Spittlebugs as vectors of Xylella fastidiosa in olive orchards in Italy

Abstract

The recent introduction of Xylella fastidiosa in Europe and its involvement in the Olive Quick Decline Syndrome
(OQDS) in Apulia (Salento, Lecce district, South Italy) led us to investigate the biology and transmission ability of the meadow spittlebug, Philaenus spumarius, which was recently demonstrated to transmit X. fastidiosa to periwinkle plants. Four xylem-sap-feeding insect species were found within and bordering olive orchards across Salento during a survey carried out from October 2013 to December 2014: P. spumarius was the most abundant species on non-olive vegetation in olive orchards as well as on olive foliage and was the only species that consistently tested positive for the presence of X. fastidiosa using real-time PCR. P. spumarius, whose nymphs
develop within spittle on weeds during the spring, are likely to move from weeds beneath olive trees to olive canopy during the dry period (May to October 2014). The first X. fastidiosa infective P. spumarius were collected in May from olive canopy: all the individuals previously collected on weeds tested negative for the bacterium. Experiments demonstrated that P. spumarius transmitted X. fastidiosa from infected to
uninfected olive plants. Moreover, P. spumarius acquired X. fastidiosa from several host plant species in the field, with the highest acquisition rate from olive, polygala and acacia. Scanning electron microscopy (SEM) revealed bacterial cells resembling X. fastidiosa in the foreguts of adult P. spumarius. The data presented here are essential to plan an effective IPM strategy and limit further spread of the fastidious bacterium.

Full text

ORIGINAL PAPER
Spittlebugs as vectors of Xylella fastidiosa in olive orchards in Italy
Daniele Cornara
1
•Maria Saponari
2
•Adam R. Zeilinger
3
•Angelo de Stradis
2
•
Donato Boscia
2
•Giuliana Loconsole
2
•Domenico Bosco
4
•Giovanni P. Martelli
1
•
Rodrigo P. P. Almeida
3
•Francesco Porcelli
1
Received: 30 December 2015 / Revised: 14 June 2016 / Accepted: 28 June 2016 / Published online: 12 July 2016
The Author(s) 2016. This article is published with open access at Springerlink.com
Abstract The recent introduction of Xylella fastidiosa in
Europe and its involvement in the Olive Quick Decline Syn-
drome (OQDS) in Apulia (Salento, Lecce district, South Italy)
led us to investigate the biology and transmission ability of the
meadow spittlebug, Philaenus spumarius,whichwasrecently
demonstrated to transmit X. fastidiosa to periwinkle plants.
Four xylem-sap-feeding insect species were found within and
bordering olive orchards across Salento during a survey car-
ried out from October 2013 to December 2014: P. spumarius
was the most abundant species on non-olive vegetation in
olive orchards as well as on olive foliage and was the only
species that consistently tested positive for the presence of X.
fastidiosa using real-time PCR. P. spumarius, whose nymphs
develop within spittle onweeds during the spring, are likely to
move from weeds beneath olive trees to olive canopy during
the dry period (May to October 2014). The first X. fastidiosa-
infective P. spumarius were collected in May from olive
canopy: all the individuals previously collected on weeds
tested negative for the bacterium. Experiments demonstrated
that P. spumarius transmitted X. fastidiosa from infected to
uninfected olive plants. Moreover, P. spumarius acquired X.
fastidiosa from several host plant species in the field, with the
highest acquisition rate from olive, polygala and acacia.
Scanning electron microscopy (SEM) revealed bacterial cells
resembling X. fastidiosa in the foreguts of adult P. spumarius.
The data presented here are essential to plan an effective IPM
strategy and limit further spread of the fastidious bacterium.
Keywords Emerging diseases Plant pathogenic bacteria 
Auchenorryncha Aphrophoridae
Key message
•Philaenus spumarius is a vector of Xylella fastidiosa,
although transmission of the bacterium to olive with
naturally infected spittlebugs has not been
demonstrated.
•The main goal of this work was to test the vector
transmission of Xylella fastidiosa to olive.
•We found that X.fastidiosa is transmitted by P.
spumarius between olive plants. Three other xylem-
sap feeders occurring in olive orchards tested negative
for X.fastidiosa.
•In 2014, the first infective P. spumarius were collected
on olive canopies, with infectivity increasing gradually
from May throughout the end of August.
•Our findings are essential for effective management of
X. fastidiosa.
Introduction
Human activities, especially the speed and volume of
transportation, have accelerated the global expansion of
invasive species because of a breakdown of natural barriers
Communicated by J. Gross.
&Francesco Porcelli
francesco.porcelli@uniba.it
1
Department of Soil, Plant and Food Sciences, University of
Bari Aldo Moro, Bari, Italy
2
Institute for Sustainable Plant Protection, National Research
Council (CNR), Bari, Italy
3
Department of Environmental Science, Policy and
Management, University of California, Berkeley, CA, USA
4
Department of Agriculture, Forestry and Food Sciences,
University of Turin, Grugliasco, Italy
123
J Pest Sci (2017) 90:521–530
DOI 10.1007/s10340-016-0793-0
Content courtesy of Springer Nature, terms of use apply. Rights reserved.

to dispersal, so much so that the distribution of invasive
species appears to be restricted primarily by climatic fac-
tors (Capinha et al. 2013). One activity highly impacted by
invasive species is agriculture, where crop diversity has
become gradually more homogeneous at the global scale
(Khoury et al. 2014), leading to a suite of shared pests and
diseases. Therefore, it is not surprising that some of the
major current and future challenges to agriculture gravitate
around the potential risks associated with the introduction
of invasive species into new regions where they are absent.
The recent establishment of the vector-borne bacterium
Xylella fastidiosa in the Salento peninsula (southern Italy)
(Saponari et al. 2013) highlights the risks associated with
the unintended introduction of organisms into new regions.
The currently available phylogenetic data indicate that the
invasive strain (named CoDiRO) belongs to the X. fastid-
iosa subspecies pauca and was possibly introduced from
Costa Rica (Giampetruzzi et al. 2015) via infected orna-
mental plant material, which has recently been shown to be
an important pathway for the long-distance dispersal of X.
fastidiosa (EFSA 2015). Because X. fastidiosa vectors are
present throughout the Mediterranean basin (EFSA 2015),
and this bacterium colonizes several crop species of eco-
nomic and cultural importance (e.g., grapevine, citrus,
almond) (Hill and Purcell 1997), the threat due to its
introduction to Europe is significant.
Despite the risks represented by the introduction of X.
fastidiosa into Italy, it is difficult to infer how fast or
widely the bacterium will spread in the region or how to
best manage infected areas and limit bacterial dispersal.
For the best-studied disease systems caused by X. fastid-
iosa, Pierce’s disease of grapevines in the USA and citrus
variegated chlorosis in Brazil, nearctic and neotropic
sharpshooter leafhoppers (Hemiptera, Cicadellidae) are the
epidemiologically relevant vectors (Redak et al. 2004).
However, in Europe this group is poorly represented,
whereas spittlebugs (Hemiptera, Cercopoidea) are the
dominant group of potential X. fastidiosa vectors (EFSA
2015). Spittlebugs have been known to transmit X. fastid-
iosa since the 1940s (Severin 1950), but only a limited
number of studies have addressed the role of these insects
on pathogen spread (Severin 1950; Purcell 1980; Sanderlin
and Melanson 2010). Although there is no documented
vector species-X. fastidiosa specificity for transmission,
and all spittlebugs should be considered potential vectors
until proven otherwise (Frazier 1965; Almeida et al. 2005),
there are significant knowledge gaps on the biology of X.
fastidiosa transmission by this group of insects.
Xylella fastidiosa is associated with disease symptoms
in olive elsewhere. Krugner et al. (2014) reported that X.
fastidiosa isolates belonging to the subspecies multiplex
were inconsistently associated with olive leaf scorch
symptoms in California, but failed to fulfill Koch’s
postulates under greenhouse conditions with isolates from
either ssp. multiplex or ssp. fastidiosa. An association of X.
fastidiosa ssp. pauca with olive scorch in Argentina and
Brazil has also been reported (Haelterman et al. 2015;
Della Coletta-Filho et al. 2016). In addition, Krugner et al.
(2014) demonstrated that a sharpshooter vector species
could transmit isolates of the ssp. multiplex and fastidiosa
from almond to olive trees. Saponari et al. (2014a) reported
the first finding of field-collected Philaenus spumarius L.
(Hemiptera: Aphrophoridae) positive by PCR for X. fas-
tidiosa; the authors carried out a first transmission experi-
ment with field-collected meadow spittlebugs from
Gallipoli (Salento region, Apulia) caged in groups of 8–10
per plant on five periwinkle (Catharanthus roseus) plants
and seven olive (Olea europea) plants for an Inoculation
Access Period (IAP) of 96 h. Eventually, two out of five
periwinkles tested positive for X. fastidiosa, whereas
transmission to olive was not achieved. Moreover, the
authors tested two Auchenorryncha species collected from
November 2013 to January 2014, Euscelis lineolatus Brulle
`
(Hemiptera: Cicadellidae) and P.spumarius, for X. fastid-
iosa by real-time PCR; only the latter tested positive for the
bacterium. Although these preliminary data should be
considered very important, the authors themselves sug-
gested that further investigation is required, especially
given i) the lack of P. spumarius transmission of X. fas-
tidiosa to olive and (ii) that a survey of the candidate vector
was limited to 3 cold months when most of the biological
cycle of the species has to be considered concluded.
Therefore, we performed a series of experiments and field
sampling to gain insights into the candidate vectors’ pos-
sible role in the X. fastidiosa epidemiology in the region.
The overall goal of this work was to generate initial
information on the role of candidate vectors, especially P.
spumarius, in X. fastidiosa spread in the Apulia epidemic.
Materials and methods
Survey of xylem-sap feeding species in and adjacent
to olive orchards
A survey of xylem-sap feeders in olive orchards was car-
ried out from October 2013 to December 2014 on 48 sites
in Lecce district, mainly around the X. fastidiosa hotspot in
Gallipoli (Fig. 1). While the six sites used in the study of
Saponari et al. (2014a) were also included in our study, no
data from the cited paper are included in our analysis. We
visited every site at least twice, once in spring-summer and
once in the fall. Four sites in the municipalities of Gallipoli
and Alezio (Fig. 1) were visited every other week. In order
to protect the anonymity of participating farmers, a detailed
geographic location of each site cannot be made publically
522 J Pest Sci (2017) 90:521–530
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available. The study was carried out on private land with
owner permission (no specific permits were required); the
field survey did not involve endangered or protected spe-
cies. The insects were collected with a sweep net contin-
uously for 1 h per field per each collection date on olive
trees, weeds and plants other than olive within and sur-
rounding the orchards. Samples were stored in 75 %
ethanol and brought to the laboratory for identification
following Ossiannilsson (1981), Holzinger (2003), Ribaut
(1952) and Della Giustina (1989). Furthermore, a collec-
tion of Auchenorryncha (insects and slide-mounted geni-
talia) used for this research has been taxonomically
identified according to the reported references and is
available at the University of Bari (DiSSPA, sezione
Entomologia e Zoologia, via Amendola 165/A, 70125 Bari,
Italy).
We used real-time PCR to detect the presence of X.
fastidiosa in all the xylem sap feeders collected during each
visit. At least 50 % of the insects were tested individually;
the remaining ones were pooled in groups of four to five
individuals of the same species. Moreover, at least 50 % of
the Auchenorryncha other than xylem-sap feeders (mainly
Cicadellidae as well as Fulgoromorpha Issidae and
Cixiidae) collected in each site was screened by real-time
PCR using individual specimens. DNA was recovered
following Marzachı
`et al. (1998) and amplification of X.
fastidiosa carried out using the primer set described by
Harper et al. (2010). Samples yielding positive reactions
were randomly selected and the purified PCR amplicons
subjected to sequence analysis. All the samples confirmed
the identity of the amplified DNA as X. fastidiosa (data not
shown).
Assessment of Philaenus spumarius abundance, host
plant shifting and infectivity
From October 2013 to December 2014, we surveyed pop-
ulations of P. spumarius (Hemiptera, Aphrophoridae) in a
1 ha. X. fastidiosa-infected olive orchard in the Gallipoli
area (N 40 012704; E 018 05341). From October 2013 to
May 2014, once every 15 days, weeds were swept con-
tinuously for 1 h during each visit. From May to the end of
October 2014, weekly, we began to sweep olive canopies
and suckers directly, in addition to weeds and plants other
than olives present in the field: the main goal was to
monitor changes in the relative abundance of adult P.
spumarius on weeds and olive trees. Each sample consisted
of ten sweeps performed on single olive plants or on wild
plants, including weeds, within and at the boundaries of the
orchard. The overall content of the sweeping net after the
ten sweeps was emptied in a plastic bag properly labeled
with the sweeping date and the swept plant, sealed, stored
in a cool-box and brought to the laboratory (located into the
infected area). Once in the laboratory, the P. spumarius
were identified and counted, and the relative abundance on
olive trees and hosts other than olive was calculated by
dividing the number of spittlebugs collected from the total
number of samples swept on the host during the 1 h sam-
pling for the total number of samples (bags) collected on
that host. P. spumarius collected in the middle of each
month were tested individually by real-time PCR as
reported above in order to assess infection prevalence in
individuals collected from olive plants and weeds, i.e., the
percentage of insects harboring the bacterium versus the
total number of collected insects on different hosts during
the season. During the November–December 2014 period,
monitoring was performed once every 2 weeks.
Olive-to-olive vector transmission of X. fastidiosa
We sought to test whether the two dominant xylem sap-
feeding species, P. spumarius and Neophilaenus cam-
pestris, could successfully transmit X. fastidiosa between
olive trees. From May to July 2014, we collected 20
Neophilaenus campestris and 20 P. spumarius individuals
weekly by sweeping olive orchards in an X. fastidiosa-free
Fig. 1 Field sites surveyed to determine the composition of a
potential Xylella fastidiosa vector community. Site with square
indicates the location where vector abundance and infectivity were
determined. Inset: map of Italy, with the studied region circled. The
maps were generated by authors using R version 3.2.0
J Pest Sci (2017) 90:521–530 523
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area in Brindisi district for a total of about 400 insects; all
these insects tested negative for X. fastidiosa by real-time
PCR. This assessment verified that our subsequent collec-
tions from these sites would likely be free of X. fastidiosa.
In July 2014, N. campestris and P. spumarius adults were
collected from the same fields and brought to an infected
olive orchard in the Gallipoli area to be caged on OQDS-
symptomatic olive branches (testing positive for X. fas-
tidiosa) for a 4-day acquisition access period (AAP). All N.
campestris were caged together on one symptomatic
branch, while P. spumarius individuals were divided into
three groups, each group caged on a different plant previ-
ously testing positive for X. fastidiosa by real-time PCR.
After the AAP, insects were taken back to the laboratory,
separated in groups of individuals and allowed a 4-day IAP
on self-rooted olive plants (Olea europaea), cultivar Cor-
atina or periwinkle plants (Catharanthus roseus), which
were maintained in an insect-proof greenhouse (Table 3).
One olive plant and one periwinkle caged devoid of insects
served as control. Insects that died during the AAP or IAP
were stored in 75 % EtOH and PCR-tested for the presence
of X. fastidiosa as described above. Three months after
insect inoculation, inoculated and uninfected control test
plants were tested for the presence of X. fastidiosa
according to Loconsole et al. (2014).
Role of host plant species on X. fastidiosa vector
transmission
To better understand the role of the host plant on the
ecology of the infections, X. fastidiosa-positive plant spe-
cies from the Gallipoli and Alezio municipalities were used
as pathogen sources for vector acquisition. In addition to
olive, numerous plant species have been shown to be col-
onized by X. fastidiosa in southern Apulia, including
almond (Amygdalus communis), oleander (Nerium olean-
der), cherry (Prunus avium), myrtle-leaf milkwort (Poly-
gala myrtifolia), coastal rosemary (Westringia fruticosa),
acacia (Acacia saligna) and broom (Spartium junceum)
(Saponari et al. 2013,2014b). We investigated acquisition
from acacia, broom, olive, almond, cherry, oleander, peri-
winkle and polygala. We identified suitable plants of each
species that were confirmed by real-time PCR to be
infected with X. fastidiosa for these experiments. No
Westringia spp. plants were available for the experiment.
One periwinkle infected by P. spumarius collected in
Gallipoli at the beginning of June and maintained in a
growth chamber was used as a positive control. In
September 2014, P. spumarius presumably free from the
bacterium were collected from the same X. fastidiosa-free
area in Brindisi as described above. Twenty P. spumarius
adults were caged as one group for a 96-h AAP on each
infected host plant species under field conditions. In
addition, five P. spumarius were caged on an uninfected
acacia plant used as a negative control. At the end of the
AAP, five or six P. spumarius were randomly selected from
each host and transferred singly to periwinkle plants for a
96-h IAP in a greenhouse. Then, the IAP spittlebugs were
stored in 75 % EtOH for real-time PCR assays. One month
after the IAP, recipient periwinkle plants were tested for
the presence of X. fastidiosa by real-time PCR (Loconsole
et al. 2014).
Scanning electron microscopy of the foregut
of spittlebugs
To search for X. fastidiosa in P. spumarius foregut lumen,
ten P. spumarius collected from diseased olive trees in
Gallipoli in September 2014 were prepared according to
Almeida and Purcell (2006), except that samples were fixed
overnight in 4 % (vol/vol) glutaraldehyde in 0.05 M cold
(4 C) phosphate buffer (Pb) at pH 7, instead of cacodylate
buffer, and observed by a Hitachi TM3000 low-pressure
scanning electron microscope.
Statistical analysis
We used repeated measures analysis of variance (ANOVA)
to test for a difference between olives and weeds in (1) P.
spumarius densities and (2) the proportion of collected P.
spumarius infected with X. fastidiosa. In both tests, the
source plant (olive or weeds; n=8 for each level) was
included as a fixed effect, and the date of collection was
included as a repeated-measures random effect (Pinheiro
and Bates 2000). In the first test, densities per sweep were
used as the response to control for differences in the
sampling effort between olives and weeds; standardized
densities were then square-root transformed to meet
assumptions of ANOVA—namely, that the error variance
is constant and the response variable is normally dis-
tributed (Oehlert 2000). For the second test, the proportion
of insects infected was arc-sine square-root transformed to
meet assumptions of ANOVA.
We also tested for differences between olive and other
host plants in X. fastidiosa acquisition and inoculation rates
by P. spumarius on periwinkle. Here logistic regression
was used with infection status as the binary response
variable and plant species—the source plant for the
acquisition experiment and test plant for the inoculation
experiment—as the sole fixed-effect explanatory variable.
No acquisition or inoculation occurred for some plant
species; this resulted in all zeros for some plant species—
called quasi-complete separation of factor levels in the
statistics literature. Standard methods of logistic regression
cannot estimate coefficients of factor levels in cases of
complete or quasi-complete separation. As a remedy, we
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used logistic regression with Firth’s bias correction (Heinze
& Schemper 2002). All analyses were conducted in R 3.2.0
(R Core Team 2015); the nlme package was used for the
repeated-measures ANOVAs (Pinheiro et al. 2014) and the
logistf package for Firth’s logistic regression (Heinze
2013).
Results
Xylem-sap feeding species found in or adjacent
to olive orchards
In all the surveyed olive fields (Fig. 1), only two spittlebug
species were collected on olive, P. spumarius and N.
campestris. One cicada species (Cicada orni) was observed
in olive orchards from June to August. We did not quantify
cicada abundance on olive, since such data on cicadas
require a dedicated study. From October 2013 to December
2014, the spittlebug population on olive plants was almost
exclusively composed of P. spumarius (98.56 % of the
overall spittlebug population collected from olive plants);
N. campestris (1.44 % of the overall spittlebug population)
was rare. Only 1 out of about 200 N. campestris that had
been collected in July 2014 tested positive for X. fastidiosa;
the single individual positive for the bacterium was col-
lected on an oleander plant. A third spittlebug, Cercopis
sanguinolenta, was collected on weeds bordering olive
orchards from March to early May; none of the 31 C.
sanguinolenta collected tested positive for X. fastidiosa.C.
orni were collected mainly on olive trees; of the 54 adults
and 18 exuviae tested, none was X. fastidiosa positive.
Twenty-four E. lineolatus found on weeds, mainly Mer-
curialis annua at the border of the fields, collected from
October to December 2014, tested negative for X.
fastidiosa.
Preliminary assessment of Philaenus spumarius
abundance, host plant shifting and infectivity
Although no more than from three up to five individuals
per collection date, adult P. spumarius were collected
continuously during the 2013–2014 winter, even during the
emergence of the first instar spittle masses in late February
2014. We observed numerous spittle masses on plants
starting from the middle of March, both inside and outside
cultivated orchards. P. spumarius adults were regularly
collected by sweeping the olive canopy in May, June and
July; in the same period, populations on weeds declined as
the ground vegetation became drier (Fig. 2a). A reversal of
this trend was observed starting from the end of July, with
P. spumarius adults collected on plants of Conyza sp. and a
reduction of the number of individuals on olive trees.
Overall, densities of P. spumarius, calculated as the total
number of individuals on the total number of bags collected
on that host as described above, were not different between
olives and weeds (n=8, F
1,7
=0.356, P=0.57).
From December 2013 to May 2014, none of the adults
collected from weeds were positive for X. fastidiosa.
During 2014, the first P. spumarius positive for X. fastid-
iosa were collected from olive canopies in the middle of
May. The percentage of infected P. spumarius adults col-
lected on olive and weeds was highest in August, but was
over 50 % in all summer (Fig. 2b). Overall, the proportion
of P. spumarius infected with X. fastidiosa (i.e., infection
prevalence) was marginally greater in olives than in weeds
(n=8, F
1,7
=5.591, P=0.050); infection prevalence in
olives was about double that in weeds (Fig. 2b).
Fig. 2 a Standardized densities (mean ±model-predicted SE) of
Philaenus spumarius from May to December 2014 in olive trees
(black) and weeds (grey) in Apulia, Italy; each sample corresponds to
ten sweeps. bP. spumarius X. fastidiosa infection rate as determined
by RT-PCR detection. Numbers below dates indicate the number of
individuals that were tested from olive trees or weeds. SEs were
calculated from ANOVA model results, predicted for each data point,
because data on P. spumarius densities per replicate were lost and
only means for each date were recovered. Model-predicted SE result
in uniform error bars
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Philaenus spumarius transmits X. fastidiosa
from olive-to-olive
All the spittlebugs collected and tested before the experi-
ment, and those not included in the experiment since death
during the transfer to infected area, tested negative to X.
fastidiosa by real-time PCR. Therefore, individuals used
for the acquisition/inoculation experiment would have been
likely free from the bacterium before the AAP. After the
4-day AAP on olive branches in the field, 16 P. spumarius
and 6 N. campestris were found dead; of these 5 P.
spumarius and 1 N. campestris individual tested positive
for X. fastidiosa. At the end of the IAP, 11 of 35 P.
spumarius and 1 of 16 N. campestris individuals were X.
fastidiosa-positive. Two of four olive plants inoculated
with P. spumarius were infected with X. fastidiosa, while
none of the two olive plants inoculated by N. campestris
tested positive (Table 3). Both olive and periwinkle plants
kept as negative controls in the greenhouse remained X.
fastidiosa-negative by real-time PCR.
Impacts of host plant species on X. fastidiosa
transmission efficiency by P. spumarius
Acquisition efficiency under field conditions, as estimated
by the percentage of X. fastidiosa real-time PCR-positive
P. spumarius, was significantly different among pathogen
source plant species (Fig. 3). Olive was the host with the
highest percentage of positive individuals; acquisition
efficiency from polygala and acacia were not statistically
different from that from olive (Table 1). However, acqui-
sition from broom, almond, cherry, oleander and periwin-
kle were all statistically lower than that from olive
(Table 1). No acquisition occurred from the negative
control (i.e., uninfected acacia). In contrast to acquisition,
there were no significant differences in vector inoculation
rates from various source plant species to periwinkle
(Table 2).
Scanning electron microscopy of the foregut
of spittlebugs
Bacterial cells resembling X. fastidiosa were detected in
two of the examined individuals (Fig. 4). We observed the
cells lining the walls of the precibarium (Purcell et al.
1979; Brlansky et al. 1983; Almeida and Purcell 2006) and
at the entrance of the cibarium along the groove of the floor
of the pump chamber. Along the precibarium, the bacterial
cells were attached polarly; cells were also noticed at the
beginning of the food duct, distally to the precibarial valve.
Moreover, sideways-attached cells (Almeida and Purcell
Fig. 3 Percent of Philaenus
spumarius acquiring X.
fastidiosa from different host
plant species (black bars) and
percent of periwinkle plants
inoculated with X. fastidiosa
from infected P. spumarius
(white bars). Numbers on the x-
axis at the base of each pair of
columns indicate the number of
P. spumarius recovered from
the source plant after AAP and
tested by PCR (left) and the
number of periwinkle plants
inoculated with single
spittlebugs randomly selected
from those recovered after the
IAP (right)
Table 1 Statistical results from bias-corrected logistic regression
testing differences between olive and other host plants in acquisition
rate by Philaenus spumarius as estimated by PCR detection
Host plant Estimate SE
a
v
2
statistic Pvalue
Intercept 0.89 0.66 2.148 0.143
Acacia (negative
control)
-3.29 1.75 7.024 0.008**
Acacia (positive control) -1.14 1.01 1.468 0.226
Broom -1.99 0.92 5.657 0.017*
Almond -4.18 1.64 14.740 \0.001***
Cherry -2.92 1.12 9.865 0.002**
Oleander -2.19 0.86 7.942 0.005**
Periwinkle -4.60 1.61 19.460 \0.001***
Polygala -0.89 0.80 1.388 0.239
The plant species name indicates the identity of the test plant
a
SE standard error of the coefficient estimate
*P\0.05; ** P\0.01; *** P\0.001
526 J Pest Sci (2017) 90:521–530
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2006) were noticed on the precibarium, proximally to the
cibarial pump floor (Table 3).
Discussion
The introduction of X. fastidiosa into Italy, and its primary
role in olive desiccation and dieback as clearly shown by
Saponari et al. (2016), has highlighted important knowl-
edge gaps for the development of management practices
aimed at reducing the spread of this pathogen in Europe.
Among these is the limited information available on spit-
tlebugs as X. fastidiosa vectors. In this study, we attempted
to address questions of immediate importance, such as
which potential vector species are present in olive orchards
in southern Apulia. P. spumarius transmitted X. fastidiosa
from olive to olive plants as well as from a range of
infected-source plant species to periwinkle test plants.
Furthermore, the spittlebug P. spumarius was the most
abundant species found in orchards on both weeds and
olive trees; X. fastidiosa prevalence in P. spumarius on
olive trees was approximately twice that of insects col-
lected from weeds, but the prevalence was very high in
both environments. Although we cannot be completely sure
that the insects used for acquisition from olive and different
source plants were X. fastidiosa-free since they were field
collected, despite the fact that all the insects tested by real-
time PCR before the experiment were devoid of the bac-
terium, transmission by P. spumarius to olive plants
together with the observed insect population shift and
vector infectivity trend represents an important break-
through in the understanding of the X. fastidiosa CoDiRO
transmission biology. In summary, P. spumarius has to be
considered an epidemiologically relevant vector species in
Salento (southern Italy, Apulia).
Although there are several species of spittlebugs (Cer-
copoidea), cicadas (Cicadoidea) and sharpshooter
leafhopper (subfamily Cicadellinae) throughout Europe
(EFSA 2015), in Apulia only four potential vector species
were collected in our surveys: three spittlebugs (P.
spumarius,N. campestris and C. sanguinolenta) and one
cicada (C. orni; this species was frequently observed dur-
ing the summer but its prevalence and abundance were not
determined in this study). Only P. spumarius was abundant
and prevalent throughout the survey period, suggesting that
it may be the most important vector in the region. How-
ever, because transmission efficiency (Daugherty et al.
2010), host plant preference throughout the year (Purcell
1981) and other factors are important in determining the
epidemiological role of individual species, all potential
vectors should be studied in the future so that their relative
roles on disease spread will be better understood. This is
especially true in the case of cicadas: there are only two
reports of cicadas as X. fastidiosa vectors (Krell et al. 2007;
Table 2 Statistical results from bias-corrected logistic regression
testing differences between olive and other plant species as sources of
Xylella fastidiosa for Philaenus spumarius followed by inoculation in
periwinkle as a shared indicator host
Host plant Estimate SE
a
v
2
statistic P-value
Intercept -0.59 0.85 0.579 0.447
Acacia (negative control) -1.81 1.83 1.479 0.224
Acacia (positive control) -1.81 1.83 1.479 0.224
Almond 0.00 1.20 0.000 1.000
Broom -1.81 1.83 1.479 0.224
Cherry -0.71 1.31 0.353 0.553
Polygala 0.92 1.24 0.670 0.413
The plant species name indicates the identity of the source plant
a
SE standard error of the coefficient estimate
Fig. 4 Scanning electron microscopy images of the abacterial cells along the precibarium and cibarium of Philaenus spumarius;bdetails of
bacterial cell aggregate at the distal area of the cibarium
J Pest Sci (2017) 90:521–530 527
123
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Paiao et al. 2002), and more research on this topic must be
done as cicadas are common on olive throughout the
Mediterranean and are among the largest and most
numerous insects within the habitat in which they occur
(Pinto-Juma et al. 2005; Patterson et al. 1997). The abun-
dance of P. spumarius on olive trees and weeds during the
sampling period did not vary statistically. Given the single
year of observation carried out with a single sampling
method, we consider the data about P. spumarius host plant
shifting and abundance presented in this work preliminary.
In addition, only one survey method was used (i.e., sweep
net). Purcell et al. (1994) suggested that a combination of
sampling methods provides a more accurate estimation of
abundance and movement of insects; these data are
important for the understanding of the role of a vector in
disease spread (Purcell et al. 1994; Irwin and Ruesink
1986).
The first finding of infective P. spumarius on olive trees
in late spring 2014, whereas all the individuals previously
collected on herbaceous plants within and outside the olive
orchard had tested negative for X. fastidiosa by PCR,
together with the gradual increase in the percentage of
infective spittlebugs collected from olive canopy and
suckers during the season suggests that this host serves as
an important source of inoculum for pathogen spread.
Furthermore, because X. fastidiosa is persistent in insect
vectors including spittlebugs (Severin 1950), vectors may
inoculate olive trees over an extended period of time. This
phenomenon may enhance disease symptom expression,
similar to what was observed in grapevines (Daugherty and
Almeida 2009). In other words, multiple and independent
infections events could lead to a reduction of the incubation
period compared to that derived from a single infection
(Daugherty and Almeida 2009). Therefore, it is possible
that olive quick decline syndrome (OQDS) symptoms are
enhanced by the incremental effects of a very large number
of independent infections. This hypothesis must be tested
as it may have important disease management
consequences.
The role of host plant species on X. fastidiosa trans-
mission by spittlebugs was tested with pathogen acquisition
performed under field conditions. This approach has both
positive and negative aspects, namely that biotic and abi-
otic factors affecting X. fastidiosa populations within plants
are realistic, while there is no control on the variation of
these parameters. X. fastidiosa vector acquisition efficiency
is correlated to bacterial populations within grapevines
(Hill and Purcell 1997), an observation that can be exten-
ded to various host plant species (Almeida et al. 2005);
therefore, it is possible that the bacterial populations in
these plant species are variable and the main driver of
observed differences. However, it should be noted that
vector behavior is also a component affecting X. fastidiosa
transmission, as demonstrated when vector species on one
plant, as well as host plant tissue within the same plant,
significantly impacted X. fastidiosa transmission (Daugh-
erty et al. 2010). It is not possible in this case to determine
whether the differences in acquisition efficiency are based
on bacterial populations within plants, which were not
measured, or vector-plant interactions. Nevertheless, the
data indicate that differences in acquisition efficiency exist
based on host plant species and these should be studied
within an epidemiological context. It was not surprising to
see no difference among host plants when inoculation was
considered as this was also previously observed with
sharpshooter vectors (Lopes et al. 2009).
While P. spumarius is likely an important vector in
Apulia, N. campestris and E. lineolatus have also been
reported as capable of acquiring X. fastidiosa (Elbeaino
et al. 2014). Our sample sizes for N. campestris and E.
lineolatus were too small to assess infection rates precisely.
Neophilaenus campestris is unlikely to be a critical vector
Table 3 Summary of transmission experiments with Philaenus spumarius and Neophilaenus campestris after a 4-day bacterial acquisition
access period on X. fastidiosa-infected olive branches in the field
Recipient plant Neophilaenus campestris Philaenus spumarius Infected plants
Insects per plant (#) X. fastidiosa infected (#) Insects per plant (#) X. fastidiosa infected (#)
Olive 3 0 No
Periwinkle 4 0 No
Olive 3 0 No
Periwinkle 2 1 No
Olive 5 1 No
Olive 4 2 Yes
Olive 4 2 Yes
Olive 4 0 No
Insects were transferred to healthy olive and periwinkle recipient plants for a 4 days inoculation access period
528 J Pest Sci (2017) 90:521–530
123
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in Apulia because it was present at low populations;
nonetheless, our data do not rule out the potential in other
nearby regions where populations may be larger. In the
case of the phloem-sap feeder E. lineolatus, however, this
species is not considered to be a potential vector (Redak
et al. 2004; Almeida et al. 2005). The leafhopper was tested
and reported in this article because Elbeaino et al. (2014)
found X. fastidiosa-positive individuals during a survey in
2013. On the contrary, Saponari et al. (2014) reported no
positive individuals out of 30 tested.
Altogether this study indicates that P. spumarius is a
commonly found vector of X. fastidiosa in Salento. P.
spumarius was capable of acquiring and inoculating X.
fastidiosa from/to different host plants, and other hosts in
the environment served as pathogen inoculum sources.
Furthermore, we demonstrated that vectors transmit X.
fastidiosa from infected olive plants in the field to test
plants maintained in greenhouse conditions; these results
are similar to those observed by Krugner et al. (2014)in
California with sharpshooter vectors and two other sub-
species of X. fastidiosa.P. spumarius was first shown to be
aX. fastidiosa vector in the late 1940s (Severin 1950); the
data presented here follow expectations based on what is
known about X. fastidiosa transmission with sharpshooter
vectors (Severin 1950; Almeida et al. 2005). It will be
important to pursue detailed studies on the biology and
ecology of P. spumarius in order to set up effective and
environmentally acceptable vector control methods. How-
ever, given that the EFSA panel considers the removal of
infected plants in a system-based approach as the only
option to prevent further spread of the pathogen to new
areas (EFSA 2016), controlling vectors alone will be use-
less if sources of X. fastidiosa are not also removed. The
data presented here suggest that X. fastidiosa-infected olive
plants are likely the main bacterium source within olive
orchards, and P. spumarius seems a major driver for X.
fastidiosa secondary spread. Our hypothesis is strengthened
by recent fulfillment of Koch’s postulate and data from
artificial inoculation of olive, besides other species, by
Saponari et al. (2016). Nevertheless, more research efforts
are urgently needed to shed light on the transmission
biology of a bacterium that threatens the Italian and
Mediterranean olive industry.
Author contribution statement
Conceived and designed the experiments: DC, MS, DB, GPM,
DB, FP. Performed the experiments: DC, MS, AdS, GL.
Analyzed the data: ARZ, DC. Contributed reagents/materi-
als/analysis tools: AdS, GPM, DB, FP, ARZ, RPPA. Wrote
the paper: DC, ARZ, RPPA. All authors read and approved the
manuscript.
Acknowledgments The authors acknowledge Enzo Manni and Fed-
erico Manni of Coop. ACLI Racale (LE) for their valuable technical
assistance and Andrea Turco and Nicola Tagarelli for help in field
activities. The authors would like to express their gratitude to
Alexander Purcell for constructive and helpful suggestions and
comments. This work is part of Daniele Cornara’s PhD dissertation at
Universita
`degli Studi di Bari (PhD program Biodiversita
`, Agricoltura
e Ambiente, cv Protezione delle Colture). This research was sup-
ported by grants from the Regional Plant Health Service of Apulia.
Funding from USDA-NIFA, CDFA-PD/GWSS and the California
Agricultural Experiment Station research programs supported the
contributions of ARZ and RPPA.
Compliance with ethical standards
Conflict of interest The authors declare no conflict of interest.
Ethical approval This article does not contain any studies with
human participants or animals performed by any of the authors.
Open Access This article is distributed under the terms of the
Creative Commons Attribution 4.0 International License (http://crea
tivecommons.org/licenses/by/4.0/), which permits unrestricted use,
distribution, and reproduction in any medium, provided you give
appropriate credit to the original author(s) and the source, provide a
link to the Creative Commons license, and indicate if changes were
made.
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