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1186 Plant Disease / Vol. 98 No. 9
Research
Evaluation of Olive as a Host of Xylella fastidiosa
and Associated Sharpshooter Vectors
Rodrigo Krugner, Mark S. Sisterson, Jianchi Chen, and Drake C. Stenger, United States Department of Agriculture–Agricultural
Research Service, San Joaquin Valley Agricultural Sciences Center, Parlier, CA 93648; and Marshall W. Johnson, Department of Ento-
mology, University of California, Riverside 92521
Abstract
Krugner, R., Sisterson, M. S., Chen, J., Stenger, D. C., and Johnson, M. W. 2014. Evaluation of olive as a host of Xylella fastidiosa and associated
sharpshooter vectors. Plant Dis. 98:1186-1193.
Olive (Olea europaea) trees exhibiting leaf scorch or branch dieback
symptoms in California were surveyed for the xylem-limited, fastidi-
ous bacterium Xylella fastidiosa. Only approximately 17% of diseased
trees tested positive for X. fastidiosa by polymerase chain reaction, and
disease symptoms could not be attributed to X. fastidiosa infection of
olive in greenhouse pathogenicity assays. Six strains of X. fastidiosa
were isolated from olive in Southern California. Molecular assays
identified strains recovered from olive as belonging to X. fastidiosa
subsp. multiplex. Pathogenicity testing of olive strains on grapevine
and almond confirmed that X. fastidiosa strains isolated from olive
yield disease phenotypes on almond and grapevine typical of those
expected for subsp. multiplex. Mechanical inoculation of X. fastidiosa
olive strains to olive resulted in infection at low efficiency but in-
fections remained asymptomatic and tended to be self-limiting. Vector
transmission assays demonstrated that glassy-winged sharpshooter
(Homalodisca vitripennis) could transmit strains of both subspp.
multiplex and fastidiosa to olive at low efficiency. Insect trapping data
indicated that two vectors of X. fastidiosa, glassy-winged sharpshooter
and green sharpshooter (Draeculacephala minerva), were active in
olive orchards. Collectively, the data indicate that X. fastidiosa did not
cause olive leaf scorch or branch dieback but olive may contribute to
the epidemiology of X. fastidiosa-elicited diseases in California. Olive
may serve as an alternative, albeit suboptimal, host of X. fastidiosa.
Olive also may be a refuge where sharpshooter vectors evade intensive
areawide insecticide treatment of citrus, the primary control method
used in California to limit glassy-winged sharpshooter populations and,
indirectly, epidemics of Pierce’s disease of grapevine.
Diseases caused by the xylem-limited, fastidious bacterium Xy-
lella fastidiosa have been a problem in California for more than
100 years, with grapevine (20,32), almond (28,41,42), and alfalfa
(43,50) being the most affected crops. X. fastidiosa is widely dis-
tributed in the Americas, causing vascular occlusion disease in
some host species while other host species remain asymptomatic
(22,26,38). Individual strains of X. fastidiosa differ in host range
and may be classified into subspecies by multilocus sequence typ-
ing (MLST) (40,53). Strains of X. fastidiosa subsp. fastidiosa
cause Pierce’s disease of grapevine (14) and are capable of causing
disease in other hosts, including almond (6). X. fastidiosa subsp.
multiplex strains do not cause disease in grapevine but are com-
monly isolated from almond expressing leaf scorch disease
(6,7,15,28), and subsp. multiplex strains also cause disease in nu-
merous perennial crop and landscape plants, including peach
(13,51), plum (37), purple-leafed plum, and sweetgum (18). In
South America, citrus variegated chlorosis and coffee leaf scorch
are caused by strains of subsp. pauca (1,5,17). Oleander leaf
scorch (8,36) is caused by strains belonging to a distinct clade
referred to as subsp. sandyi.
X. fastidiosa is transmitted by xylem-sap-feeding leafhoppers
(28,33). Historically, the key native vectors in California were the
blue-green sharpshooter (Graphocephala atropunctata Signoret) in
Napa Valley (35) and green sharpshooter (Draeculacephala mi-
nerva Ball) in the San Joaquin Valley (12,34,43). In the late 1980s,
the glassy-winged sharpshooter (Homalodisca vitripennis (Ger-
mar)) was introduced to California (4,45,46) and is now well estab-
lished in Southern California and the southern San Joaquin Valley.
Glassy-winged sharpshooter, a known vector of X. fastidiosa (2), is
polyphagous (47) and highly mobile (3,10,25). Establishment of
glassy-winged sharpshooter in citrus was a primary factor in recent
epidemics of Pierce’s disease in Southern California (31) and the
southern San Joaquin Valley (29,30,44).
California is the sole producer of olive (Olea europaea L.) in the
United States, with approximately 17,800 ha planted and a produc-
tion value estimated at $130 million per annum (49). Although
most commercial production is located in the San Joaquin and
Sacramento Valleys, olive trees are commonly planted throughout
California as ornamental trees in urban and rural areas. In recent
years, leaf scorch and branch dieback symptoms in olive became a
concern to growers, homeowners, and landscape managers in Cali-
fornia. Such symptoms are typical of those caused by X. fastidiosa,
prompting us to investigate the role of X. fastidiosa in olive leaf
scorch or branch dieback disease. Recently, multiple fungal species
have been isolated from symptomatic trees in California, with
pathogenicity of some fungal isolates demonstrated by experi-
mental inoculation of olive (48). However, those experiments did
not address pathogenicity of X. fastidiosa to olive. Furthermore,
limited information is available concerning the role of olive as a
source of X. fastidiosa (9,19) and sharpshooter vectors (11). Here,
Corresponding author: R. Krugner, E-mail: rodrigo.krugner@ars.usda.gov
Mention of trade names or commercial products in this publication is solely
for the purpose of providing specific information and does not imply rec-
ommendation or endorsement by the United States Department of Agricul-
ture (USDA). USDA is an equal opportunity provider and employer.
Accepted for publication 20 March 2014.
http://dx.doi.org/10.1094/PDIS-01-14-0014-RE
This article is in the public domain and not copyrightable. It may be freely
reprinted with customary crediting of the source. The American Phytopatho-
logical Society, 2014.
Plant Disease / September 2014 1187
we describe studies designed to evaluate (i) prevalence of X. fas-
tidiosa infection of olive in California, (ii) molecular typing of X.
fastidiosa strains isolated from olive, (iii) pathogenicity of X. fas-
tidiosa strains from olive, (iv) transmission of X. fastidiosa to olive
by glassy-winged sharpshooter, and (v) activity of known insect
vectors of X. fastidiosa in olive orchards.
Materials and Methods
Sampling olive for X. fastidiosa. Sampling sites in urban areas
and commercial orchards were determined by visual inspection to
identify trees displaying leaf scorch or branch dieback symptoms.
Symptoms were documented by photography. From October 2008
to September 2012, tissue samples were collected from 198 symp-
tomatic olive trees found in Southern California (i.e., ornamental
trees in San Diego, Orange, Riverside, Los Angeles, and Ventura
Counties), the San Joaquin Valley (i.e., trees in commercial or-
chards in Kern, Tulare, and Fresno Counties), and the Sacramento
Valley (i.e., ornamental trees in Yolo County). Samples were trans-
ported to the laboratory and screened for presence of X. fastidiosa
by polymerase chain reaction (PCR) using primers RST31 and
RST33, as described (27), and by culturing on PW medium (13).
Axenic cultures recovered from olive and validated as X. fastidiosa
by molecular methods (see below) were added to the United States
Department of Agriculture–Agricultural Research Service San
Joaquin Valley Agricultural Sciences Center X. fastidiosa collec-
tion stored at –80°C.
Molecular typing of X. fastidiosa strains. For molecular iden-
tification, genomic DNA was extracted (6) from 7- to 14-day-old
cultures. Preliminary identification of subspecies affiliation was
accomplished using the four-primer PCR assay (6), in which am-
plicon profile of subsp. fastidiosa may be distinguished from that
of subsp. multiplex. Known strains of subsp. fastidiosa (Temecula)
and multiplex (Dixon) were used as standards. Further characteri-
zation was accomplished using MLST (40). Genomic DNA of
select olive-infecting strains isolated in this study (LM10, RH1,
and Fillmore) and the RC75 strain isolated by others (kindly
provided by A. Purcell) was used as a template for PCR amplifi-
cation of pilU and seven housekeeping genes (cysG, gltT, holC,
malF, leuA, nuoL, and petC). Amplified products were cloned into
pGEMT-easy. A consensus sequence was determined for each
amplicon based on sequences of three independent clones. For
MLST, consensus sequences of all eight amplicons were con-
catenated into a single sequence (7,480 nucleotides) for each
strain. Concatenated sequences of X. fastidiosa strains isolated
from olive were aligned with concatenated sequences of select
reference strains available in GenBank. The resulting multiple
alignment was used as input data to generate a neighbor-joining
tree based on 1,000 bootstrap replications. Nodes with bootstrap
support of less than 50% were collapsed to polytomies.
Concatenated sequences for X. fastidiosa subsp. pauca strain 9a5c
was used as an outgroup to root the tree.
Mechanical inoculation of X. fastidiosa to olive. To determine
whether strains of X. fastidiosa isolated from olive persist and
cause disease in olive, six strains of X. fastidiosa isolated from
olive were grown in pure culture and mechanically inoculated (21)
to 1-year-old (approximately 30 cm in height) greenhouse-reared
olive plants. Inocula were prepared from 7- to 10-day-old cultures
as a turbid cell suspension of approximately 108 cells/ml in water.
A small drop of inoculum (7 µl) (or water, for negative controls)
was placed at three different locations of the main stem (bottom,
middle, and top); the stem was pierced with a needle through each
drop. In total, 30 plants (O. europaea L.) of each olive cultivar
(‘Mission’, ‘Manzanillo’, ‘Sevillano’, ‘Arbequina’, ‘Arbosana’,
‘Koroneiki’, and ‘Barouni’) were inoculated with X. fastidiosa
strain RH1 a total of four times between March and September
2009. In October 2009, a second group of 30 plants (Manzanillo)
was inoculated with X. fastidiosa strain Fillmore. A third group of
plants (Arbequina) was inoculated with strains RH1 (2 plants),
LM10 (14 plants), and Fillmore (3 plants) in June 2011. A fourth
group of plants (Arbequina) was inoculated with RH1 (5 plants),
LM10, (10 plants), Fillmore (5 plants), and Oceanside (5 plants) in
October 2012. Each group of inoculated plants included mock-
inoculated plants as controls. All test plants were kept in an insect-
free greenhouse and monitored over a period of 1 year for symp-
tom development and presence of X. fastidiosa by PCR (using
primers RST31 and RST33) and culturing.
Mechanical inoculation of X. fastidiosa to grapevine and al-
mond. Pathogenicity tests in almond and grapevine were con-
ducted to complement MLST typing. Strains identified as belong-
ing to subsp. fastidiosa were expected to cause disease in
grapevine and almond, whereas strains identified as subsp. multi-
plex were expected to cause disease in almond but not grapevine.
X. fastidiosa strains isolated from olive in this study (RH1, LM10,
Fillmore, and Oceanside) were mechanically inoculated to
grapevine (Vitis vinifera L. ‘Chardonnay’), almond (Prunus dulcis
(Mill.) D.A. Webb ‘Sonora’), and olive using the same inocula
described above (i.e., fourth group of olive plants inoculated in
October 2012). As positive controls, strains Temecula and Stag’s
Leap (subsp. fastidiosa) were inoculated to grapevine and strains
M12 and Dixon (subsp. multiplex) were inoculated to almond.
Strain M23 (subsp. fastidiosa) was inoculated to both grapevine
and almond. Inocula were prepared and delivered to test plants as
described above, except that test plants were inoculated only on a
single date. As a negative control, groups of 2 to 10 plants per host
species were mock inoculated with water. All test plants were kept
in an insect-free screenhouse and monitored for 1 year for
symptom development and presence of X. fastidiosa by PCR and
culturing.
Glassy-winged sharpshooter transmission assays. Transmis-
sion assays were conducted to confirm that glassy-winged sharp-
shooter is a vector of X. fastidiosa strains isolated from olive.
Laboratory colonies of glassy-winged sharpshooter were
established and maintained as described by Krugner (23). Plants
infected by mechanical inoculation (described above) were used as
acquisition source plants. Source plants for reference strains
Temecula and Stag’s Leap (subsp. fastidiosa) were infected grape-
vines (Chardonnay). Source plants for reference strains Dixon and
M12 (subsp. multiplex) were infected almond plants (Sonora).
Almond was used as a source plant for strain M23 (subsp. fastidi-
osa). Source plants used for X. fastidiosa strains isolated from
olive in this study were almond (strains RH1 and Fillmore) or
Arbequina olive (strain LM10). Source plants for X. fastidiosa
olive strain Oceanside were not available, because no mechanically
inoculated plants became infected. All source plants were verified
as infected by PCR and culturing. Colony-reared adult glassy-
winged sharpshooters were given a 96-h acquisition access period
(AAP) on source plants. At the end of the AAP, insects were
transferred in groups of 10 to test plants (Arbequina olive grown in
3.8-liter pots) for a 96-h inoculation access period (IAP).
Following the IAP, insects were removed; test plants were treated
with a foliar application of insecticidal soap (Safer Brand Insect
Killing Soap; Woodstream Corp.) and the systemic insecticide
imidacloprid (Admire Pro; Bayer CropSciences) as a soil drench.
To verify that colony insects were not contaminated with X.
fastidiosa, groups of 10 adults not given an AAP on source plants
were caged on test plants for a 96-h IAP. To verify that test plants
were not contaminated with X. fastidiosa from the source nursery,
test plants not exposed to glassy-winged sharpshooters also were
maintained in the same insect-free greenhouse as inoculated test
plants. Inoculated and control test plants were maintained at 23 to
27°C and 22 to 25% relative humidity under natural light during
summer and supplemented with artificial light during early spring,
late fall, and winter. Plants were assayed for presence of X.
fastidiosa at 12 and 24 weeks post IAP using PCR and culturing
methods described above. On each sampling date, one leaf with
petiole intact was collected from the bottom, middle, and top of
each plant and subjected to analysis.
Insect vector activity in olive orchards. To document activity
of insect vectors of X. fastidiosa in olive orchards, yellow card
sticky traps (14 by 22.9 cm; Seabright Laboratories) were moni-
1188 Plant Disease / Vol. 98 No. 9
tored in two olive orchards. Both orchards were located in Fresno
County, CA. Site A was planted in 2003 and consisted of Arbe-
quina, Arbosana, and Koroneiki. Site B was planted in 2009 and
consisted of Arbequina and Arbosana. Site A was located within a
known glassy-winged sharpshooter-infested zone. Site B was lo-
cated outside the glassy-winged sharpshooter-infested zone, near
permanent pastures known to harbor populations of green sharp-
shooters (43). Traps were placed at canopy height and replaced
biweekly. At time of collection, the number of glassy-winged
sharpshooters and green sharpshooters on each trap were recorded.
Trapping at site A was initiated in March 2010 and ended in Sep-
tember 2013. Trapping at site B was initiated in March 2011 and
ended August 2013. Traps at site A were placed along five tran-
sects, with a total of 26 traps placed in the orchard. At site B, 14
traps were placed around the perimeter of the orchard. Traps were
removed from both sites in late October through November of each
year during harvest. No insecticides were applied to either orchard
during the study period. Herbicides and fungicides were applied in
accordance with standard grower practices.
Results
Prevalence of X. fastidiosa in olive in California. Samples
were collected from olive trees displaying branch dieback (Fig.
1A) or leaf scorch (Fig. 1B) symptoms. Symptoms observed on
ornamental trees in urban areas in southern California were similar
to symptoms observed on trees in commercial orchards in the San
Joaquin Valley and Yolo County. In total, samples were collected
from 198 olive trees, with X. fastidiosa detected by PCR in 16.6%
of samples (Table 1). Prevalence of X. fastidiosa was greater in
Southern California than in the San Joaquin Valley or Yolo County.
Specifically, 38.5% (30 of 78) of symptomatic trees sampled in
Southern California tested positive for X. fastidiosa by PCR,
whereas only 2.5% (3 of 121) of symptomatic trees sampled in the
San Joaquin Valley and Yolo County tested positive for X. fastidi-
osa by PCR (Table 1).
In total, six strains of X. fastidiosa were isolated from olive trees
at several locations in Southern California: La Mirada (strains
LM10 and LM14), Rolling Hills (strains RH1 and RH2), Fillmore
(strain Fillmore), and Oceanside (strain Oceanside) (Table 1). The
Tab le 1 . Survey of olive for Xylella fastidiosa in California
Collection date County Location Samples positive by PCRa Number of X. fastidiosa strains isolated
May 2009 Yolo Davis 0/4 0
June 2010 Fresno Fresno 0/10 0
July 2011 Fresno 0/20 0
July 2011 Fresno 0/6 0
July 2012 Clovis 0/26 0
August 2008 Tulare Porterville 0/3 0
August 2008 Terra Bella 0/2 0
August 2009 Ducor (site 1) 0/10 0
August 2009 Ducor (site 2) 3/10 0
June 2011 Woodlake 0/3 0
June 2011 Lemon Cove 0/4 0
June 2011 Lindsay 0/16 0
August 2008 Kern Bakersfield 0/7 0
August 2009 Ventura Piru 2/5 0
August 2009 Fillmore 1/7 1
August 2009 Ventura 3/3 0
October 2008 Los Angeles Rolling Hills 0/5 1
April 2009 Rolling Hills 3/3 1
March 2009 Rancho Bernardo 3/3 0
May 2011 La Mirada 6/17 2
May 2009 Orange Newport Beach 2/8 0
May 2009 Costa Mesa 1/7 0
May 2009 Riverside Riverside 0/9 0
August 2009 Riverside 5/6 0
May 2009 San Diego Carlsbad 3/3 0
October 2012 Oceanside 1/1 1
a Numerator denotes number of plants positive by polymerase chain reaction (PCR) and denominator denotes number of plants tested.
Fig. 1. A, Branch dieback and B, leaf scorch symptoms observed in olive trees
sampled for Xylella fastidiosa in California. Note that X. fastidiosa was cultured from
both samples shown but correlation of X. fastidiosa with symptoms in olive was low.
Plant Disease / September 2014 1189
Fillmore strain was isolated from an olive tree present in a single
row located between two mature citrus orchards; all other olive-
infecting strains were isolated from ornamental trees located in
urban settings. Each culture was verified as X. fastidiosa based on
PCR (with primers RST31 and RST33) using genomic DNA
extracted from cultures. Attempts to isolate strains of X. fastidiosa
from olive trees in the San Joaquin Valley or Yolo County were
unsuccessful.
Fig. 2. Xylella fastidiosa strains isolated from olive in California belong to subspecies multiplex. Presented is a neighbor-joining tree showing relationships of X. fastidiosa
strains from olive (RH1, LM10, Fillmore, and RC75; designated with boxes) with characterized reference strains of X. fastidiosa. Input data were a multiple alignment o
f
concatenated sequences (7,480 nucleotides) for seven housekeeping genes and pilU. Multiple taxa appearing on a single branch were 100% identical in sequence. Colored
lines denote individual clades, some of which have been assigned as subspecies. Bootstrap values (based on 1,000 replications) are shown adjacent to nodes; X. fastidiosa
subsp. pauca strain 9a5c is designated with an asterisk and was used as an outgroup to root the tree. Scale bar at bottom left indicates a genetic distance of 0.01.
1190 Plant Disease / Vol. 98 No. 9
X. fastidiosa strains from olive belong to subsp. multiplex.
Preliminary typing using the four-primer assay (6) indicated that
the six X. fastidiosa strains isolated from olive in this study were
indistinguishable from reference strains of subsp. multiplex and
distinct from reference strains of subsp. fastidiosa (data not
shown). MLST was performed on a subset of strains isolated from
olive (LM10, RH1, and Fillmore) in this study and strain RC75
(provided by A. Purcell), also isolated from olive in California.
Sequences determined in this study were deposited in GenBank as
accessions KF954183 to KF954214. Alignment of concatenated
sequences for the eight loci examined revealed that LM10, RH1,
and RC75 were 100% identical to each other and to subsp.
multiplex reference strain M12; the Fillmore strain differed from
M12 at only 2 base positions (99.97% identity). In the neighbor-
joining tree (Fig. 2) based on an alignment of concatenated
sequences, the four olive-infecting strains clustered with known
strains of subsp. multiplex in a clade distinct from subsp. fas-
tidiosa, subsp. sandyi, and a clade containing strains from mul-
berry.
X. fastidiosa strains from olive yield disease phenotypes on
almond and grapevine typical of those expected for subsp. mul-
tiplex. Inoculation of almond with X. fastidiosa strains isolated
from olive or reference strains of subsp. multiplex resulted in a
high correlation among symptoms (leaf scorch), detection by PCR,
and isolation of the bacterium by culturing (Table 2). In contrast to
reference strains of subsp. fastidiosa, inoculation of grapevine with
X. fastidiosa strains from olive did not produce symptoms. Further-
more, all grapevines inoculated with X. fastidiosa olive strains
were negative by PCR and did not yield live cultures. Collectively,
pathogenicity tests using grapevine and almond as test hosts sup-
ported placement of X. fastidiosa strains isolated from olive in
subsp. multiplex.
X. fastidiosa infection of olive does not correlate with disease
symptoms. Field surveys resulted in detection of X. fastidiosa
from approximately 17% of symptomatic trees tested. In green-
house studies (Table 2), only 8 of 284 olive trees inoculated with X.
fastidiosa strains RH1, Fillmore, LM10, or Oceanside tested posi-
tive by PCR for X. fastidiosa. However, no olive test plants positive
for X. fastidiosa by PCR developed symptoms. Leaf scorch
symptoms were observed on 22 olive test plants: 18 test trees
inoculated with X. fastidiosa (but PCR-negative) and 4 test trees
mock inoculated with water. Even after multiple attempts, X.
fastidiosa was not detected by PCR or cultured from any olive test
plant displaying symptoms. Collectively, the results indicate that
leaf scorch or branch dieback symptoms of olive were not well
correlated with X. fastidiosa infection in the field and were not
reproducible by inoculation of X. fastidiosa to olive under
controlled conditions in the laboratory.
X. fastidiosa infection of olive may be transient rather than
chronic. Among test plants inoculated with X. fastidiosa olive
strain RH1, two, one, one, and two test plants of Arbequina,
Arbosana, Mission, and Barouni, respectively, tested positive for
presence of X. fastidiosa via PCR conducted at 12 weeks post
inoculation. However, no test plants inoculated with strain RH1
tested positive for the bacterium at 24 weeks and 1 year after
inoculation. In addition, none of the plants inoculated with the
Fillmore strain tested positive for the bacterium via PCR at 12
weeks, 24 weeks, and 1 year after inoculation. Two test plants
(Arbequina) inoculated with strain LM10 tested positive by PCR
for X. fastidiosa at 12 weeks, 24 weeks, and 1 year after inocula-
tion. Despite repeated detection by PCR of the LM10 strain over a
1-year period, both infected test plants did not express branch die-
back or leaf scorch symptoms. No other olive test plants were posi-
tive for X. fastidiosa via PCR. All attempts to reisolate X. fastidi-
osa from inoculated olive test plants failed. Collectively, these
results suggest that X. fastidiosa infection of olive may be self-
limiting, such that chronic infection may be uncommon.
Glassy-winged sharpshooter transmits X. fastidiosa to olive
at low efficiency. On average, 9.6 (range 6 to 10) insects per plant
were alive at the end of the 96-h IAP on olive test plants. At 24
weeks after the IAP, branch dieback symptoms were observed in
nine test plants, including one control plant that was not exposed to
insects. However, all symptomatic test plants were negative for
presence of X. fastidiosa via PCR. At 24 weeks after the IAP, X.
fastidiosa was detected in a total of 6 of 145 test plants (Table 3).
Among X. fastidiosa-positive test plants, one had been exposed to
insects that acquired strain RH1, three plants had been exposed to
insects that acquired the subsp. multiplex reference strain Dixon,
and two plants had been exposed to insects that acquired the subsp.
fastidiosa reference strain M23. No other asymptomatic plant
tested positive for presence of X. fastidiosa via PCR and all at-
tempts to reisolate the bacterium from glassy-winged sharpshooter-
inoculated olive test plants failed.
Tab l e 2. Mechanical inoculation of Xylella fastidiosa strains to three test
speciesa
Strain (subspecies), assay Grapevine Almond Olive
Temecula (fastidiosa)
Symptoms 1/5 NT NT
PCR 1/5 NT NT
Culture 1/5 NT NT
Stag’s Leap (fastidiosa)
Symptoms 9/20 NT NT
PCR 7/20 NT NT
Culture 7/20 NT NT
M23 (fastidiosa)
Symptoms 12/20 3/10 NT
PCR 8/20 3/10 NT
Culture 9/20 3/10 NT
Dixon (multiplex)
Symptoms NT 8/10 NT
PCR NT 8/10 NT
Culture NT 8/10 NT
M12 (multiplex)
Symptoms NT 12/14 NT
PCR NT 9/14 NT
Culture NT 9/14 NT
LM10 (multiplex)
Symptoms 0/5 1/9 3/24b
PCR 0/5 1/9 2/24c
Culture 0/5 1/9 0/24
RH1 (multiplex)
Symptoms 0/5 7/14 15/217b
PCR 0/5 6/14 6/217c
Culture 0/5 6/14 0/217
Fillmore (multiplex)
Symptoms 0/8 3/8 0/38
PCR 0/8 1/8 0/38
Culture 0/8 1/8 0/5
Oceanside (multiplex)
Symptoms 0/1 0/4 0/5
PCR 0/1 0/4 0/5
Culture 0/1 0/4 0/5
a Numerator denotes number of plants with symptoms or positive for
X
. fastidiosa by polymerase chain reaction (PCR) or culturing; denom-
inator denotes number of plants tested; NT = not tested.
b Expressing leaf scorch or branch dieback symptoms but negative for
X
. fastidiosa by PCR.
c Positive for X. fastidiosa by PCR but asymptomatic.
Tab l e 3. Glassy-winged sharpshooter transmission assays of Xylella fas-
tidiosa strains to olive test plants
Strain (subspecies) Acquisition source Test plant PCRa
Temecula (fastidiosa) Grapevine 0/11
Stag’s Leap (fastidiosa) Grapevine 0/25
M23 (fastidiosa) Almond 2/26
Dixon (multiplex) Almond 3/26
M12 (multiplex) Almond 0/27
LM10 (multiplex) Olive 0/4
RH1 (multiplex) Almond 1/26
aNumerator denotes number of plants positive for X. fastidiosa by polymer-
ase chain reaction (PCR) and denominator denotes number of plants tested.
Plant Disease / September 2014 1191
Sharpshooter vectors are active in olive orchards. Yellow
sticky trap counts revealed presence and activity of glassy-winged
sharpshooter and green sharpshooter in two olive orchards located
in Fresno County. Although glassy-winged sharpshooters were
common at site A (Fig. 3A), only three green sharpshooters were
caught (2 June 2010, 13 December 2010, and 20 June 2011) during
the 4-year sampling period. Therefore, only data for glassy-winged
sharpshooters are presented in Figure 3A. At site A, glassy-winged
sharpshooters were common between June and August, and peaked
with an average (± standard error of the mean) of 0.08 ± 0.02 (n =
29), 0.10 ± 0.03 (n = 55), 0.03 ± 0.01 (n = 10), and 0.03 ± 0.01 (n
= 11) adults per trap per day in 2010, 2011, 2012, and 2013, re-
spectively. Site B was not located inside a known glassy-winged
sharpshooter-infested zone and, as expected, no glassy-winged
sharpshooters were observed. Site B was located adjacent to a
permanent pasture known to harbor green sharpshooters. The
number of green sharpshooters caught at site B was low, with no
clear seasonal patterns (Fig. 3B), a result similar to that reported
for green sharpshooter movement into almond orchards and vine-
yards (12). In total, 17, 12, and 1 green sharpshooter were caught
on sticky traps in 2011, 2012, and 2013, respectively. Green sharp-
shooters prefer grasses over woody perennials (34). Accordingly,
movement of green sharpshooters into olive is likely transient and
may have been due to proximity of a permanent pasture serving as
a green sharpshooter source habitat.
Discussion
The role of X. fastidiosa in etiology of olive leaf scorch and
branch dieback in California. Correlation of X. fastidiosa infec-
tion with leaf scorch or branch dieback symptoms of olive was
poor in both the field survey and greenhouse pathogenicity or vec-
tor transmission assays. These observations indicated that X. fas-
tidiosa strains reported here did not cause leaf scorch or branch
dieback disease in olive maintained under the described conditions.
Furthermore, in greenhouse assays, there was no overlap in olive
test plants expressing symptoms with olive test plants in which
presence of the pathogen was detected by PCR (Table 2). Several
explanations for the presence of symptoms in test plants (including
mock-inoculated plants) lacking detectable X. fastidiosa are plausi-
ble: some nursery-grown test plants may have been infected with a
pathogen capable of causing disease in olive (48), or symptoms
observed in some test plants were due to abiotic stress (drought)
during the extended post inoculation incubation period. Such con-
founding issues are not unknown in studies of pathogens (such as
X. fastidiosa) of perennial hosts that have a long incubation period
and cause symptoms by interruption of xylem sap flow.
Under greenhouse conditions, establishment and multiplication
of X. fastidiosa strains in olive by mechanical inoculation methods
or insect transmission occurred at low frequency. By comparison,
inoculation of grape or almond with reference strains and select
olive-infecting strains resulted in a much higher correlation of
symptom expression, pathogen detection by PCR, and recovery by
culturing. These observations indicate that the strains of X.
fastidiosa isolated from olive behaved as expected for subsp.
multiplex genotypes. Therefore, limited infection rates, lack of
persistence of the pathogen over time, and absence of disease
symptoms in olive may be considered the result of pathogen–host
interactions specific to olive. Because some olive plants were
inoculated with the same inocula used to infect almond, low infec-
tivity and lack of disease symptoms in olive cannot be explained as
a general loss of pathogenicity in culture. Thus, olive may be con-
sidered a host in which X. fastidiosa acts similar to an endosymbi-
ont, as has been shown for common riparian plants (35) and Ara-
bidopsis thaliana (38), in which infected plants remain asymp-
tomatic and bacterial populations are limited. However, additional
data on the fate of X. fastidiosa in olive trees under field conditions
over a period of years are needed to address this hypothesis.
Three species of fungi were identified as causal agents of olive
twig and branch dieback in California (48). However, nothing is
known about the causal agent of leaf scorching symptoms. Because
X. fastidiosa was found in trees showing twig and branch dieback,
leaf scorching, or both symptoms, further studies are needed to
evaluate whether co-infection by fungal pathogens and X. fastidi-
osa alters symptom expression.
The role of olive in epidemiology of diseases caused by X.
fastidiosa. Although X. fastidiosa did not cause olive leaf scorch or
branch dieback disease, olive may, under certain circumstances,
serve as a reservoir for X. fastidiosa. In Southern California, X.
fastidiosa infection of olive was common (Table 1), such that olive
may contribute to incidence of X. fastidiosa in ornamental and
landscape perennials susceptible to infection by strains of subsp.
multiplex. Higher incidence of X. fastidiosa in olive in Southern
California may be attributed to high population levels of the
glassy-winged sharpshooter, which has a restricted range in the
San Joaquin Valley. In the San Joaquin Valley, where most almond
production occurs, infection of olive by X. fastidiosa was uncom-
mon (Table 1). This observation suggests a limited contribution of
olive as a source of X. fastidiosa inocula for the expansive almond
industry in the San Joaquin Valley, even though strains of X. fas-
tidiosa isolated from olive can experimentally cause almond leaf
scorch disease (Table 2).
The glassy-winged sharpshooter can reproduce (R. Krugner, un-
published) and overwinter (11) on olive. Although population den-
sities of glassy-winged sharpshooter can be considerably variable
among host plant species (52), population dynamics of glassy-
winged sharpshooter observed in olive was similar to those re-
ported in other hosts and locations in California. In general, there
Fig. 3. Sharpshooter vector activity in olive orchards. A, Number of glassy-winged
sharpshooters captured per trap per day from 2010 to 2013 at site A. Three green
sharpshooters also were captured at site A during the trapping period (2 June 2010,
13 December 2010, and 20 June 2011). B, Number of green sharpshooters
captured per trap per day from 2011 to 2013 at site B. No glassy-winged
sharpshooters were caught at site B.
1192 Plant Disease / Vol. 98 No. 9
is an increase in adult sharpshooter levels from late June to a peak
in mid- to late July consistent with the appearance of first-genera-
tion adult emergence (24). Furthermore, trapping data from Fresno
County (Fig. 3A) clearly indicate that glassy-winged sharpshooters
are active in olive plantings. Thus, olive may contribute indirectly
to the epidemiology of Pierce’s disease by providing a refuge for
glassy-winged sharpshooters. In contrast, green sharpshooter
movement into olive was transient (Fig. 3B), suggesting that olive
is unlikely to serve as a source of green sharpshooter.
California represents a mosaic of urban areas and regions of in-
tensive, diverse agriculture in which X. fastidiosa, numerous hosts,
and associated sharpshooter vectors are widely distributed, compli-
cating management of vector populations and sources of inocula.
Under such conditions, potential benefits of vector or pathogen
control in olive are difficult to predict with respect to reduced inci-
dence of disease and increase in crop yield or quality. Elsewhere,
the situation may be different. In 2013, X. fastidiosa infection of
olive in Italy was reported (39) but details on pathogen genotype,
prevalence, and means of spread are limited. Because the European
Union (16) considers X. fastidiosa an exotic, high risk pathogen,
there is intense interest in olive as a host of X. fastidiosa and the
role of olive in epidemiology of diseases caused by X. fastidiosa.
Acknowledgments
This research was supported by a California Olive Committee grant awarded
to R. Krugner and by the United States Department of Agriculture–Agricultural
Research Service appropriated project 5302-22000-010-00D. We thank T. de la
Torre, M. Venegaz, and A. J. Salyers for assisting in plant and insect mainte-
nance; G. Phillips for maintaining bacterial cultures and assisting isolation; P.
Dwyer for collection of field trapping data; and Duarte nursery for donating
grapevines.
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