The Candidatus Liberibacter-Host Interface: Insights Into Pathogenesis Mechanisms and Disease Control

PY55CH20-Wang ARI 13 June 2017 13:36
Review in Advance first posted online

V I E W
E on June 21, 2017. (Changes may
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still occur before final publication
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online and in print.)
C E The Candidatus
I N
A
D V A
Liberibacter–Host Interface:
Insights into Pathogenesis
Mechanisms and Disease
Control
Nian Wang,1 Elizabeth A. Pierson,2
Annu. Rev. Phytopathol. 2017.55. Downloaded from www.annualreviews.org
Access provided by Columbia University on 06/22/17. For personal use only.
João Carlos Setubal,3 Jin Xu,1 Julien G. Levy,2

Yunzeng Zhang,1 Jinyun Li,1 Luiz Thiberio Rangel,3
and Joaquim Martins Jr.3
1
Citrus Research and Education Center, Department of Microbiology and Cell Science,
Institute of Food and Agricultural Sciences, University of Florida, Lake Alfred, Florida 33850;
email: nianwang@ufl.edu
2
Department of Horticultural Sciences, Texas A&M University, College Station, Texas 77843
3
Department of Biochemistry, Institute of Chemistry, University of São Paulo, São Paulo,
SP, 05508-000, Brazil
Annu. Rev. Phytopathol. 2017. 55:20.1–20.32 Keywords

The Annual Review of Phytopathology is online at Huanglongbing, zebra chip, citrus, potato, pathogenicity, disease control
phyto.annualreviews.org
https://doi.org/10.1146/annurev-phyto-080516- Abstract
035513
“Candidatus Liberibacter” species are associated with economically devas-
Copyright c 2017 by Annual Reviews. tating diseases of citrus, potato, and many other crops. The importance of
All rights reserved
these diseases as well as the proliferation of new diseases on a wider host
range is likely to increase as the insects vectoring the “Ca. Liberibacter”
species expand their territories worldwide. Here, we review the progress
on understanding pathogenesis mechanisms of “Ca. Liberibacter” species
and the control approaches for diseases they cause. We discuss the Liberib-
acter virulence traits, including secretion systems, putative effectors, and
lipopolysaccharides (LPSs), as well as other important traits likely to con-
tribute to disease development, e.g., flagella, prophages, and salicylic acid
hydroxylase. The pathogenesis mechanisms of Liberibacters have been dis-
cussed. Liberibacters secrete Sec-dependent effectors (SDEs) or other vir-
ulence factors into the phloem elements or companion cells to interfere
with host targets (e.g., proteins or genes), which cause cell death, necro-
sis, or other phenotypes of phloem elements or companion cells, leading
to localized cell responses and systemic malfunction of phloem. Receptors
20.1
Changes may still occur before final publication online and in print
on the remaining organelles in the phloem, such as plastid, vacuole, mitochondrion, or endoplas-
mic reticulum, interact with secreted SDEs and/or other virulence factors secreted or located on
the Liberibacter outer membrane to trigger cell responses. Some of the host genes or proteins
targeted by SDEs or other virulence factors of Liberibacters serve as susceptibility genes that
facilitate compatibility (e.g., promoting pathogen growth or suppressing immune responses) or
disease development. In addition, Liberibacters trigger plant immunity response via pathogen-
associated molecular patterns (PAMPs, such as lipopolysaccharides), which leads to premature cell
death, callose deposition, or phloem protein accumulation, causing a localized response and/or
systemic effect on phloem transportation. Physical presence of Liberibacters and their metabolic
activities may disturb the function of phloem, via disrupting osmotic gradients, or the integrity
of phloem conductivity. We also review disease management strategies, including promising new
technologies. Citrus production in the presence of Huanglongbing is possible if the most promis-
ing management approaches are integrated and approaches are discussed in the context of local,
area-wide, and regional Huanglongbing/Asian Citrus Psyllid epidemiological zone. For zebra chip
disease control, aggressive psyllid management enables potato production, although insecticide
resistance is becoming an issue. Meanwhile, new technologies such as clustered regularly inter-
spaced short palindromic repeat (CRISPR)-derived genome editing provide an unprecedented
opportunity to provide long-term solutions.
INTRODUCTION
Diseases caused by “Candidatus Liberibacter” species have gained recent worldwide notoriety.
Initially, this was due to the rapid and devastating proliferation of citrus Huanglongbing (HLB),
which nearly destroyed the citrus industry in Florida in the United States and heavily impacts
citrus production in most citrus-producing regions of the world. Outbreaks of zebra chip (ZC)
on potatoes in the Americas and New Zealand and of diseases on other economically impor-
tant vegetable crops further raised awareness of the importance of “Ca. Liberibacter” species as
pathogens. Currently, “Ca. Liberibacter” species are associated with diseases of crops in a limited
number of families (Table 1). Although many different isolates have been sequenced or otherwise
distinguished, e.g., by 16S rRNA sequencing, to date only one member of the genus, Liberibacter
crescens, has been cultured. Despite Koch’s postulates not having been satisfied because of the
inability to culture the putative pathogens, ample evidence has established Liberibacters as the
causative agents of HLB, ZC, and certain other plant diseases (Table 1). Hereafter, we refer to all
candidate members of the genus as Liberibacters. In all cases to date, specific psyllids have been
identified as the Liberibacter vectors (Table 1).
The dependence of Liberibacters on a highly mobile insect vector dramatically complicates
disease epidemiology and management. The development of diseases caused by Liberibacters
relies on more than the temporal and spatial convergence of a virulent pathogen, susceptible
host, and disease-conducive environment, which is referred to as the disease triangle (56). The
epidemiological outcomes for Liberibacter-associated plant diseases are determined by how vari-
ous environmental factors affect plant-Liberibacter-psyllid interactions and collectively are better
described by a disease pyramid, with the insect vector as an additional obligatory component
(Figure 1). It is now apparent that Liberibacters caused HLB in citrus well before their
twentieth century identification as the cryptic assassin. New diseases caused by Liberibacters,
such as ZC, are being discovered with alarming frequency. It is likely that deeper investigation
20.2 Wang et al.
Table 1 Diseases caused by Liberibacters, their hosts, and distributions

Pathogen Diseases Plant hosts Insect vector Distribution Reference
Candidatus HLB Citrus and citrus Asian citrus Widespread in most 20
Liberibacter relatives psyllid citrus-producing areas
asiaticus (Las) (Diaphorina of Asia, Africa, and the
citri ) Americas
Ca. L. africanus HLB Citrus and citrus African citrus South Africa 58, 82
(Laf ) relatives psyllid (Trioza
erytreae)
Ca. L. africanus Unknown Cape chestnut African citrus South Africa 59
subsp. capensis (Calodendrum capense) psyllid
(LafC) (T. erytreae)
Ca. L. africanus Unknown Horsewood (Clausena) African citrus South Africa 147
subsp. clausenae psyllid
(LafCl) (T. erytreae)

Ca. L. africanus Unknown Small forest knobwood African citrus South Africa 148
subsp. zanthoxyli (Zanthoxylum) psyllid
(LafZ) (T. erytreae)
Ca. L. africanus Unknown White ironwood (Vepris) African citrus South Africa 148
subsp. vepridis psyllid
(LafV) (T. erytreae)
Ca. L. africanus Unknown Flaky cherry-orange African citrus South Africa 148
subsp. tecleae (Teclea gerrardii ) psyllid
(LafT) (T. erytreae)
Ca. L. americanus HLB Citrus and citrus Asian citrus Brazil 167
(Lam) relatives psyllid (D. citri )
Ca. L. solanacearum ZC Solanaceous crops Bactericera Central America, 66, 100,
(Lso) cockerelli western Mexico, 128
Haplotype A western United States,
New Zealand
Lso haplotype B ZC Solanaceous crops B. cockerelli Eastern Mexico, central 128
United States
Lso haplotype C Yellows decline Carrot Trioza apicalis Finland, Sweden, 128
and vegetative France, Norway,
disorders Netherlands, Germany
Lso haplotype D Yellows decline Carrot Bactericera Spain, Morocco 129
and vegetative trigonica
disorders
Lso haplotype E Vegetative Celery and carrots B. trigonica Spain, France, Morocco 166, 169,
disorders (likely) 170
Ca. L. europaeus Asymptomatic Rosaceae plants, Cacopsylla pyri Italy, Hungary 143
(Leu) including apple (Malus (L.)
domestica), blackthorn
(Prunus spinosa),
hawthorn (Crataegus
monogyna), and pear
(Pyrus)
(Continued )
www.annualreviews.org • Liberibacter Pathogenesis Mechanisms and Disease Control 20.3
Table 1 (Continued )
Pathogen Diseases Plant hosts Insect vector Distribution Reference
Leu Stunted growth Scotch broom Broom psyllid New Zealand, Australia 173
of shoots, (Arytainilla (?), Europe (?)
shortened spartiophila)
internodes, leaf
dwarfing, and
leaf tip chlorosis
Liberibacter crescens Unknown Mountain papaya (Carica Unknown Puerto Rico 94
(Lcr) stipulata × C. pubescens)
Abbreviations: HLB, Huanglongbing; ZC, Zebra chip.
will reveal more Liberibacter species and that the inevitable introduction of Liberibacters into
more plant/psyllid vector combinations will continue to give rise to new diseases in other crops.
Expansion of Liberibacter distribution and associated diseases has been and will continue to be
facilitated by the introduction of the vector and/or susceptible plant species to new geographic
regions, such as occurred with the introduction of the potato/tomato psyllid into New Zealand
(172). Future disease expansions also may be aided by increases in the geographic and host ranges
of the psyllids, which are associated with the movement of plants and changes in psyllid behaviors,
as described below.
The rapid proliferation of plant diseases caused by Liberibacters and the devastating economic
consequences associated with them create urgency and present challenges for disease control.
The proliferation of Liberibacter diseases also presents unique opportunities to study plant-insect-
pathogen coevolution contributing to their origin and the plant response, and pathogenesis mech-
anisms underlying disease symptomology. Tremendous progress has been made in understanding
the ecological and evolutionary underpinnings of the Liberibacter disease pyramid. Many of these
observations have been summarized previously in several excellent reviews (20, 37, 61, 63, 65,
103, 181). In particular, Bové (20) has provided an insightful historical review of HLB. In this
review, we focus our efforts on the biology of Liberibacters within the plant host. We include a
genomic comparison of Liberibacters and summarize current findings on potential pathogenicity
mechanisms with an aim toward improvements in disease control.
Plant host
(e.g., citrus and potato)
Psyllid
Environment
Candidatus Liberibacter
Figure 1
The disease pyramid for Candidatus Liberibacter–associated plant diseases.
20.4 Wang et al.
DISEASES CAUSED BY LIBERIBACTERS AND WORLDWIDE

DISTRIBUTION
Multiple species of “Candidatus Liberibacter” have been identified from plants grown worldwide
(Table 1). These include “Ca. L. asiaticus” (Las), “Ca. L. africanus” (Laf ) (82), and “Ca. L.
americanus” (Lam) (167), which affect citrus and cause HLB (also called citrus greening); “Ca. L.
solanacearum” (Lso; a.k.a. “Ca. L. psyllaurous”), which causes ZC of potatoes and diseases on many
other solanaceous hosts (66, 100, 101) as well as on carrots in Finland, Sweden, Norway, Spain,
and Morocco and on celery in Spain (118, 120); “Ca. L. europaeus” (Leu), which causes disease
on scotch broom (173) and is also found in multiple Rosaceae plants, including apple, blackthorn,
hawthorn, and pear, without causing disease symptoms (143); and Liberibacter crescens (Lcr) found
in the tropical Babaco plant (also known as the hybrid mountain papaya) (94) (Table 1).
Among the diseases caused by “Ca. Liberibacter” spp., HLB and ZC spread worldwide and
caused dramatic economic losses, whereas other diseases may just be starting to gain such promi-
nence. We focus on HLB and ZC and the Liberibacters responsible for these diseases.
Huanglongbing
HLB is caused by Las, Lam, and Laf, with Las and Lam transmitted by the Asian citrus psyllid
(ACP; Diaphorina citri ) and Laf by the African citrus psyllid (Trioza erytreae). How Las estab-
lished its association with citrus remains unknown. It has been hypothesized that Laf was present
on the African continent before the introduction of citrus, possibly in an indigenous Rutaceous
species in Africa such as Calodendrum capense, Clausena anisate, Vepris lanceolate, or Zanthoxylum
capense (59, 135, 148). Liberibacters closely related to Laf, i.e., LafC, LafCl, LafV, and LafZ,
have been identified from the aforementioned native rutaceous hosts of T. erytreae in South Africa
(Table 1) (59, 148). Similarly, Lam might have evolved from a Liberibacter resembling Leu in-
troduced into Brazil from Europe via infected plants, considering ACP was first reported in Brazil
in 1942, whereas Lam was first found on citrus in Brazil in 2004. As shown in Figure 2, Lam
and Leu are closely related. The establishment of T. erytreae, which can transmit both Las and
Laf (20), in mainland Europe (30) raises the question of whether Leu or other Liberibacters will
ultimately evolve to infect citrus in the Mediterranean area, one of the major citrus-producing
areas yet without HLB.
Even though Las, Laf, and Lam have followed different evolutionary paths to establish their
associations with citrus and their genomic sequences are quite different (41, 105, 185), the symp-
toms caused by the three Liberibacters on citrus are similar. Las was reported to cause more severe
symptoms than Laf (31). Characteristic symptoms associated with HLB include yellow shoots;
blotchy mottling leaves; upright, hardened, and small leaves; leaves showing zinc deficiency and
corky vein; twig dieback; stunted growth, and tree decline. The canopy of HLB-diseased trees be-
comes thinner. Off-season flowering, smaller or lopsided fruit with aborted seeds, and preharvest
fruit drop are frequently observed with HLB-diseased trees. Root degradation is also commonly
observed (87).
HLB, D. citri, and T. erytreae have expanded rapidly into most citrus-producing areas world-
wide. Currently, HLB is confirmed in most citrus-producing areas in Asia, the Americas, and
Africa, although HLB has not been identified in the Mediterranean or Australian citrus-producing
areas. However, the presence of Las and ACP in Papua New Guinea (39) and Indonesia poses a
threat to the Australian citrus industry. Establishment of Las in Ethiopia, Mauritius, and Réunion
in Africa also threatens the African citrus industry. African citrus psyllids have been found in Spain
(30). T. erytreae also can transmit Las (115). In both Brazil and the United States, citrus psyllids
invaded the citrus groves and became established, followed by Las invasion.
Agrobacterium tumefaciens (D14500.1)

Rhizobium leguminosarum SEMIA 2083 (FJ025096.1) Outgroup
Rhizobium etli CFN 42 (NR074499.1)
Lcr BT0
Lcr
NR102476.1
FJ263693.1
Lam
13
FN678792.1
JX244258.1
JX244259.1 Leu
JX244260.1
27 Laf
GQ468843.1
100 Lso
GU991651.1
FJ871059.1
JN245974.1
DQ431997.1
DQ432004.1
6
23
2
KP063235.1
FJ811891.1
FJ811892.1 Las
FJ811893.1
FJ811894.1
JN245975.1
FJ811895.1
JN245978.1
276
0.01
Tree scale JQ867418.1
EU265646.1
EU371107.1
JN245979.1
Figure 2
The maximum-likelihood (ML) phylogenetic tree of the Candidatus Liberibacter genus based on 16S rDNA sequences. The high-
quality 16S rDNA sequences (>1,000 bp, totaling 478 sequences, including the three outgroups) extracted from the NCBI (National
Center for Biotechnology Information) nucleotide database were used for phylogenetic analysis. The sequences were aligned using
MUSCLE (multiple sequence comparison by log-expectation) (44), and the generated alignment was trimmed using Gblocks Version
0.91b (25). The ML tree was constructed using FastTree 2 (141) with gtr (general time reversible) and gamma options. The tree file was
midpoint rooted using iTOL (interactive tree of life) (95). The branches with lengths shorter than 0.01 were collapsed, with exceptions
[such as Lcr and Ca. L. europaeus (Leu clade), and the node number inside the collapsed branch was indicated. The bootstrap supports
for the branches were colored red to green (0–1). “Ca. Liberibacter psyllaurous” was located in the “Ca. L. solanacearum” (Lso) clade.
20.6 Wang et al.
Zebra Chip and Other Diseases Caused by “Candidatus Liberibacter

solanacearum”
ZC is the best characterized disease caused by Lso, and currently all commercial potato (Solanum
tuberosum) varieties are susceptible. The name describes the major tuber symptom, which is a
brown discoloration of the phloem and medullary rays that is often apparent upon slicing fresh
tubers but intensifies with frying. This defect results in potato tubers that are unsuitable for
production of potato chips or French fries. ZC was reported first in Mexico in 1994, in Texas
in 2000, and in the Pacific Northwest region of the United States in 2010 (34, 36). ZC is also a
problem in Central America, including Guatemala and Honduras, and in New Zealand (1, 100).
The symptoms of ZC are visually apparent first in the aboveground parts of the plants and include
curling, purpling, and/or chlorosis of leaves. As the disease progresses, plants appear stunted,
develop swollen internodes, and may form aerial tubers. In addition to the tuber defects, severe
disease reduces tuber production and often causes plant death (118). The bacteria may be passed
to the next generation of plants via infection of tubers used as seed pieces (137). Lso was confirmed
as the causative agent of ZC based on graft transmission (35, 152).

Currently, five Lso haplotypes have been identified (Lso A, Lso B, Lso C, Lso D, and Lso E)
infecting different crops (8, 104, 120, 169) (Table 1). The Lso haplotypes were identified using
single nucleotide polymorphism genotyping of the 16S rRNA, 16S/23S ISR, and the 50S rplJ and
rplL ribosomal protein genes (128); multilocus sequence typing markers (60); and simple sequence
repeats (104). Lso A and Lso B are vectored by the tomato/potato psyllid Bactericera cockerelli and
cause ZC (101, 102). Both haplotypes are present in the Americas and may be found separately or
together in vectors and plant hosts. Lso A currently is the only haplotype found in New Zealand.
In addition to ZC, Lso A and B cause diseases of other economically important solanaceous hosts,
including tomato, pepper, eggplant, tamarillo, tobacco, and cape gooseberry (Physalis peruviana) in
North and Central America and New Zealand and are probably transmitted to these solanaceous
hosts by B. cockerelli. Lso may be seed transmitted in most of these crops, having been demonstrated
to do so in chili pepper (23). Other weedy solanaceous species are known to host Lso A and B,
showing reduced or no disease symptoms (101, 171, 182).
Lso C and Lso D cause disease in carrot in Europe and Africa, where they are vectored by the
carrot psyllids Trioza apicalis and Bactericera trigonica, respectively (8, 120). Haplotype E causes
disease in carrot and celery in Spain but no confirmed psyllid vector has been described (169).
Recent reports suggest that in Spain, B. trigonica, Bactericera tremblayi, and Bactericera nigricornis
as well as another Bactericera species carry Lso and could be vectors (170). Lso can also be seed
transmitted in both carrot and celery (16, 169).
LIBERIBACTERS IN PLANTA: CLUES TO UNDERSTANDING

LIBERIBACTER PATHOGENICITY
One of the hallmarks of diseases caused by Liberibacter is the relatively consistent symptomology
that occurs among different symptom-expressing hosts. Interestingly, this pattern of symptomol-
ogy is regularly observed in citrus and potato when infected by other phloem-limited pathogens.
For example, citrus stubborn disease caused by Spiroplasma citri (158) and citrus disease caused by
“Ca. Phytoplasma” (168) produce symptoms similar to HLB (20). Recognition of ZC in potato is
complicated by the similarities of the early symptoms to potato purple top caused by “Ca. Phyto-
plasma” (93) and potato leafroll virus (35). Heavy psyllid feeding itself causes a condition referred
to as psyllid yellows that also results in leaf chlorosis and purpling, shortened internodes, the for-
mation of aerial tubers, and reduced tuber yields (154). These observations support the hypothesis
that disruption of phloem function, regardless of the pathogen, mechanism, or host, may result in
the expression of consistent disease symptoms.
Another hallmark of diseases caused by Liberibacters is the cryptic nature of the pathogen
and the inconsistent relationship between pathogen titer in a particular tissue and symptomology.
Indeed much effort has been invested in the detection of this often obscure pathogen in planta.
Understanding the movement of Liberibacters in planta and the relationship between Liberib-
acter titer and disease symptomology is critical to unveiling how Liberibacters cause disease and
designing suitable disease control strategies. Evidence suggests that in plants, Liberibacters reside
solely within phloem tissues. For example, Las has been reported to be restricted to the inside of
sieve tubes (54, 73); however, one study also found Las in companion cells (57). Other Liberib-
acters such as Lso also live inside the sieve elements but have not been observed in associated
companion or mesophyll cells (152, 169).
Systemic infection of the host plant follows the direction of phloem sap translocation, moving
from sources (e.g., mature leaves) to sinks (e.g., roots, tubers, young leaves, flushes, and fruit),
although how this is accomplished and the speed of movement are subject to debate. Despite the
presence of structural genes for the production of pili and flagella in the genomes of all Liberibacter
(20, 41), these structures have yet to be observed on any Liberibacter in planta. This suggests that
passive movement occurs with the mass flow of phloem sap, which explains the temporal patterns
of downward and upward movement to roots and flushes and young leaves and fruit, respectively.
Microscopic analysis of Las in planta has shown that Las cells float freely in the phloem sap without
attaching to the sieve tube cell walls or each other (i.e., without forming biofilms) (73, 90, 91).
Similar observations have been made of Lso in planta (131). Circumstantial evidence suggests that
Liberibacter movement occurs primarily in the vertical direction along the sieve tubes through the
sieve pores rather than horizontally to adjacent sieve tubes. This hypothesis was first suggested by
observations of the uneven colonization of Las in dodder, where adjacent phloem vessel elements
were observed to be either completely full of Las or free of the pathogen (70). It is likely that sieve
plate pores with diameters ranging from <1 μm to ≈14 μm (46) do not prevent Liberibacters
from passing between sieve elements. For example, the diameter of Las has been reported to be
approximately 0.1 to 0.3 μm in citrus phloem (20, 73). Both Las and Lso have been observed
traversing the sieve plate pores (54, 131, 159).
The direction and rate of movement of Liberibacters within the host plant have been studied.
In growth chamber experiments with potato and tomato plants, Levy et al. (96) found that Lso
spread from the site of Lso infection upon psyllid feeding to the main stem within seven days.
Subsequent work demonstrated Lso dispersal out of the infected leaf occurred within 24 hours
(24). The movement of the bacteria within the plant followed the direction of translocation of
carbohydrates from the source leaves to the sinks, resulting in bacterial titers that were measurable
first in the newly developing leaves. Rapid accumulation of Lso following infection also occurred
in other sinks, including the roots and tuber stolons (182). After Lso inoculation of potato, Lso
was confirmed in the inner phloem above the export leaf and in the outer phloem below the
export leaf, consistent with upward movement of phloem sap in the inner phloem and downward
movement of phloem sap in the outer phloem (33) of the bicollateral vascular system of solanaceous
plants.
In citrus, it has been estimated that the speed at which sap moves in the phloem approximates
3.94 μm/s, or 0.34 m/day (47). However, Las movement has been estimated to be approximately
2–3 cm/day in citrus, i.e., at reduced speed compared to the mass flow of phloem sap (S. Lopes,
personal communication). The apparent difference in Las movement and mass flow of phloem sap
is a topic that needs to be addressed. Detection of Las in citrus foliar tissues has been problematic
because of the spatial and seasonal patterns of pathogen movement in the plant. Once Las arrives
20.8 Wang et al.
at the roots, it multiplies and serves as a source of inoculum to young flushes or fruit and is
a relatively reliable site of pathogen detection (87). The movement of Las within the plant is
limited by plant sectoriality, resulting from the subdivision of physically coherent vasculature.
Many plants exhibit sectoriality when physiological activities are integrated within some parts of
their structures, but the same activities display a strong degree of independence at other structural
scales because of vascular structure (179). Sectoriality leads to segmented patterns of infection and
symptom development typically observed in early citrus infections. When large populations of
ACP infect multiple branches simultaneously, the sectorial pattern of HLB symptoms disappears.
The expression of disease symptoms typically lags plant infection by several months to several
years, which is very problematic for disease management.
In field-grown potatoes, although there is typically a 2–3-week lag between plant infection and
symptom development, there is a strong correlation between the timing of plant infection and
the severity of tuber disease symptoms. Symptoms may be evident in the tubers within 3 weeks of
exposure of the plants to Lso-infected psyllids, which may proceed or coincide with the initiation
of foliar symptoms (22, 96). The cessation of tuber development typically coincides with the
development of disease symptoms, and tubers may not form if the plants are infected before tuber
initiation (22). However, even late-season infections are economically important for growers.
Rashed et al. (145) demonstrated that tubers of plants infected just four days before harvest may
become infected despite the plants and tubers being asymptomatic and testing negative for Lso at
harvest. Bacterial titers were found to increase in many of these tubers to detectable levels during
cold storage, and tubers after storage were as defective in sprouting as tubers showing symptoms
before storage (145). Of significance, recent findings demonstrated that plants infested with psyllids
only 48 hours before vine kill still developed symptoms in tubers left to mature in the ground for
one month (149). Collectively, these findings demonstrate the rapidity of systemic infection by Lso
and the susceptibility of plants to infection throughout the growing season. They also demonstrate
that disease symptoms in specific tissues such as tubers may develop independently from other
plant organs, as demonstrated by the progressive infection and symptom development in tubers
after vine kill and in cold storage.
The previous discussion demonstrates that Liberibacters probably live solely within phloem
tissues and are capable of rapid, systemic infection that is consistent with the sectorality of the plant
vasculature. Even though most symptoms caused by Liberibacters seem to result from disruption
of phloem function, organ-specific plant response to infection has been observed (e.g., symptom
development in detached tubers).
GENOMIC FEATURES OF LIBERIBACTERS

Genome analysis of Liberibacters has helped understand the biology, evolution, and virulence
mechanism of the pathogens. To date, 18 genomes of 5 Liberibacter species, i.e., Las (8 genomes),
Laf (1 genome), Lam (2 genomes), Lso (5 genomes), and Lcr (2 genomes) have been sequenced
(Supplemental Table 1). The genome sizes of Liberibacters range from 1.1 to 1.45 Mb with
a low GC content, approximately 35.3%. The average nucleotide identity (ANI) values between
different strains of the same species are 99.6% to 99.97%, demonstrating highly conserved genomic
sequence among these strains. However, among the different species, the ANI values were only
67.94% to 80.79%, indicating the highly diverse genomic traits among the different Liberibacter
species.
The pan-genome size of all Liberibacters is 2,394 genes, and the core-genome size is 305 genes.
The pan-genome plot does not reach a plateau, suggesting an open pan-genome and the need
for additional genome sequencing. Some Lso haplotypes infecting carrot or celery and the Leu
genome have yet to be sequenced. Sequencing of those aforementioned Liberibacter species will
help us gain a better understanding of the genomic features and their virulence mechanisms.
Among the 2,394 shared genes, only 619 have at least one representative in each of the
five species, which suggests a relatively low level of inter-species gene sharing. This is also sug-
gested by the high level of gene specificity: 1,506 genes of the pan genome are species specific,
meaning that almost two-thirds of all genes in the pan genome are not shared among different
species of the genus. Even though there is variation in gene specificity (the two extremes being
45.5% in Lcr and 16.0% in Laf ), we believe a general hypothesis for the high level of specificity
is the obligate intracellular lifestyle of all members of the genus, which creates a barrier for gene
exchange (138). The different hosts also represent different environments, and each one could
have specific selective pressures.
By using the clustered regularly interspaced short palindromic repeat (CRISPR) recognition
tool (17), possible CRISPR arrays have been found in most of the 18 sequenced Liberibacter
genomes (Supplemental Table 1) ( J. Xu & N. Wang, unpublished data) (194). The CRISPR
system is a prokaryotic immune system that confers resistance against phages and plasmids and
is present in most archaea and many bacteria (13). A complete CRISPR/Cas system consists of a
CRISPR array, which contains short direct repeats separated by short variable spacer sequences,
and diverse Cas genes located adjacent to this array (13). The CRISPR array and Cas genes have
been predicted in Las (194). Six of the 18 genomes contain a type I CRISPR/Cas system. Although
most CRISPR/Cas systems have been found in chromosomes, the CRISPR/Cas systems of many
Liberibacters are located on a prophage, especially in Las, thus suggesting that CRISPR/Cas might
contribute to the dominance of a single prophage in Las strains (194), as described in below. The
limited number of spacers in the CRISPR/Cas system is consistent with the limited exposure of
Liberibacters to phage and plasmids either in the phloem or in psyllids.
PATHOGENESIS MECHANISMS OF LIBERIBACTERS

With the exception of Lcr (94), Liberibacters have not been cultured in artificial media; therefore,
traditional molecular and genetic analyses have been difficult to perform. This difficulty has greatly
hampered efforts to understand the virulence mechanisms of Liberibacters. To date, most insights
into Liberibacter pathogenesis mechanisms have been derived from analyses of Liberibacter ge-
nomic sequences (41, 48, 94, 105, 185), studies using surrogates such as Sinorhizobium (176) and
E. coli (140), and expression in planta as well as indirectly from host transcriptomic, proteomic,
and metabolomic responses to Las, Lam, and Lso infection (50, 81, 112–114, 160, 190, 195, 196).
In this section, we summarize the virulence traits and pathogenicity mechanisms of Liberibacters.
The host responses to Liberibacter infection based on transcriptome, proteome, and metabolome
approaches were recently reviewed (37, 181) and are not reviewed in detail here.
Virulence Traits of Liberibacters

Owing to the lifestyle in the phloem and reductive evolution, Liberibacters contain very few
virulence traits compared to free-living plant-pathogenic bacteria such as Xanthomonas and Pseu-
domonas. We focus on the recent progress in the study of Liberibacter secretion systems and
effectors, lipopolysaccharides, flagella, prophage, and salicylic acid, which have been suggested to
play important roles in causing disease symptoms, contributing to bacterial survival in planta, and
suppressing plant defenses.
20.10 Wang et al.
Extracellular space
Autotransporters β-barrel proteins OM vesicles
OM
β
Chaperones
N
Molecules
C other than
mature SDE
Periplasm
N proteins
Mature
SDE proteins C
SPase
IM YajC SecF SecD SecYEG YidC

SecA
ADP + Pi
SP
SecB ATP
Cytoplasm Preprotein
Figure 3
Candidatus Liberibacter spp. contain basic components of the Sec machinery. Putative Sec-dependent
effector (SDE) proteins (brown) have been predicted to be translocated via the Sec machinery. SecB is the
Sec-system-specific chaperone that channels the preprotein to the Sec translocation pathway, binding to
SecA. The preprotein-carrying SecA binds to the membrane. The SecYEG complex constitutes a channel
for polypeptide movement. Continued translocation requires ATP hydrolysis to provide energy force. Las
may utilize some noncanonical mechanisms to secrete proteins across the outer membrane. B-barrel proteins
in the outer membrane may have adopted the ability to transport proteins to the extracellular space. Some
SDEs proteins are autotransporters. In addition, some of these SDE proteins might be secreted in outer
membrane vesicles. Abbreviations: IM, inner membrane; OM, outer membrane; SP, signal peptide; SPase,
signal peptidases.
Secretion systems and putative effectors. Systems capable of secreting bacterial proteins called
effectors into host cells are among the most important virulence factors of bacterial pathogens.
Protein effectors often suppress plant defenses or manipulate developmental processes within the
host to benefit the pathogen (88). Interestingly, Liberibacters contain type I secretion systems
(T1SSs) and a complete general secretory pathway (Sec) (Figure 3) but lack other secretion
systems (41, 48, 105, 185). In addition to Sec, Lcr contains the twin arginine transport system,
which is not present in other Liberibacters.
Genes encoding T1SSs consisting of TolC, HlyD, and PrtD have been reported in Liberibac-
ters (41, 105). Based on analyses of 18 sequenced Liberibacter genomes (Supplemental Table 1),
a complete T1SS has been identified in Las, Laf, and Lso but not in Lam or Lcr. Putative T1SS
substrates, e.g., genes encoding serralysin and hemolysin, have been identified in Liberibacter
genomes (41, 105). A putative serralysin gene has been found in most sequenced Las, Lso, and
Laf genomes but not in Lam or Lcr, whereas a predicted hemolysin gene has been identified in
all sequenced Liberibacters. The functionality and contribution of T1SSs and their substrates to
the virulence of Liberibacters remain to be determined.
The Sec machinery facilitates the majority of protein transport across the cytoplasmic mem-
brane (Figure 3) and is essential for bacterial viability (153). The Sec apparatus also secretes
important virulence factors in some plant-pathogenic bacteria. For example, in phloem-dwelling
phytoplasmas, the Sec pathway is critical for the secretion of vital virulence factors, including
SAP54, SAP11, and Tengu, into plant cells (76, 111, 163). Importantly, phytoplasmas cause similar
symptoms as do Liberibacters (168). The genome sequences and virulence factors of phytoplasmas
that infect citrus remain to be determined but may reveal common effectors that are important
for the symptom development in diseases caused by both groups of pathogens.
It is unknown how Liberibacters translocate proteins from periplasm to the outside of the outer
membrane, where these proteins may be directly involved in host interactions. Interestingly, outer
membrane vesicles (OMVs) have been observed on the surface of Lso (131). OMVs have been
shown to secrete various virulence factors in other pathogens (19). It is probable that Liberibacters
use OMVs to secrete Sec-dependent effectors (SDEs) (Figure 3) and other putative virulence
factors.
Signal peptide (SP)-containing extracytoplasmic proteins (SPEPs), which are potential sub-
strates of the Sec apparatus, have been predicted for Las—i.e., Las_psy62 (166 SPEPs), Las_gxpsy
(168 SPEPs) and Las Ishi-1 (164 SPEPs) strains, Lam (133 SPEPs), Laf (141 SPEPs), Lso (171
SPEPs), and Lcr (214 SPEPs) (140). The three Las strains from the United States (Las_psy62),
China (Las_gxpsy), and Japan (Las Ishi-1) show high uniformity in their predicted Sec-dependent
extracytoplasmic proteins, with 151 proteins overlapping in all three strains. However, signifi-
cant differences have been observed among Las, Laf, and Lam, even though they all cause HLB.
Only 45 Sec-dependent extracytoplasmic proteins show homology between these species. To ex-
perimentally validate the predicted extracytoplasmic proteins, E. coli–based alkaline phosphatase
(PhoA) gene fusion assays have been conducted. A total of 86 of the 166 predicted Las proteins
were experimentally validated to contain an SP (140). In addition, 16 and 86 putative SDEs also
have been predicted by Pitino et al. (136) and Cong et al. (32), respectively. Among the genes
encoding SP-containing proteins, 36 have been shown to be highly expressed in planta compared
with in psyllids, whereas eight others are highly expressed in psyllids compared with in planta
(188), thus suggesting that those proteins may play critical roles in Liberibacter adaptation to
living in the plant and insect hosts.
Phenotypic changes have been observed via transient or transgenic expression of multiple
SDEs in planta, including in Nicotiana benthamiana and citrus (136) (N. Wang, unpublished data).
Specifically, Las5315 is localized in the chloroplast and induces cell death when it is transiently
expressed in N. benthamiana. It also induces callose deposition. How Las5315 contributes to the
virulence of Las in citrus remains unknown. Notably, homologs of Las5315 have not been found
in Lam (136) and other sequenced Liberibacters.
In addition to the SDEs, two type V autotransporters, LasAI and LasA, also rely on the Sec
machinery for transport and have been proposed to exist in Las. Neither LasAI nor LasA contains
a typical SP domain for Sec-dependent translocation (67). It remains to be determined whether
either protein is involved in the virulence of Las.
Lipopolysaccharides. Lipopolysaccharides (LPSs), which are composed of lipid A, an oligosac-

charide core, and an O-antigen, are critical components of the outer membranes of gram-negative
20.12 Wang et al.
bacteria. LPSs contribute to bacterial fitness and virulence by maintaining the cell structural in-
tegrity and protecting bacteria from certain chemical stresses, such as antimicrobial compounds.
Lipid A is somewhat conserved, whereas the oligosaccharide core and O-antigen are much more
variable. LPSs of bacterial pathogens often trigger plant immune responses, such as oxidative
bursts, salicylic acid (SA) accumulation, and callose deposition during pathogen-plant interactions
(197), whereas those of symbiotic bacteria may suppress plant defense responses as they invade
their partner host (7). Las induces callose deposition, which might be partially responsible for
phloem blockage in citrus (51, 90, 91). Whether the LPSs of Liberibacters trigger callose deposi-
tion remains to be determined. Interestingly, the Las genes encoding LPS synthesis and transport
are highly expressed in planta but not in the psyllid vector (188).
LPSs are synthesized on the inner membrane and typically require transporters, such as the
two ABC transporters MsbA and LptB2FG to be transported to the outer membrane (132). The
genes involved in the biosynthesis of the three LPS components are found in all the sequenced
Liberibacters except Lam (185). A majority of the LPS synthesis genes are missing in Lam, and
only two genes, lpxC and lpxD, are present (185). Loss of LPS has been observed previously in some
bacteria and may allow them to adapt to stressed niches (164). The different LPSs of Las, Lam,
and Laf might affect their induction of host defenses. LPSs have been reported to activate two of
the biosynthetic steps of the phenolic conjugates coumaroyltyramine (CT) and feruloyl-tyramine
(FT) (130). Lso causes an increase in the level of phenolics in infected potato tubers, which might
result from potato cells recognizing the LPSs (126). Las-induced phenolics in citrus also have
been reported (72). A lack of LPSs might allow Lam to avoid recognition by plant cells but may
also make Lam more susceptible to externally applied or plant-derived antimicrobial compounds.
Flagella. Bacterial flagella are responsible for rotational propulsion, promote host colonization
via adherence, and induce plant immunity. A nearly complete set of genes involved in the flagellar
synthesis pathway are present in the sequenced Liberibacter genomes (41, 48, 94, 105, 185).
Flagella have been observed on Lso cells in psyllids (29). Expression of all of these genes has
been documented in the transcriptome of Liberibacter-infected psyllids (53). For Las, flagella
have not been observed on the surface of bacterial cells in planta (20). The expression of genes
involved in flagellar assembly, including fliF, flgI, and flgD, and of the motB gene involved in motor
function is upregulated in planta. In contrast, flgL, flgK, and fliE are overexpressed in the psyllid
(188). The flagellar expression pattern suggests that Las may have a functional flagellum in some
environments. The Lso flagellin gene is also highly expressed in B. cockerelli (190). Unlike Las,
Lso, and other Liberibacters, the Lam genome does not contain flbT. FlbT functions as a key
regulatory checkpoint for flagellin gene expression, and flagellin is not expressed in flbT mutants
(52). The lack of flbT in Lam suggests that flagellar genes might not be expressed so as not to
activate plant defenses in planta (185).
Liberibacter flagella have been reported to stimulate plant defense. Las encodes a flagellin
that contains a conserved 22 amino acid domain (Flg22). The synthetic Flg22Las peptide does not
induce plant cell death in tobacco but has retained the ability to induce callose deposition at a
concentration of 20 μM or above (198). Transient expression of FlaLso in tobacco induces a delayed
increase in reactive oxygen species (ROS) production and slow necrotic cell death. The flg22Lso
peptide does not induce ROS production in tobacco, tomato, or potato but does induce callose
deposition in them (68). Flg22Lso and the flagellin FlaLso induce expression of genes associated
with PAMP-triggered immunity (PTI) in N. benthamiana (68).
Prophages. A prophage is a temperate bacteriophage genome that has integrated into the bac-
terial chromosome or exists as a plasmid. There are currently two known types of Las prophages,
Type 1 (SC1 or FP1-like) and Type 2 (SC2 or FP2-like) (41, 192). SC1 becomes replicative in
planta, and when lytic genes are expressed, bacteriophage particles are readily formed in periwin-
kle (192) and citrus (57). By contrast, SC2 is a replicative excision plasmid that lacks lytic genes.
An analysis of the 18 sequenced Liberibacter genomes (Supplemental Table 1) has revealed that
either Type 1 or 2 prophage is present in each strain. Interestingly, although most Las strains
contain a single prophage and a few strains contain both prophages, strain Ishi-1 from Japan (89)
does not contain any prophages. The pattern of a single prophage present in most Las strains also
has been observed in an extensive survey of Las isolates in Thailand and southern China (142,
194). Among strains observed in an extensive survey of Las isolates in China, it was typical for
Las to have a single prophage, with Guangdong isolates harboring mainly the type 2 prophage,
whereas isolates from Yunnan are dominated by the type 1 prophage (194). Notably, the Lso NZ1
contains three prophages (174).
Prophages have been suggested to assist Las in suppressing plant defenses (83). For exam-
ple, SC2-gp095 encodes a ROS-scavenging peroxidase, which is a predicted lysogenic conversion
factor. SC2-gp095 expression is suppressed in psyllids, but it is expressed at a high level in peri-
winkle. Expression of the peroxidase in Lcr, but not in E. coli, results in enhanced resistance to
H2 O2 . Nonclassical secretion has been predicted for SC2-gp095, and its secretion from Lcr has
been confirmed by enzymatic and western blot analyses. Transient expression of the peroxidase
in planta results in strong transcriptional downregulation of RbohB, the key gatekeeper of the
H2 O2 -mediated defense signaling in plants. It has been suggested that Las evades early detection
in the phloem by virtue of its secreted peroxidase activity, enabling it to build up a high titer
in citrus over several years before manifesting disease symptoms, thus resulting in the long in-
cubation period often associated with HLB progression (83). However, the lack of a prophage
in many Las strains does not appear to relate to the lack of HLB symptoms because Ishi-1 and
the Guangdong isolates, which do not contain any prophages, induce similar HLB symptoms as
isolates containing prophages (89, 194). Overall, this evidence suggests that prophages contribute
to bacterial virulence but are not required for Las pathogenicity.
Salicylic acid hydroxylase. Plant immunity responses involve the perception of the invading
pathogen and the activation of specific defense signal transduction pathways that lead to activation
of PTI, effector-triggered immunity (ETI), and systemic acquired resistance (SAR) (42, 88). SA
and its derivatives play a central role in plant defense by mediating responses against pathogens
in many plant species, and they are important for activating PTI, ETI, and SAR (42). SA is an
endogenous defense signal, and its derivative MeSA (methyl salicylate) is one of the signals for SAR
(134). Interestingly, all Liberibacters contain an SA hydroxylase protein encoded by sahA ( J. Li &
N. Wang, unpublished results). It has been suggested that Las uses SA hydroxylase to degrade SA
and suppress plant defenses. Purified SahA displays strong enzymatic activity to degrade SA and its
derivatives. In transgenic tobacco plants, SahA abolishes SA accumulation and the hypersensitive
response induced by incompatible pathogens. Foliar sprays of 2,1,3-benzothiadiazole (BTH) and
2,6-dichloroisonicotinic acid (INA), SA functional analogs not degraded by SahA, are moderately
effective in suppressing Las population growth and HLB disease progression in infected citrus
trees under field conditions ( J. Li & N. Wang, unpublished results).
Mechanistic View of Liberibacter Pathogenicity

Based on current knowledge of Liberibacters and plant interactions, we suggest that the pathogen-
esis mechanisms of Liberibacters can be summarized in the following model (Figure 4). Liberib-
acters actively secrete SDEs or other virulence factors into the phloem, causing disease symptoms,
20.14 Wang et al.
Xylem Source
SE
CC
Water RFO Symplastic

High- pathway
pressure
Sucrose Apoplastic
pathway
Organelles (e.g., ER
Sucrose or mitochondria)
Polyols and receptors on
RFO the membranes of
the organelles
Some putative effectors

might affect expression
of target genes directly
or indirectly.
Las and PAMPs
on cell surface
Transport phloem
Release phloem
Low-
pressure
Symplastic
pathway
Sucrose Apoplastic
pathway
Sink
Figure 4
Illustration of Liberibacter virulence mechanisms in plant. Liberibacters (brown ovals) live in the phloem
elements. Phloem is responsible for transportation of carbohydrates from source to sink. Nutrients are
transmitted into phloem via a symplastic pathway or an apoplastic pathway. Liberibacters might secrete
Sec-dependent effectors (SDEs) or other virulence factors (represented by a blue dot, maroon triangle, red dot, or
orange trapezoid ) into the phloem sieve elements (SEs) or companion cells (CCs) to interfere with host
targets (proteins or genes, shown as enlarged), which cause cell death, necrosis, or other phenotypes of
phloem elements or companion cells, leading to localized cell responses and systemic malfunction of
phloem. Receptors on the remaining organelles in the phloem, such as the plastid, vacuole, mitochondrion,
or endoplasmic reticulum (ER), might be able to interact with secreted SDEs and/or other virulence factors
secreted or located on Liberibacter outer membrane to trigger cell responses. Some putative SDEs might
affect the expression of target genes directly or indirectly. In addition, Liberibacter might trigger plant
immunity response via pathogen-associated molecular patterns (PAMPs), such as lipopolysaccharides and
outer membrane proteins, leading to premature cell death, callose deposition, or phloem protein
accumulation, which lead to a localized response and/or systemic effect on phloem transportation. The
physical presence of Liberibacters and their metabolic activities may disturb the function of phloem via
disrupting osmotic gradients, or the integrity of phloem conductivity. Abbreviations: RFO, raffinose family
oligosaccharide.
most likely by interfering with host targets either in the phloem or companion cells (e.g., proteins
or genes). Because Liberibacters live in the phloem elements, an intracellular environment, the
receptors on the remaining organelles in the phloem, such as the plastid, vacuole, mitochondrion,
or endoplasmic reticulum (71), might be able to interact with secreted SDEs and/or other viru-
lence factors secreted or located on the Liberibacter outer membrane. Some of the host genes or
proteins targeted by SDEs or other virulence factors of Liberibacters might serve as susceptibility
genes that facilitate compatibility (e.g., promoting pathogen growth or suppressing immune re-
sponses) or disease development. One of the potential effects of the effectors or virulence factors
is phloem malfunction, e.g., due to destructive effects on components of sieve tubes and elements
themselves or interacting with companion cells, which supply proteins and transcripts to the sieve
elements (109). Investigation of Liberibacter secretory systems, potential effectors, and suscepti-
bility genes might lead to improvements in understanding Liberibacter pathogenicity and disease
control. Genome editing of susceptibility genes or their promoter region has been previously
shown to successfully generate disease-resistant citrus varieties (84, 86).
In addition, Liberibacters cause disease symptoms by eliciting self-destructive plant responses

and/or affecting phloem function, which, in turn, contributes to the development of disease symp-
toms. Liberibacters cause malfunctions in phloem loading, unloading, and transport, which even-
tually lead to disease symptom development. The physical presence of Liberibacters and their
metabolic activities may disturb the function of phloem, via disrupting osmotic gradients, or
the integrity of phloem conductivity. In addition, plant recognition of PAMPs (e.g., flagella and
LPSs) of Liberibacters might cause self-destructive plant responses, including the accumulation
of phloem proteins and callose in citrus phloem in response to Las infection as well as the defense
responses in the tubers to Lso.
Interestingly, sieve tubes full of Lso cells have been observed in carrot (131). For Las, fully
occupied sieve tubes have been observed in sink tissues, including young leaves, seed coat, and
roots, but not in collection or transportation phloem. The fully occupied phloem is unlikely to
be functional, resembling the loss of function of Xylella-occupied xylem (26). The fully occupied
sieve tubes at sink tissues might affect the integrity of the phloem and disrupt the osmotically
generated pressure gradient that drives the phloem mass flow. Alternatively, as mentioned earlier,
Liberibacters contain many PAMPs (41, 105, 185). Liberibacter PAMPs have been known to
induce plant defense, although mechanistically how host plants respond to these PAMPs remains
to be determined. Plant immunity responses observed previously in Las-infected citrus include the
production of secondary metabolites, such as phenolics, and accumulation of phloem proteins and
callose (51, 72, 90, 91). Lso causes an increase in phenolic levels in infected potato tubers, which
may result from their recognition of PAMPs (126). Additionally, Liberibacters consumption of
photoassimilates may disrupt osmotic gradients, thus affecting phloem loading, unloading, and
transport. Liberibacters are unable to take up and metabolize sucrose but utilize other sugars such
as glucose in the phloem. This may cause an imbalance in carbohydrate partitioning, such as the
accumulation of sucrose in the source leaves, in turn giving rise to the inhibition of photosynthesis
and subsequent physiological disorders (49).
Currently, it is unclear which factor plays the most important role in disrupting the phloem
function, i.e., whether it is due to the virulence factors, the host responses to the PAMPs, the
impact of the metabolic activity of the pathogens, or the physical occupation of the phloem
by the pathogens. It is important to acknowledge that despite not knowing the exact causes,
malfunction of phloem loading, unloading, and transport can explain many initial phenotypical
changes in plants infected with Liberibacter. We postulate that many later symptoms are probably
the cumulative and indirect effects of root decline, reduced photosynthesis, and reduced nutrient
transportation associated with phloem malfunction rather than the direct outcome of Liberibacter
20.16 Wang et al.
infection. In HLB-affected trees, phloem malfunction may explain the blotchy mottle symptoms
and root decline; later symptoms such as zinc deficiency and twig dieback may be the end result
of the decline process. For ZC, malfunction of phloem loading, unloading, and transportation
likely explain the starch accumulation in leaves and decreased starch levels in the tubers (9, 22).
However, it is important to note that Liberibacter titers and ZC symptoms increased in tubers
following cold storage and in tubers on vine-killed plants, i.e., under conditions in which tubers
are no longer influenced by whole-plant translocation processes. These observations demonstrate
that the development of symptoms in specific organs may occur independently of the condition
of the rest of the plant, suggesting the local symptoms are due to either action of virulence factors
or plant responses to PAMPs.
DISEASE CONTROL
Knowledge of the biology of Liberibacters and the mechanisms underlying bacterial pathogenicity
and symptom development has led to the design of a variety of management strategies. For all
Liberibacter-caused diseases in citrus, potato, and other crops, disease control currently focuses
on psyllid management and inoculum removal, owing to the universal absence of disease resistance
in all crops. However, successful long-term disease management depends on designing effective
control strategies based on an understanding of the characteristics of and interactions among the
crops, insects, and Liberibacters.
Huanglongbing Disease Management

For various citrus-producing areas worldwide, different strategies are needed, depending on the
threat level of Las and ACP and the stage of HLB or ACP epidemiology (e.g., initial entry or
endemic). Quarantine measures have been adopted to keep HLB away from Australia and the
Mediterranean citrus-producing areas. For many newly infected citrus-producing areas such as
California, quarantine and eradication measures are being implemented. Whether those newly
infected areas can prevent HLB from becoming endemic not only depends on the strict implemen-
tation of quarantine and eradication measures but also relies on the control effort in neighboring
citrus-producing areas of the HLB/ACP epidemiological zone, as detailed below. For newly in-
fected areas, early and accurate HLB diagnosis and Las detection are critical for inoculum removal.
Readers are referred to the review by Arredondo Valdés et al. (10) on HLB diagnosis and Las de-
tection. For HLB-endemic regions, economic and profitable citrus production is still possible
by integrating the most promising HLB and ACP management approaches. Both chemical and
biological psyllid control are critical components of HLB management. Readers are referred to
several excellent reviews on psyllid control (18, 63). In addition, RNAi has been suggested to be
a promising approach to control ACP, and readers are referred to a review by Hunter et al. (80).
Huanglongbing/Asian citrus psyllid epidemiological zone. Based on the frequency of ACP

immigration and transmission of Las within the components of the citrus-producing areas, the
citrus-producing areas can be classified into three levels: local (no geographical separation or bar-
rier to prevent ACP from traveling between citrus groves); area wide (clear separation between the
citrus groves prevents psyllids from flying between groves, but the psyllids can still be introduced
via wind or other mild weather events); and regional (geographical separation or barriers prevent
ACP from flying between the citrus-producing areas, and ACP rarely moves among the different
citrus-producing areas unless there are major weather events such as hurricanes). The United
States, Mexico, and countries in the Caribbean and Central America belong to a single regional
HLB/ACP epidemiological zone, as evidenced by the timing of HLB occurrence and detection
of the same haplotype of Las in this region. HLB was found in Florida in 2005, Cuba in 2007, the
Dominican Republic in 2008, and Belize and Puerto Rico in 2010, and was found almost simulta-
neously in Costa Rica, Nicaragua, Honduras, Guatemala, Mexico, and Jamaica. Haplotype A Las
has been found in the United States, Mexico, and countries in the Caribbean and Central America
(116). In addition, de León et al. (40) discovered that ACP in Florida, Texas, and California in
the United States and in Mexico, Costa Rica, and Belize belongs to the same group, whereas ACP
in Brazil belongs to a different group, indicating Brazil belongs to a separate regional HLB/ACP
epidemiological zone from the United States, Mexico, and countries in the Caribbean and Cen-
tral America. Boykin et al. (21) reported that there is no relationship between the United States
and Caribbean introductions of the vector. However, studies of the Las population data support
a relationship among the introductions in the United States, Mexico, and countries in Central
America and the Caribbean. Both Mexico and the United States have haplotype B Las, thus sug-
gesting a separate introduction from haplotype A (116). Within local and area-wide HLB/ACP
epidemiology zones, coordinated HLB/ACP management is being implemented. For the different
citrus-producing areas in the regional HLB/ACP epidemiology zones, HLB/ACP management
is difficult to coordinate but is needed. Quarantine measures have been implemented to limit the
spread of HLB-positive plant materials and ACP from one location to another within the regional
HLB/ACP epidemiology zones.
Plant resistance to Huanglongbing and Asian citrus psyllid. No HLB-resistant commercial

citrus cultivars have been identified to date (55), although tolerance has been observed in trifoliate
orange (Poncirus trifoliata L. Raf.) and some of its hybrids (6). P. trifoliata is a trifoliate species
that is graft compatible with Citrus spp. and is used as a rootstock. In the presence of HLB, well-
defined HLB disease symptoms are absent in trifoliate orange and some of its hybrids (6). Albrecht
et al. (4) compared the metabolomics of whole leaves of Las-infected and healthy greenhouse-
grown seedlings of the tolerant cultivar US-897 Citrus reticulata Cleopatra × Poncirus trifoliata) and
susceptible cultivar Cleopatra mandarin (C. reticulata). The number of metabolites differentially
produced in Las-infected versus uninfected leaves was greatest in the susceptible cultivar Cleopatra
mandarin and lowest in the tolerant cultivar US-897, where few differences in the metabolite
profiles were found. Tolerance to HLB did not appear to be associated with the accumulation
of higher amounts of protective metabolites in response to Las infection (4). Ramadugu et al.
(144) conducted a six-year field trial of various Citrus varieties under natural disease challenge
conditions in an HLB-endemic region in Florida. This study included 65 Citrus accessions and
33 accessions from 20 other closely related genera. On the basis of Las titers and disease symptoms,
the authors have tentatively categorized 2 accessions as immune, 6 as resistant, and 14 as tolerant.
Resistance and tolerance may be attributed to multiple factors, including psyllid colonization
ability and the absence of Las multiplication. Specifically, this study used seedlings, thus avoiding
the possible influence of rootstocks on the HLB response. Many citrus relatives appear to support
the replication of Las for a short period of time before subsequently suppressing them completely.
Indian wood apple (Limonia acidissima) is a transient host of Las (79). Murraya species support
only very low titers of Las and are possible transient hosts of Liberibacters (180). Those citrus
relatives showing HLB resistance/tolerance, such as Eremocitrus and Microcitrus, which are
sexually compatible with citrus, may be useful in future breeding to impart HLB resistance to
cultivated citrus.
Some newly released citrus scion varieties, such as Sugar Belle, have been suggested to display
HLB tolerance (F. Gmitter, personal communication). Tolerance of scions to HLB is affected by
rootstocks. The foliar HLB symptoms of scions on US-897 are fewer than those on many other
20.18 Wang et al.
rootstocks. Scions grafted on Volkamer lemon also show less canopy damage (5). These results
suggest that optimization of rootstock and scion combination is a useful strategy for control of
HLB.
Plant resistance to ACP includes the prevention of spread and multiplication of ACP in a
plant, thus providing an effective, economical, environmentally safe, and sustainable method of
controlling ACP (146). Westbrook (183) surveyed 87 accessions, primarily in the Rutaceae orange
subfamily Aurantioideae, in the field for an abundance of ACP eggs, nymphs, and adults. Very low
abundances of all life stages of ACP were found on two accessions of P. trifoliata. Richardson &
Hall (146) have further tested the resistance of 46 accessions of P. trifoliata to ACP, and observed
no eggs on 36% of the P. trifoliata accessions. P. trifoliata appears to have antixenosis and antibiosis
resistance to ACP.
Enhanced nutrient program. Foliar applications of micronutrients have been widely used in
Florida to mitigate HLB-induced mineral deficiencies and counter the debilitating effects of the
disease. The nutrient program often includes micronutrients combined with SA and/or phos-
phite. The effectiveness of these treatments has been controversial (62, 157, 161). In some studies,
no differences have been observed in tree health, Las titer, and fruit quality and yield between
trees treated with an enhanced nutritional program and control trees (62). In contrast, Shen et al.
(157) reported that the enhanced nutrient program has reduced Las content and increased leaf
size and weight after at least three years of application. A positive effect on citrus fruit yields by
foliar nutrients was also observed (161). However, Xia et al. (187) surveyed nutritional approaches
to HLB management in China and found no consistent evidence to support the nutritional ap-
proach as an effective means of maintaining grove productivity or slowing disease progression in
HLB-diseased plants. The authors suggest, however, that the HLB-diseased trees may remain
productive for several years under a good nutritional program when combined with other cultural
practices such as good irrigation. SAR inducers and other plant defense inducers are regularly
included in such nutrient programs. Spraying of HLB-diseased trees with plant defense inducers,
including β-aminobutyric acid, BTH, and INA, individually or in combination slows Las pop-
ulation growth in planta and decreases HLB disease severity by approximately 15% to 30%, as
compared with the negative control (98). Notably, the application of plant defense inducers via
foliar spraying did not reduce Las titers (98). Overall, these studies have suggested that foliar nutri-
ents promote tree growth in asymptomatic trees or in those planted in citrus groves with poor soil
nutrients at an early stage of infection and that plant defense inducers can confer a moderate level
of disease control but cannot decrease Las titers in infected trees. However, the enhanced nutrient
program appears to have no significant effect on HLB-positive trees at advanced stages of infection.
Thermotherapy. Thermal therapy treatments have been used for decades to prevent or eliminate
diseases in plants (92). Continuous thermal exposure to 40◦ C to 42◦ C for a minimum of 48 h has
been reported to be sufficient to significantly decrease Las titers or eliminate Las entirely in
HLB-affected citrus seedlings (74). Hoffman et al. (74) indicated that heat treatment enhances
the vigor of Las-infected citrus and promotes new root growth. Ehsani and colleagues (45) also
reported a decrease in HLB symptoms after heat treatment of citrus trees in groves. The current
challenge with thermotherapy is that although sufficiently high temperatures may be reached
in the aboveground parts of the plant, killing temperatures are unlikely to be reached in roots
where the heat treatment is buffered by the soil (108). Thus, thermotherapy is unlikely to redue
Las populations in the roots, which then serve as reservoirs for reinfection of the canopy during
flushes. The effectiveness of thermotherapy on reducing Las populations in roots needs to be
improved for it to become a viable component of integrated HLB management.
Replanting. Owing to the loss of trees to HLB in HLB-endemic citrus-producing areas, re-
planting is critical to sustaining citrus production. Certified HLB-free nursery stock is required
for replanting. Younger orchards express symptoms much more quickly than older trees, normally
within 6 to 12 months after planting (61). Aggressive psyllid control on young trees can prevent
psyllid feeding and transfer of Las to the young seedlings (155). In addition, instead of replanting
one-year-old seedlings, planting two- or three-year-old seedlings allows profitable production
sooner after replantation, and the older trees are more tolerant of HLB compared to younger
replantation (G. Zhong, personal communication). Growing HLB-free trees in nurseries now
typically requires the production of citrus within totally enclosed structures to prevent contact
with ACP. This practice has been expanded to include the growth of mature trees. Citrus un-
dercover production systems (CUPSs), which utilize an enclosed structure to grow mature trees
for citrus production (151), are now being used commercially on a small scale in both the United
States and China.
Bactericides. Bactericides, including ampicillin, carbenicillin, cephalexin, oxytetracycline

(OTC), penicillin, rifampicin, streptomycin sulfate, and sulfadimethoxine, have been shown to
be highly effective in eliminating or suppressing Las in infected trees (191). Streptomycin sulfate,
OTC hydrochloride, and OTC calcium complex have been approved for use in controlling HLB
via foliar spray in Florida since 2016. The benefits and efficacy of these bactericides in the Florida
citrus industry via foliar spray remain to be determined.
Trunk injection of different bactericides, including OTC, has some efficacy for HLB control
(78). However, the challenges of trunk injection include potential damage to trees and the lack of a
suitable device for injecting trees at a commercial scale, thus leading to low efficacy and high labor
costs. Optimization of trunk injections and development of an efficient trunk injector are required
before the application of trunk injection of bactericides to control HLB can be considered a viable
commercial disease management tool.
It remains to be determined whether Las will develop resistance against bactericides. The
emergence of bactericide resistance typically results from horizontal gene transfer (HGT) of
mobile genetic elements that carry the resistance genes among co-occurring bacteria (178) or
by mutation of bacterial targets. HGT is widespread among bacteria. For example, tetracycline
resistance genes encoding either efflux or ribosomal protection proteins in many bacteria are
suggested to have been acquired by HGT from the tetracycline-producing streptomycetes (28).
HGT is thought to be less common in intracellular bacteria, including Liberibacters, because
they are sequestered from external microbial populations (117, 175). Several bacterial symbionts,
including Wolbachia spp., Carsonella DC, and “Candidatus Profftella armatura” (124), have been
discovered in the ACP gut and may potentially serve as donors of resistance genes. For Las, only
lysE has been proposed to have been acquired from “Ca. Profftella armatura” via HGT (124).
It remains to be determined whether any of the gut microbes contain genes for resistance to
bactericides such as OTC or streptomycin. Notably, Las contains the ABC transporters Msb1
and MsbA2, which share high identity with E. coli MsbA and have been predicted to confer
multidrug resistance to Las (99, 150). It remains to be determined whether MsbA-like genes may
contribute to the development of resistance to OTC, streptomycin, or other bactericides and
whether long-term use of bactericides will lead to antimicrobial resistance of Las in the future.
Efforts are being made to improve the application of bactericides and identify novel bactericides
to control HLB. Nanoemulsion formulations of ampicillin have been found to eliminate Las
in HLB-diseased citrus in planta more efficiently than non-nanoformulation controls (189). A
structure-based design approach has been implemented to identify small antimicrobial molecules
targeting SecA, an essential component of the Sec machinery (2, 3, 77). In addition, it has also been
20.20 Wang et al.
reported that benzbromarone inhibits LdtR, a Las homolog of the multidrug resistance regulator
MarR (133).
Area-wide Huanglongbing/Asian citrus psyllid management. One successful HLB manage-

ment approach in the United States, China, and Brazil is area-wide HLB/ACP management. As
reported by Bassanezi et al. (14), area-wide management decreases ACP populations compared
with those in groves not under such management, and relatively few immigrant psyllids that car-
ried Las were found in the study. The beginning of the HLB epidemic for a new plantation of
sweet orange under area-wide management was delayed by 10 months. Approximately five years
after planting, the disease incidence for the new plantation under area-wide management was
decreased by 90% compared with controls (14). Successful area-wide HLB/ACP management has
also been reported by Belasque et al. (15).
Genetic improvement of citrus against Huanglongbing. Breeding disease-resistant varieties

is the most efficient and sustainable approach to controlling most plant diseases. Traditional
breeding to generate HLB-resistant citrus varieties has been hindered by a lack of HLB-resistant
germplasm, polyembryony, pollen-ovule sterility, sexual and graft incompatibilities, and extended
juvenility (38). Transgenic approaches have been used to improve citrus resistance against HLB
by overexpressing NPR1 (nonexpressor of pathogenesis related genes 1), a key regulator in the
signal transduction pathway that leads to SAR (43), and overexpression of antimicrobial proteins
(e.g., thionins and plant defensins) (69) or antimicrobial peptides (e.g., cecropin B) (162). Citrus
varieties expressing spinach defensins developed by Erik Mirkov (Texas A&M University) have
been granted an experimental use permit by the US Environmental Protection Agency for field
testing of resistance against HLB. However, the regulatory hurdles to register transgenic citrus and
consumer acceptance of transgenic citrus may present many challenges to the commercialization
of such varieties.
Recent developments in targeted genome-editing technologies, however, have the potential
to facilitate the process to produce genetically modified citrus cultivars that lack transgenes (86).
Targeted genome-editing technologies based on zinc finger nucleases, transcription activator-like
effector nucleases, or CRISPR/Cas9–single guide RNA (sgRNA) (hereafter, Cas9-sgRNA) provide
a promising path to improve crops, including generating disease-resistant plants. Cas9-sgRNA
technology has been successfully used for genome editing in many plants (127, 156). The citrus
genome has been successfully modified by using Cas9-sgRNA (84, 85). Importantly, genome
editing via CRISPR technology has been used to generate disease-resistant citrus. Specifically,
the promoter and coding regions of the disease susceptibility gene CsLOB1, which favors citrus
bacterial canker disease, caused by Xanthomonas citri, have been modified to generate canker-
resistant plants (84, 86).
Nontransgenic approaches that generate plants without introducing foreign genes into the
genome are likely to have wider acceptance and an easier regulatory pathway. Nontransgenic,
foreign DNA–free, genome-modified plants have been generated by transfecting pre-assembled
complexes of purified Cas9 protein and sgRNA into plant protoplasts of Arabidopsis thaliana,
tobacco, lettuce, and rice to achieve targeted mutagenesis in regenerated plants at frequencies of
up to 46% (184). Nontransgenic wheat plants have also been generated with CRISPR technology
by regeneration of plants from callus cells transiently expressing CRISPR/Cas9 introduced as DNA
or RNA (193). Nontransgenic, genome-edited plants made using Cas9-sgRNA may provide an
unprecedented opportunity to generate HLB-resistant/tolerant citrus varieties.
Zebra Chip Disease Management

The control strategy of choice for managing ZC remains application of a rotating collection of
chemical insecticides, including imidacloprid, abamectin, spiromesifen, and spinosad. However,
insecticides can have difficulties in reaching their target because psyllids spend most of their time
within the plant canopy on the underside of leaves. Moreover, the efficacy of application may
vary depending on agronomical practices and growing conditions (139). The heavy usage and
dependence on insecticides have led to the development of resistance by psyllids to imidacloprid
(106), and, more recently, resistance to abamectin was discovered in the San Luis Potosı́ vicinity in
Mexico (27). Insect resistance to pesticides can lead to larger insect populations, with consequences
for greater disease incidence and the spread of disease to new crops. Although management sys-
tems, as currently practiced in commercial potato production fields, have been successful in man-
aging psyllid populations, effective rotation of insecticides with different chemistries, regionally
coordinated pesticide applications, and use of materials and practices that are friendlier to bene-
ficial arthropods are needed to extend the useful life of existing pesticides. Future strategies also
must take into account the growing evidence for the role of persistent, resident psyllid popula-
tions in areas where they were not previously thought to overwinter (75, 119, 121, 177) as well
as differences in the relative abundance, physiology, and behavior of insect haplotypes (107, 122,
123, 165). Importantly, field studies clearly demonstrate that plants remain susceptible to ZC until
harvest and that vine kill following infection does not protect tubers (149).
An additional complicating factor in the long-term management of ZC is the observation that
potato psyllids may carry and transmit one or both of Lso A and B to the host. Recent studies
reveal life-stage-specific effects of different Lso haplotypes on the fitness of the vector (190). These
observations have implications for inoculum dosage (24) as well as field-scale epidemiology. Both
Lso haplotypes individually kill potato plants, although the relationship between the severity of ZC
symptoms and Lso haplotype is unclear. Symptoms in Lso A–infected tomato are typically milder
(reduced growth) than symptoms in Lso B–infected tomato, which frequently displays dramatic
symptoms leading to plant death (C. Tamborindeguy, personal communication; E. Tienebo,
J. Levy, and E. Pierson, unpublished results).
Breeding for resistance to Lso, the psyllid vector, or both remains the most promising long-
term strategy for managing Lso-associated diseases. However, to date no resistance to ZC in any
commercial breeding lines has been identified. Several wild solanaceous species have been identi-
fied that show resistance to insect feeding. For example, Solanum habrochaites, a wild solanaceous
plant that is both antixenosis (repellent) and toxic to the whitefly Bemisia tabaci, was evaluated
for its potential to similarly affect B. cockerelli (97). Despite these effects, in choice studies with
Solanum lycopersicum at least 50% of the S. habrochaites plants tested positive for Lso, indicating
this resistance is not sufficient to protect solanaceous plants against Lso infection. Additionally,
unique germplasm has been derived from wild potato species with Solanum tuberosum, Solanum
berthaultii, Solanum raphanifolium, Solanum chacoense, and Solanum guerreroense in their pedigree
(110). Some of these exhibited antixenosis to B. cockerelli, and some displayed no Lso infection in
no-choice transmission assays. Further testing and selection for agronomical traits acceptable to
the industry are required.
In addition, phage-based therapies targeting the pathogen are promising approaches (12).
Strategies for genetic modifications incorporating disease resistance in potato may eventually
benefit from research in the more commercially valuable citrus crops. Finally, gene-silencing
strategies such as RNA interference and CRISPR-based gene editing may provide ways to control
insect populations or modulate host response to Lso infection. For example, Wuriyanghan et al.
(186) recently demonstrated that double-stranded RNAs and siRNAs, when expressed in the gut of
20.22 Wang et al.
B. cockerelli via injection or oral acquisition, were able to induce mortality in the recipient psyllids.
Ongoing investigations to identify genes that are important for maintaining endosymbionts (81)
may facilitate efforts to reduce Lso infection in psyllids.
CONCLUDING REMARKS
Liberibacters and their insect vectors are expanding their distribution worldwide, which represents
an intensifying global threat to the production of citrus and many other crops. Importantly,
weather-driven physiologically based demographic models predict that Egypt, Israel, Cyprus,
Sicily, Southern Spain, and Portugal are at great risk for ACP establishment and the spread of
HLB (64). Spatial models also predict that southern Australia is at risk of HLB introduction in
the future (11). Both models predict that climates in large areas of Africa, Latin America, and
North Australia are well suited to ACP and Las expansion (125). Additionally, recent findings
confirm the suspicion that psyllids may overwinter in colder conditions than initially predicted
and are using weeds as green bridges between seasons, possibly explaining recent expansions in
the geographic range of ZC in the United States and New Zealand (75, 119, 121, 177). Europe is
one of the major potato-producing areas in the world currently free of ZC. There is concern that
Lso haplotypes found in Europe, but not currently infecting potato, can or will jump to potato
via a psyllid vector utilizing potato as a host. Significant progress has been made in understanding
the pathogenesis mechanisms of Liberibacters, which will continue to be useful in the design of
effective disease management strategies. Currently, potato production is managed by aggressive
psyllid control. Citrus production in the presence of HLB is also possible if the most promising
management approaches are integrated. Meanwhile, new technologies such as CRISPR/Cas9 may
provide an unprecedented opportunity to provide long-term management solutions. There are
many similarities in the pathogenicity mechanisms of different Liberibacters. Thus, we believe
that there is much that can be learned by working together across commodities and disciplines to
tackle these scientifically intriguing yet economically destructive crop diseases.
DISCLOSURE STATEMENT
The authors are not aware of any affiliations, memberships, funding, or financial holdings that
might be perceived as affecting the objectivity of this review.
ACKNOWLEDGMENTS
We thank Drs. Dennis Gross, Leland Pierson, Charlie Rush, and Cecilia Tamborindeguy for
valuable discussions. This work has been supported by Florida Citrus Research and Development
Foundation, USDA-APHIS MAC program, USDA-SCRI, USDA-Specialty Block Grant, and
Florida Citrus Initiative program.
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The Candidatus Liberibacter-Host Interface: Insights Into Pathogenesis Mechanisms and Disease Control

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PY55CH20-Wang ARI 13 June 2017 13:36

Review in Advance first posted online

still occur before final publication

João Carlos Setubal,3 Jin Xu,1 Julien G. Levy,2

Annu. Rev. Phytopathol. 2017. 55:20.1–20.32 Keywords

20.2 Wang et al.

Table 1 Diseases caused by Liberibacters, their hosts, and distributions

(LafCl) (T. erytreae)

www.annualreviews.org • Liberibacter Pathogenesis Mechanisms and Disease Control 20.3

Abbreviations: HLB, Huanglongbing; ZC, Zebra chip.

20.4 Wang et al.

DISEASES CAUSED BY LIBERIBACTERS AND WORLDWIDE

www.annualreviews.org • Liberibacter Pathogenesis Mechanisms and Disease Control 20.5

Agrobacterium tumefaciens (D14500.1)

20.6 Wang et al.

Zebra Chip and Other Diseases Caused by “Candidatus Liberibacter

as the causative agent of ZC based on graft transmission (35, 152).

LIBERIBACTERS IN PLANTA: CLUES TO UNDERSTANDING

www.annualreviews.org • Liberibacter Pathogenesis Mechanisms and Disease Control 20.7

20.8 Wang et al.

GENOMIC FEATURES OF LIBERIBACTERS

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PATHOGENESIS MECHANISMS OF LIBERIBACTERS

Virulence Traits of Liberibacters

20.10 Wang et al.

Autotransporters β-barrel proteins OM vesicles

IM YajC SecF SecD SecYEG YidC

www.annualreviews.org • Liberibacter Pathogenesis Mechanisms and Disease Control 20.11

Lipopolysaccharides. Lipopolysaccharides (LPSs), which are composed of lipid A, an oligosac-

20.12 Wang et al.

www.annualreviews.org • Liberibacter Pathogenesis Mechanisms and Disease Control 20.13

Mechanistic View of Liberibacter Pathogenicity

20.14 Wang et al.

Water RFO Symplastic

Some putative effectors

In addition, Liberibacters cause disease symptoms by eliciting self-destructive plant responses

20.16 Wang et al.

Huanglongbing Disease Management

Huanglongbing/Asian citrus psyllid epidemiological zone. Based on the frequency of ACP

www.annualreviews.org • Liberibacter Pathogenesis Mechanisms and Disease Control 20.17

Plant resistance to Huanglongbing and Asian citrus psyllid. No HLB-resistant commercial

20.18 Wang et al.

www.annualreviews.org • Liberibacter Pathogenesis Mechanisms and Disease Control 20.19

Bactericides. Bactericides, including ampicillin, carbenicillin, cephalexin, oxytetracycline

20.20 Wang et al.

Area-wide Huanglongbing/Asian citrus psyllid management. One successful HLB manage-

Genetic improvement of citrus against Huanglongbing. Breeding disease-resistant varieties

www.annualreviews.org • Liberibacter Pathogenesis Mechanisms and Disease Control 20.21

Zebra Chip Disease Management

20.22 Wang et al.

www.annualreviews.org • Liberibacter Pathogenesis Mechanisms and Disease Control 20.23

20.24 Wang et al.

www.annualreviews.org • Liberibacter Pathogenesis Mechanisms and Disease Control 20.25

20.26 Wang et al.

www.annualreviews.org • Liberibacter Pathogenesis Mechanisms and Disease Control 20.27

20.28 Wang et al.

Europe. J. Econ. Entomol. 103(4):1060–70

www.annualreviews.org • Liberibacter Pathogenesis Mechanisms and Disease Control 20.29

20.30 Wang et al.

www.annualreviews.org • Liberibacter Pathogenesis Mechanisms and Disease Control 20.31

20.32 Wang et al.

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