DNA in fragments.
Apoptosis can be determined for cells
by looking for DNA fragmentation,
measuring protein and enzyme levels,
and inspecting the cells using electron
microscopy, for example.
We have observed that the degree
of apoptosis is lessened in SIT males
after they ingest the beneficial gut
bacterium, P. agglomerans. Bacterial
agella, structures that allow bacteria
1
2
3
0
0.01
0.02
0.03
0.04
0.05
0.06
The graph shows
the means of
Caspase 3 levels
in pools of:
1 = nonirradiated
factory males;
2 = sterile males on
standard diet;
3 = sterile males on
standard diet plus
agellar protein.
Y axis = absorbance
at 405 nm
to swim or move, have been reported to
inhibit apoptosis as part of preserving
a host cell for pathogen invasion. P. ag-
glomerans also possess agella. Thus, we
hypothesized that the agella of P. ag-
glomerans were inuencing the amount
of apoptosis in irradiated gut cells.
If agella are the responsible entity
for decreasing the amount of apoptosis
in the SIT midgut, and thus improving
the gut condition, then an important
question is: at what point in the apop-
totic process are the bacteria inhibiting
reactions? Based on earlier work in our
laboratory, and part of this research
project, we hypothesized that levels
of Cytochrome C and Cytochrome C
Oxidase from mitochondria or Caspase
3 levels in cell cytoplasm are reduced
in SIT males that ingest agella. DNA
fragmentation should be also lessened
after ingestion of agella because those
enzymes indirectly affect the integrity
of DNA.
Methods: Briey, fragella from and
whole cells of living Pantoea agglo-
merans and in the form of an extract
were provisioned to irradiated and
nonirradiated Medies through their
diet. Similarly, commercially prepared
agellin, the agellar protein, was also
provisioned to the adult flies. After
two days, ies were sacriced and were
examined for the amount of DNA
fragmentation of their midgut cells,
and levels of Cytochrome C Oxidase ,
Cytochrome C, and Caspase 3, all pro-
teins associated with apoptosis. Midgut
epithelial cells were also scrutinized
visually using transmission electron
microscopy (TEM).
Results: Fragmentation of DNA ap-
peared less in sterile males that fed on
agellar proteins than sterile males that
fed on standard diet lacking agella
or agellin. Micrographs of irradiated
male medies that fed on preparations
containing whole cells of Pantoea ag-
glomerans, agellar extract from P. ag-
glomerans, or commercial agellin did
not show evidence of apoptosis. Mito-
chondria were captured in the process
of dividing, suggestive of normal cell
activity. Nuclei were densely stained
with nuclear material.
Caspase 3 enzymatic levels were
assayed for both sterile and non-irradi-
ated factory medies on diets with and
without agellar protein. Flagellar pro-
tein decreased the amount of Caspase
3 activity within irradiated ies, a key
enzyme that is involved in apoptosis.
We compared Cytochrome c oxi-
dase levels for sterile and non-irradi-
ated factory Medies using quantita-
tive PCR (qPCR) and found Cycle
threshold (Ct) levels to differ by as
much as 2 points (sterile medies avg.
Ct value, 16.076 vs. nonirradiated avg.
Ct value, 18.171). Therefore, the sterile
males demonstrated an increase in gene
expression for Cytochrome c oxidase
beyond that seen in the nonirradiated
ies, indicative of apoptosis. Although
we found differences that serve as our
foundation for the study, we are repeat-
ing our experiments currently with the
advent of recent technical improve-
ments in the assays.
In conclusion, we have found that
whole cells of Pantoea agglomerans,
flagellar extracts of P. agglomerans
and commercially prepared agellin all
improve the cellular status of irradiated
male Medies (not all data shown). Fla-
gellar extracts and agellin improved
cellular organelles.
One reason why a probiotic diet
has not yet been put into mass rearing
is the perceived difculty in adding a
living component to the diet. As our
research continues, if agellar proteins
are determined to produce the same ef-
fect on sterile males that living bacteria
do, and our results suggest they do, then
this concern can be eliminated.
While we still consider our data
preliminary at this point, we are cau-
tiously optimistic that we are mak-
ing gains toward understanding the
mechanism behind radiation damage
improvements for fruit ies that ingest
a probiotic diet and that further work
is merited. A fuller understanding of
this dynamic should lead to practical
improvements in sterile male tness
and strengthening of the Preventative
Release Program.
Acknowledgements: I gratefully
acknowledge the contributions of
CSUEB graduate students Jacque-
line Louie and Sima Bahadori; Dr.
Susan McCombs of USDA- APHIS,
Waimanalo, HI; and Tina Carvalho of
the University of Hawaii at Manoa,
Honolulu, HI. This research was made
possible through the generous support
of the Citrus Research Board.
Project Leader Carol Lauzon,
Ph.D., is Professor, Department of
Biological Sciences, California State
University East Bay.
References:
Dorstyn, L., Read, S., Cakouros, D.,
Huh, J.R., Hay, B.R., and S. Kumar. 2002.
The role of cytochrome c in caspase
activation in Drosophila melanogaster
cells. J. Cell Biol. 156(6):1089-1098.
Lauzon, C.R. Symbionts of Teph-
ritids. In: Insect Symbioses, A CRC
publication. Bourtzis and Miller, eds.
Feb. 2003. p. 115-130.
Lauzon, C.R., S.D.McCombs and
S.E. Potter. 2009 Vertical passage of
Enterobacter agglomerans and Kleb-
siella pneumoniae in Ceratitis capitata
Weidemann, the Mediterranean fruit
y. Ann. Entomol. Soc. Amer. 102(1):
85-95.
Niyazi, N., C.R. Lauzon, and T.E.
Shelly. 2004. Effect of probiotic adult
diets on tness components of sterile
male Mediterranean fruit ies (Diptera:
Tephritidae) under laboratory and
eld conditions. J. Econ. Entomol. 97:
1570-1580.

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