ARTHROPODS IN RELATION TO PLANT DISEASES
Localization of ‘Candidatus Liberibacter solanacearum’ (Rhizobiales:
Rhizobiaceae) in Bactericera cockerelli (Hemiptera: Triozidae)
W. RODNEY COOPER,1 VENKATESAN G. SENGODA,
AND
JOSEPH E. MUNYANEZA
USDAÐARS-Yakima Agricultural Research Laboratory, 5230 Konnowac Pass Road, Wapato, WA 98951
Ann. Entomol. Soc. Am. 107(1): 204Ð210 (2014); DOI: http://dx.doi.org/10.1603/AN13087
ABSTRACT ÔCandidatus Liberibacter solanacearumÕ is a pathogen of solanaceous crops (Solanales:
Solanaceae) that causes zebra chip disease of potato (Solanum tuberosum L.) and plant dieback in
tomato (S. lycopersicum L.) and pepper (Capsicum spp.). This pathogen is vectored by the potato/
tomato psyllid Bactericera cockerelli (Šulc) (Hemiptera: Triozidae), but little is known about the
interactions between B. cockerelli and ÔCa. Liberibacter solanacearum.Õ Fluorescence in situ hybridization was used to assess the incidence of ÔCa. Liberibacter solanacearumÕ in the hemolymph,
bacteriomes, alimentary canals, and salivary glands of B. cockerelli. Liberibacter was observed in 66%
of alimentary canals, 39% of salivary glands, and 40% of bacteriomes dissected from adult psyllids.
Compared with adults, the organs of Þfth instars appeared less likely to harbor Liberibacter, which
was observed in 52% of alimentary canals, 10% of salivary glands, and 6% of bacteriomes dissected from
the nymphs. Results of real-time polymerase chain reaction conÞrmed that fewer Þfth instars were
infected with Liberibacter compared with adults and indicated that Þfth instars were less likely to
transmit the pathogen to noninfected host plants. These observations of the localization of ÔCa.
Liberibacter solanacearumÕ in the organs and tissues of B. cockerelli adults and nymphs will aid the
study of LiberibacterÐpsyllid interactions and the epidemiology of ÔCa. Liberibacter solanacearum.Õ
KEY WORDS potato psyllid, tomato psyllid, mycetome, mycetocyte, Liberibacter psyllaurous
ÔCandidatus Liberibacter solanacearumÕ (Rhizobiales:
Rhizobiaceae) is an important pathogen of solanaceous crops (Solanales: Solanaceae) in North America
and New Zealand (Munyaneza et al. 2007, Hansen et
al. 2008, Liefting et al. 2008). This pathogen is associated with zebra chip disease of potato tubers (Solanum
tuberosum L.) and causes chlorosis, leaf curling, and
plant decline in potato, tomato (S. lycopersicum L.),
peppers (Capsicum spp.), and tobacco (Nicotiana tabcum L.; Abad et al. 2009; Munyaneza et al. 2009a,b,
2013). The primary insect vector of ÔCa. Liberibacter
solanacearumÕ among solanaceous plants is the potato/tomato psyllid, Bactericera cockerelli (Šulc)
(Hemiptera: Triozidae) (Munyaneza et al. 2007).
Many aspects of the interactions between ÔCa. Liberibacter solanacearumÕ and B. cockerelli are poorly
understood, including the mechanisms of acquisition
and transmission of the pathogen by the psyllid. B.
cockerelli presumably acquires Liberibacter via an oral
route (Munyaneza 2012) and transmits the pathogen
to host plants with discharged saliva (Pearson et al.
2010). Liberibacter may also be transovarially transMention of trade names or commercial products in this article is
solely for the purpose of providing speciÞc information and does not
imply recommendation or endorsement by the United States Department of Agriculture. USDA is an equal opportunity provider and
employer.
1 Corresponding author, e-mail: [email protected].
mitted (Hansen et al. 2008). Results of preliminary
studies suggested that Liberibacter colonized the psyllid bacteriomes (W.R.C., unpublished data), which
are specialized organs that facilitate the acquisition,
cultivation, and transovarial transmission of both obligate and transient symbiotic bacteria (Baumann
2005). These previous reports suggest that Liberibacter migrates from the psyllidÕs alimentary canal to
colonize the ovaries, salivary glands, and bacteriomes.
However, the frequency with which Liberibacter colonizes the various internal organs is not known, and
the colonization of Liberibacter or other insect-vectored plant pathogens within insect bacteriomes has
not been previously documented.
The documentation of Liberibacter in speciÞc organs of B. cockerelli is critical to our understanding of
the epidemiology of this pathogen. Notably, it will
clarify how factors such as insect life stages inßuence
the retention and transmission of Liberibacter by its
insect vector (Buchman et al. 2011, Casteel et al.
2012). The overall goal of our study was to document
the infection of speciÞc organs of B. cockerelli adults
and Þfth instars by ÔCa. Liberibacter solanacearum.Õ
We used ßuorescence in situ hybridization to observe
Liberibacter in the hemolymph, reproductive organs,
bacteriomes, alimentary canals, and salivary glands of
nymph and adult B. cockerelli.
January 2014
COOPER ET AL.: LIBERIBACTER IN Bactericera cockerelli
Materials and Methods
Experimental Insects. B. cockerelli infected with
ÔCa. Liberibacter solanacearumÕ were obtained from a
laboratory colony maintained on infected potato
ÔRanger Russet.Õ Noninfected psyllids were obtained
from a separate colony maintained on noninfected
tomato ÔMoneymaker.Õ The pathogen originated from
insects collected from Þelds of potato near Weslaco,
TX, in 2007, and the insects were collected from Þelds
of potato near Weslaco, TX, and Prosser, WA, from
2007 to 2012. To ensure that all adults were reproductively mature, adults from both colonies were maintained on noninfected tomato ÔMoneymakerÕ at 25⬚C
with a photoperiod of 16:8 (L:D) h and ⬇50% relative
humidity (RH) for 7 d before initiating experiments.
Periodic screening of colony insects for ÔCa. Liberibacter solanacearumÕ using polymerase chain reaction
(PCR) indicated that 80 Ð100% of adults reared on
Liberibacter-infected plants, but none of the insects
reared on noninfected plants, were infected with Liberibacter.
Fluorescence In Situ Hybridization. Five wax-barrier circles in each of two rows were drawn on a Tissue
Tack Microscope slide (Polysciences Inc., Warrington, PA) using an Aqua hold barrier Pap pen
(ScientiÞc Devise Laboratory, Des Plaines, IL). An
adult psyllid (⬎7 d after adult emergence) anesthetized with CO2 was placed on a separate glass microscope slide, and a small hole was made in the ventral
abdominal plate between the third and fourth segments using number 5 forceps (DÕOutils Dumont SA,
Montignez, Switzerland) at 32⫻ magniÞcation under
a dissecting microscope (Olympus SZX7, Olympus
America Inc. Central Valley, PA). Hemolymph was
then collected and smeared on the Tissue Tack microscope slide within one of the circles made with
the Aqua hold barrier Pap pen. Psyllids were then
dissected at 32⫻ magniÞcation. The insects were
mounted with their ventral sides facing up to a glass
microscope slide using double-sided tape. A drop of
phosphate-buffered saline (Fisher, Pittsburgh, PA)
was placed over the insect and held in place by cohesion. Using two number 5 forceps, the ventral plate
of the abdomen was removed and the reproductive
organs, bacteriome, and alimentary canal were each
transferred to separate Aqua hold barrier circles on
the Tissue Tack microscope slide. The salivary glands
of psyllids are located directly dorsal to the procoxae
(Cicero et al. 2009). The glands were dissected from
the insects by removing the prothoracic legs, which
weakened the cuticle of the prothorax, and then
gently pulling the head away from the body. Both pairs
of primary and accessory salivary glands were then
transferred to an Aqua hold barrier circle. Thus, the
hemolymph, reproductive organs, bacteriome, alimentary canal, and salivary glands from a single insect
were positioned in each row of Þve Aqua hold barrier
circles on the Tissue Tack slides. Experiments with
adult insects were conducted Þve times (trials) with
different cohorts of 10 insects from the Liberibacterinfected colony and 10 insects from the noninfected
205
colony. In total, 100 adults with equal numbers of
males and females were dissected and prepared for
ßuorescence in situ hybridization of Liberibacter.
In separate experiments, Þfth instars of B. cockerelli
were dissected and prepared for ßuorescence in situ
hybridization as described for adults, except that the
hemolymph and reproductive organs were not included in our assessments. The gender of each nymph
was determined based on the appearance of the bacteriome observed through the cuticle (Carter 1961).
Experiments with nymphs were conducted three
times (trial) with different cohorts of insects. The Þrst
trial included 10 control insects (7 females, 3 males)
and 10 insects from the Liberibacter-infected colony
(6 females, 4 males); the second trial included 6 control insects (2 females, 4 males) and 6 insects from the
infected colony (4 females, 2 males); and the third trial
included 6 control insects (4 females, 2 males) and 16
insects from the infected colony (7 females, 9 males).
Methods for ßuorescence in situ hybridization were
modiÞed from the report by Ammar et al. (2011). The
dissected tissues were air-dried at room temperature.
The slides were then maintained for 3Ð5 min on a slide
warmer set at 50⬚C to adhere the tissues to the slides.
Samples were Þxed in CarnoyÕs solution (Electron
Microscopy Sciences, HatÞeld, PA) for 1 h, brießy
rinsed in 100% ethanol, and washed three times for 20
min in hybridization buffer consisting of 20 mM TrisHCl (pH 8.0; Fisher), 0.9 M NaCl (Fisher), 0.01%
sodium dodecyl sulfate (IndoÞne Chemical Company,
Hillsborough, NJ), and 30% formamide (Fisher). Samples were hybridized overnight with 250 picomol/ml
of high performance liquid chromatography-puriÞed
oligonucleotide probe labeled with Alexa Fluor 488 on
the 5⬘ end (Invitrogen, Carlsbad, CA) and dispersed in
hybridization buffer. The probe sequence was GCC
TCG CGA CTT CGC AAC CCA T (Jagoueix et al.
1996, Crosslin et al. 2011). No probe controls (hybridized in buffer without the ßuorescent probe) were
included in one adult trial with Þve insects from each
treatment (control and infected) and in one nymph
trial with four insects from each treatment. During
probe hybridization, samples were kept under humid
conditions within an environmental chamber (Percival ScientiÞc, Inc., Perry, IA) maintained at 25 ⫾
0.5⬚C with the lights off. After hybridization, samples
were brießy washed in hybridization buffer, followed
by two washes for 20 min in hybridization buffer, and
one 20-min wash in Tris-buffered saline (Fisher). The
presence of Liberibacter was detected at 200 or 400⫻
using a ßuorescence microscope (Zeis Axioskop 40
FL, Carl Zeiss USA, Thornwood, NY) with Zeiss Þlterset 09 (excitation wavelength ⫽ 450 Ð 490 nm, beam
splitter ⫽ 510 nm, and emission wavelength ⫽ 515 nm;
Fig. 1). The noninfected tissues of the alimentary
canal ßuoresced yellow using Zeis Þlter-set 09 (Fig.
1A). Boundaries of the other organs and tissues were
observed as blue auto-ßuorescence with Zeiss Þlterset 05 (excitation wavelength ⫽ 395Ð 440, beam splitter ⫽ 460, and emission wavelength ⫽ 470; Fig. 1).
Occasionally, low-level white light was used to position the slides or to verify the absence of cuticle
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ANNALS OF THE ENTOMOLOGICAL SOCIETY OF AMERICA
Vol. 107, no. 1
Fig. 1. Observation of ÔCa. Liberibacter solanacearumÕ infection (green ßuorescence) in (A) alimentary canal, (B)
hemolymph, (C and D) salivary glands, and (E and F) bacteriomes dissected from B. cockerelli adults. Blue or yellow
auto-ßuorescence shows organ boundaries.
fragments, which auto-ßuoresced green and appeared
similar to the ßuorescence of the Alexa Fluor 488
probe. Samples were photographed using a DP25 camera mounted to the microscope and operated using the
CellSens software (Olympus America Inc.).
A contingency table analysis (PROC FREQ, SAS
Institute 2012) was conducted to determine whether
insect gender inßuenced infection by the pathogen,
regardless of speciÞc tissue infection. Separate anal-
yses were conducted for adults and nymphs. Both
analyses controlled for trial, and statistical differences
between genders were assessed based on the CochranÐMantelÐHaenszel row mean score statistic
(Stokes et al. 2000). The probability of Liberibacter
infection was compared among psyllid tissues using
logistic regression (PROC LOGISTIC, SAS Institute
2012). Separate analyses were conducted for tissues
from adults and nymphs, and control insects were
January 2014
COOPER ET AL.: LIBERIBACTER IN Bactericera cockerelli
excluded from the analyses. Psyllid tissue and the
tissue by gender interaction were included as the Þxed
effects, and the trial was included as a covariate. The
FIRTH option was included in the MODEL statement
to correct for small sample sizes. Wald 95% CIs for
odds ratios were used to determine signiÞcance in
probabilities of infection among psyllid tissues when
analyses indicated a signiÞcant main effect.
PCR Detection of Liberibacter in Psyllid Bacteriomes. Bacteriomes were dissected from ⬎7-d-old B.
cockerelli adults (Þve from the Liberibacter-infected
colony, and eight from the noninfected colony) and
were transferred to 100 ␮l of phosphate-buffered saline. DNA was extracted from psyllid bacteriomes
using the cetyltrimethylammonium bromide method
(Zhang et al. 1998). Each sample was placed in 500 ␮l
of buffer consisting of 2% cetyltrimethylammonium
bromide, 1.4 M NaCl, 20 mM EDTA, 100 mM Tris-HCl
(pH 8.0), and 0.2% mercaptoethanol. The samples
were incubated in 65⬚C for 30 min and then maintained at room temperature for 3 min before adding
500 ␮l of ice-cold chloroform. After centrifugation at
16,000 ⫻ g for 10 min, the aqueous layer was transferred to 0.6 volume of isopropanol with 1 ␮l of glycogen. Samples were held on ice for 20 min to precipitate DNA. After centrifugation, the resulting pellet
was washed with ice-cold 70% ethanol, air-dried, and
dissolved in 50 ␮l of sterile water. DNA was quantiÞed
using Quanti-iT PicoGreen dsDNA reagent kit (Invitrogen) using the manufacturerÕs protocol.
Real-time PCR was used to determine whether bacteriomes from psyllids were infected with ÔCa. Liberibacter solanacearum.Õ Real-time PCR was conducted
with 12.5 ␮l of TaqMan Universal PCR Master Mix
(Applied Biosystems; Roche Diagnostics, Indianapolis, IN), 2.5 ␮l of each primer (9 ␮M), 2.5 ␮l of labeled
probe (2.5 ␮M), and 5 ␮l of sample extract. The PCR
primers were CL-ZC-F-TCG GAT TTA GGA GTG
GGT AAG TGG and CL-ZC-R-ACC CTG AAC CTC
AAT TTT ACT GAC, and the labeled probe was CLZC-P 6Fam-TTG GCA CCA TGA ACC GCA GAA
ACA CTA AT-Tamra (Crosslin et al. 2011). Reactions
were ampliÞed on a Chromo4 Real-Time System (BioRad Laboratories, Hercules, CA) with the following
cycling conditions: 50⬚C for 2 min, 95⬚C for 10 min,
then 40 cycles of 95⬚C for 15 s and 60⬚C for 60 s.
Liberibacter Transmission Rates. Tomato ÔMoneymakerÕ plants were grown in 900-cm3 pots in an environmental room maintained at 25⬚C with a photoperiod of 16:8 (L:D) h and 50% RH. Plants with at least
two fully expanded leaves were used to compare Liberibacter transmission rates by adult (⬎7-d-old) and
Þfth instar potato psyllids. A single insect was conÞned
to each plant for 24 h using a plastic cylinder cage with
mesh windows. The inoculated plants were placed in
growth chambers (model I30BLL, Pervical ScientiÞc
Inc. Perry, IA). Each chamber was programmed with
a photoperiod of 16:8 (L:D) h with the photophase
beginning at 0600 hours (PaciÞc daylight time [PDT])
and the following 24-h temperature regime: 27.5⬚C at
0600 hours, 30⬚C at 1200 hours, 27⬚C at 1800 hours, and
25⬚C at 2400 hours (PDT). This temperature regime
207
reportedly facilitates the development of visual symptoms of ÔCa. Liberibacter solanacearumÕ infection in
plants within 2Ð3 wk after pathogen inoculation (Munyaneza et al. 2012). The temperature within each
chamber was monitored using a HOBO Data Logger
(Onset Computer Corporation, Pocasset, MA). Insects were removed after 24 h and tested for Liberibacter infection using real-time PCR, as described
above. The plants were maintained in the growth
chambers for 27 d and then tested for Liberibacter
using real-time PCR, as described above for the assessment of Liberibacter in psyllid bacteriomes. The
experiment was conducted twice with different plants
and cohorts of insects. The Þrst trial consisted of 15
plants per combination of insect life stage and infection status. In the second trial, 50 plants were exposed
to insects from the Liberibacter-infected colony (25
per life stage) and 10 plants were exposed to insects
from the noninfected colony (5 per life stage).
A contingency table analysis (PROC FREQ, SAS
Institute 2012) was used to determine whether insect
life stage (Þfth instars vs. adults) inßuenced the occurrence of Liberibacter in whole insects based on
PCR. A separate contingency table analysis was used
to determine whether insect life stage inßuenced the
ability for the insects to vector the pathogen to noninfected tomato. Control insects and plants were excluded from both analyses. Analyses controlled for
insect gender and trial, and statistical differences
among treatments were assessed based on the CochranÐMantelÐHaenszel row mean score statistic
(Stokes et al. 2000).
Presence of Liberibacter in Psyllid Excrement. To
collect psyllid excrement, Þve psyllid adults that were
obtained directly from the insect colonies were conÞned to single leaves on each of six tomato plants.
Three plants were infested with psyllids reared on
Liberibacter-infected plants, and three plants were
infested with psyllids reared on noninfected plants.
The single-leaf cages were constructed of 100-mmdiameter petri dishes (BD Falcon, Franklin Lakes,
NJ), each with a 3-cm-diameter mesh window. A small
opening on the edge of the petri dish that was sealed
with foam provided an entry for the leaf petiole. The
two halves of the petri dish were held together using
paraÞlm (Pechiney Plastic Packaging Company, Chicago, IL). After 7 d, excrement was collected in 1.5-ml
microfuge tubes by rinsing the leaves twice with 100
␮l of phosphate-buffered saline. The experiment was
repeated by conÞning 10 adult psyllids on individual
leaves. In the second experiment, the excrement was
collected on the end of a pipette tip and transferred to
200 ␮l of phosphate-buffered saline. In a third experiment, excrement was collected directly from the colony plants (three samples from each colony) using the
methods described for the second experiment. When
collecting excrement, care was taken to prevent the
transfer of psyllid eggs or exuviae. In each experiment
(excrement from leaves with 5 or 10 psyllids and directly from colony plants), the presence of ÔCa. Liberibacter solanacearumÕ in excrement was assessed
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ANNALS OF THE ENTOMOLOGICAL SOCIETY OF AMERICA
using real-time PCR as described in “PCR Detection
of Liberibacter in Psyllid Bacteriomes.”
Results and Discussion
Fragments of cuticle or cuticular wax that were
occasionally present on the microscope slides exhibited bright green ßuorescence, regardless of whether
the psyllids were obtained from the Liberibacter-infected or noninfected colonies. The use of white light
aided in the discernment between cuticle remnants
and other tissues. These fragments of cuticle were
mostly observed in hemolymph smears; therefore,
the incidence of ÔCa. Liberibacter solanacearumÕ in
the hemolymph may be imprecise. In addition to the
green ßuorescence of the cuticle fragments, the sperm
of both infected and noninfected psyllids ßuoresced
bright green after hybridization with the ßuorescent
probe. This was also observed in preliminary experiments using a probe that targeted a different region of
the Liberibacter genome (W.R.C., unpublished data).
The sperm from insect reproductive organs that were
not hybridized with the probe did not exhibit ßuorescence, indicating that the probe was not completely washed from the sperm. The sperm was often
observed as long Þlaments in the seminal vesicles of
males and in the spermatophore in females and as
ßuorescence on the walls of mature oocytes. We could
not accurately distinguish between the ßuorescence
of sperm and Liberibacter infection of the reproductive organs. Therefore, Liberibacter infection of reproductive organs was not included in our analyses.
These two potential sources of false-positive ÞndingsÑfragments of cuticle and spermÑwere carefully
considered when rating the incidence of Liberibacter
infection in psyllid tissues.
Contingency table analyses did not indicate that
infection differed between male and female adults
(row mean score ⫽ 0.4; df ⫽ 1; P ⫽ 0.52, 81% of females
infected, 71% of males infected) or nymphs (row
mean score ⫽ 0.1; df ⫽ 1; P ⫽ 0.94, 50% of females
infected, 67% of males infected). In total, ÔCa. Liberibacter solanacearumÕ was observed in 76% of psyllid
adults and 38% of Þfth instars that had developed from
eggs on Liberibacter-infected plants but was not observed in insects that were reared on noninfected
plants. Results of real-time PCR of whole insects used
for transmission studies indicated that 100% of adults
and 86% of nymphs that were reared on Liberibacterinfected plants were infected. This inconsistency in
the incidence of infection detected using the two
different methods suggests that PCR may detect lower
bacteria titers in psyllids compared with ßuorescence
in situ hybridization.
Using ßuorescence in situ hybridization, ÔCa. Liberibacter solanacearumÕ was clearly observed in the
alimentary canal (Fig. 1A), hemolymph (Fig. 1B),
salivary glands (Fig. 1C and D), and bacteriome (Fig.
1E and F) of dissected psyllids. Liberibacter may also
have been observed in the oocytes, but infection of the
reproductive organs could not be unambiguously discerned from ßuorescence of the sperm. Logistic re-
Vol. 107, no. 1
Table 1. Probabilities (95% CI) of ‘Ca. Liberibacter solanacearum’ infection observed in specific organs and tissues of
adults and fifth instars of B. cockerelli using fluorescence in situ
hybridization
Psyllid tissue or
organ
Hemolymph
Bacteriome
Alimentary canal
Salivary glands
Tissue
Tissue ⫻ gender
Adults
Fifth instars
0.23 (0.12Ð0.38)b
0.40 (0.27Ð0.55)b
0.66 (0.51Ð0.78)a
0.39 (0.25Ð0.55)b
␹2 ⫽ 16.6; df ⫽ 3;
P ⬍ 0.01
␹2 ⫽ 1.4; df ⫽ 3;
P ⫽ 0.72
Ña
0.06 (0.02Ð0.22)b
0.52 (0.34Ð0.69)a
0.10 (0.03Ð0.28)b
␹2 ⫽ 15.9; df ⫽ 1;
P ⬍ 0.01
␹2 ⫽ 2.3; df ⫽ 1;
P ⫽ 0.31
Values within columns followed by different letters are signiÞcantly
different at the ␣ ⫽ 0.05 interval.
a
Not assessed for the presence of Liberibacter.
gression analyses indicated that the probability of Liberibacter infection differed signiÞcantly among the
different organs of both adults and nymphs (Table 1).
The lack of signiÞcant tissue ⫻ gender interactions in
these analyses indicated that the probability of Liberibacter occurring in each speciÞc organ did not
differ between male and female insects, regardless of
the insect life stage. In both adults and nymphs, Liberibacter was observed more often in the alimentary
canal compared with other organs (Table 1). The
pathogen occurred throughout the alimentary canal
including the midgut loop, the Þlter chamber, and in
each of the four midgut appendages. Liberibacter was
detected in psyllid excrement (Table 2), indicating
that it was also present in the hindgut. Some observations gave the appearance that Liberibacter had
adhered to or was migrating through the midgut wall
(Fig. 1A), but further studies are required to conÞrm
these observations.
Fluorescence in situ hybridization revealed that the
salivary glands of nearly 40% of adult psyllids that were
reared on Liberibacter-infected plants were colonized
with the pathogen (Table 1; Fig. 1C and D). Liberibacter appeared to infect both types of glands (primary
and secondary), but it was difÞcult to identify the
different gland tissues after they had adhered to the
slides and were subsequently processed for ßuorescence in situ hybridization. Our observation that ÔCa.
Liberibacter solanacearumÕ colonizes the salivary
Table 2. Occurrence of ‘Ca. Liberibacter solanacearum’ in
excrement and bacteriomes of B. cockerelli detected using realtime PCR
Samples
Control insectsa
Infected insectsa
Excrement
Trial 1, 5 adults per leaf b
Trial 2, 10 adults per leaf b
Directly from colonyc
Bacteriomesd
0/3
0/3
0/3
0/8
1/3
3/3
1/3
5/5
a
Number of positive samples per total samples.
Collected from tomato leaves on which groups of adults were
conÞned for 7 d.
c
Collected from leaves with groups of adults and nymphs.
d
Dissected from individual B. cockerelli adults.
b
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COOPER ET AL.: LIBERIBACTER IN Bactericera cockerelli
glands of B. cockerelli supports the hypothesis that
Liberibacter is transmitted during salivary discharge
(Pearson et al. 2010). ÔCa. Liberibacter solanacearumÕ
requires a latent period (the period of time between
the acquisition and transmission of the pathogen by
the insect) of 2Ð3 wk at 20 Ð30⬚C (Sengoda et al. 2013).
This latent period might represent the amount of time
required for the pathogen to translocate from the
alimentary canal to the salivary glands.
Although Liberibacter was observed infecting the
salivary glands of 40% of adult psyllids, the pathogen
was observed in only 10% of salivary glands from Þfth
instars (Table 1), suggesting that nymphs are less
likely to transmit the bacterium than adults. Previous
studies that investigated pathogen transmission by
groups of adult and nymph B. cockerelli produced
conßicting conclusions. One study indicated that B.
cockerelli nymphs were less efÞcient vectors of ÔCa.
Liberibacter solanacearumÕ compared with adults
(Buchman et al. 2011), while another study indicated
the opposite (Casteel et al. 2012). Results of our experiments on Liberibacter transmission by single
adults and nymphs are consistent with the report by
Buchman et al. (2011). Compared with Þfth instars,
signiÞcantly more adult psyllids tested positive for
Liberibacter using real-time PCR (row mean score ⫽
5.0; df ⫽ 1; P ⫽ 0.03, 86% of Þfth instars, 100% of
adults), and more adults transmitted Liberibacter to
their host plants (row mean score ⫽ 8.8; df ⫽ 1; P ⬍
0.01, 21% of Þfth instars, 56% of adults). Our Þndings
indicate that Liberibacter is less likely to colonize the
salivary glands of nymphs than that of adults, thus
reducing the probability that nymphs can transmit the
pathogen.
Liberibacter was observed infecting bacteriomes of
40% of adult psyllids and 6% of nymphs by ßuorescence in situ hybridization (Table 1). Results of realtime PCR corroborate our observations that the bacteriomes were colonized by Liberibacter (Table 2).
The obligate symbiont of psyllids, ÔCa. Carsonella ruddiiÕ (gammaproteobacteria), primarily inhabits the
bacteriocytes (round cells visible in Fig. 1E and F),
whereas facultative symbionts are occasionally located within the syncytium tissues (area between the
bacteriocytes in Fig. 1E and F; Chang and Musgrave
1969, Fukatsu and Nikoh 1998, Subandiyah et al. 2000).
Liberibacter appeared to occasionally inhabit bacteriocytes (Fig. 1E), but a few observations clearly
showed Liberibacter was only present in the syncytium (Fig. 1F). The bacteriomes of female psyllids are
larger than those of males (Cooper and Horton 2013),
but these differences in size did not inßuence the
probability of Liberibacter inhabiting the bacteriomes
of females and males (Table 1). Based on our results,
we cannot explicate whether colonization of the bacteriomes by Liberibacter is incidental, or if colonization of the bacteriomes represents an important phase
in Liberibacter infection of psyllids. The colonization
of bacteriomes by Liberibacter may enable interactions between the pathogenic and beneÞcial symbionts. For example, colonization of the bacteriomes by
Liberibacter may displace beneÞcial symbionts, or the
209
presence of facultative symbionts in the bacteriome
may reduce the probability of Liberibacter infection.
In summary, this study documents the presence of
ÔCa. Liberibacter solanacearumÕ in different organs
and tissues of nymph and adult B. cockerelli. Our results support previous Þndings, which suggested that
Liberibacter is persistently transmitted by the psyllid
(Hansen et al. 2008, Pearson et al. 2010). Our results
also suggest that Liberibacter more readily infects the
organs and tissues of adults than those of nymphs,
which may explain why nymphs are less infective than
adults. Our report is the Þrst to document the colonization of psyllid bacteriomes by Liberibacter. Further research is needed to 1) identify the factors responsible for reducing Liberibacter infection in
nymphs, 2) identify the mechanisms in which Liberibacter migrate from the alimentary canal to colonize
other organs and tissues of B. cockerelli, and 3) explore
the ecological implications of the infection of psyllid
bacteriomes by Liberibacter.
Acknowledgments
Pauline Anderson, Heather Headrick, and Millie Heidt
provided technical assistance. This research was supported in
part by the Washington State Potato Commission (Moses
Lake, WA).
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Received 31 May 2013; accepted 15 September 2013.