Huanglongbing - Pathogen Detection System For Integrated Disease Management

Journal of the Saudi Society of Agricultural Sciences (2014) xxx, xxxxxx
King Saud University
Journal of the Saudi Society of Agricultural Sciences

www.ksu.edu.sa
www.sciencedirect.com
REVIEW ARTICLE
Huanglongbing: Pathogen detection system

for integrated disease management A review
Yasir Iftikhar
a
b
c
a,*
, Saeed Rauf b, Umbreen Shahzad c, Muhammad Awais Zahid
Department of Plant Pathology, University College of Agriculture, University of Sargodha, Pakistan

Department of Plant Breeding and Genetics, University College of Agriculture, University of Sargodha, Pakistan
Institution of Horticultural Sciences, University of Agriculture, Faisalabad, Pakistan
Received 15 January 2014; revised 14 April 2014; accepted 22 April 2014
KEYWORDS
Huanglongbing;
Breeding;
Genetic diversity;
Pathogen detection system;
Management
Abstract Huanglongbing (HLB) is a major threat to citrus sustainable yield and production.
Therefore, various strategies are discussed in this review to provide solutions for the control of
the disease. These include phyto-sanitory techniques to reduce pathogen inoculum in the eld which
are based on several approaches such as the presence of a reliable pathogen detection system, control over vector populations, cultural practices, chemotherapy and nally the production of diseasefree propagating material. In addition to phytosanitory techniques, efforts to introduce resistant
genes into cultivatable germplasm are also needed and are thus also discussed in this review.
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Contents
1.
2.
3.
Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
Disease vector: population dynamics . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
Pathogen detection systems . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
3.1. Variability of results due to primer, probes and enzymes . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
3.2. Bacterial gene sequences/genetic diversity . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
4. Management practices to control the pathogen . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
4.1. Cultural Practices . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
4.2. Biological control of the vector population . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
4.3. Chemotherapy . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
* Corresponding author. Tel.: +92 3009686405; fax:
483703665.
E-mail address: yasiriftikhar@uos.edu.pk (Y. Iftikhar).
Peer review under responsibility of King Saud University.
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1658-077X 2014 King Saud University & Saudi Society of Agricultural Sciences. Production and hosting by Elsevier B.V. All rights reserved.
http://dx.doi.org/10.1016/j.jssas.2014.04.006
Please cite this article in press as: Iftikhar, Y. et al., Huanglongbing: Pathogen detection system for integrated disease management A review.
Journal of the Saudi Society of Agricultural Sciences (2014), http://dx.doi.org/10.1016/j.jssas.2014.04.006
Y. Iftikhar et al.
4.4. Breeding for huanglongbing disease resistance . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

4.4.1. Genetic variation for resistance . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
4.5. Molecular breeding . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
4.5.1. Reverse genetics: a strategy for disease resistance breeding . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
4.5.2. Single nucleotide polymorphism (SNP): a way to screen germplasm and segregating generations . . . . . . . . . .
4.5.3. Genetic transformation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
5. Conclusion . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
References. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
1. Introduction
Diseases caused by different pathogens like fungi, prokaryotes,
nematodes, viroids, viruses and probable viruses, are one of
the most potential factors to shrink the yield from citrus
groves. Among them citrus greening, a prokaryotic disease,
is one of the devastating diseases prevailing in citrus orchards
all over the world (Batool et al., 2007). This disease has been
extensively reviewed by many scientists (Batool et al., 2007;
Bove, 2006; Graca, 1991). Gottwald (2010) reviewed the epidemiological understanding of huanglongbing and showed that
pathogen co-evolved as insect endo-symbiont which later on
also moved to the plants. It was also showed that disease vector can transmit it to a very long distance. On average, the disease can cause 30100% in yield losses depending upon the
severity of the disease. It takes 25 years for a tree to become
unproductive from the rst appearance of the symptoms and
the total life span of the tree is reduced to 710 years, although
signicant quantitative data on fruit yield and quality reduction due to this disease are absent.
HLB has been established itself in more than 40 countries
(Brlansky, 2007). Disease is caused by the fastidious Gram-negative uncultivable bacterium belonging to the a-subdivision of
the phylum Proteobacteria (Garnier et al., 1984; Jagoueix
et al., 1994). Three major strains of this bacterium, Asiaticus,
Africanus and Americanus have been differentiated on the basis
of environmental conditions and insect vector (Coletta et al.,
2004; Garnier et al., 2000). Symptomology of the disease has
been elaborated by many scientists (Graca, 1991; Bove, 2006
and Batool et al., 2007). Typical disease symptoms include small
and upright leaves and chlorotic mottling, Zn deciency symptoms, severe vein yellowing and greening of mature fruits (Das,
2004). The pathogen has not restricted itself to Citrus species.
Several other hosts have also been identied (Table 1).
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disease. Two species, Diaphorina citri and Trioza erytreae, are

known as vectors of specic strains such as Asiaticus, Americanus and Africanus of bacterial inoculum, respectively. These
species can be differentiated on the basis of their sensitivity to
temperature. Transmission of the pathogen has been described
and reviewed in detail by Manjunath et al. (2008). Pathogen
population inside the host tree releases specic volatile chemical
methyl salicylate which attracts the vector population to feed on
the infected tree and thus pathogen is also injected in the vector
(Mann et al., 2012). Methyl salicylate based attractant for vector has been formulated for commercial exploitation (Stelinski
et al., 2013, US patent 13/774,112).
The pathogen was found in both nymph and adult stages
but was absent in the eggs or in offspring produced by infected
females. The insect vector may carry the pathogen for 12 weeks
(Hung et al., 2004). On the other hand, Das et al. (2002) noted
the presence of an abundant vector population during
February through April, being lower during OctoberJanuary.
Factors like temperature and humidity, weeds and cultural
practices also affect the vector population. Higher temperature
and saturation are negatively related to the growth of the African psyllid population (Tamesse and Messi, 2004). They also
observed that adult psyllids showed variable infection on three
hosts i.e. 100% in lime, 97% in lemon and 76% in mandarin.
Vector population control was considered key in the management of disease. Grafton-Cardwell et al. (2013) reviewed the
management strategies for the control of pest and showed
the chemical control as primary management strategy of
insect. However it was shown that chemical control induced
the resistance against the insecticides. Therefore integrated
strategies for the management of disease were recommended.
Host and environmental factors effects the spatial distribution of the vector, therefore DNA based diversity and seasonal
abundance in the various populations of vector has been summarized in Table 2.
2. Disease vector: population dynamics

3. Pathogen detection systems
HLB is transmitted through different means; infected branches,
cascuta and insect vector in nature. Citrus psylla has been identied the most potent insect vector for the transmission of the
Table 1
Traditionally, HLB is scored on the basis of variable disease

symptoms. However, HLB is a fastidious organism that can
List of alternative host of Candidatus Liberibacter.
Pathogen
Alternative hosts
Location
References
Candidatus Liberibacter asiaticus
Cleome rutidosperma, Pisonia aculeate,

Trichostigma octandrum
Murraya paniculata
Tomato and Pepper
Tomato and Potato
Wampee (Clausena lansium Skeels)
Jamica
Brown et al. (2011)
Florida
New Zealand
California
China
Damsteegt et al. (2010)

Liefting et al. (2009)
Hansen et al. (2008)
Deng et al. (2007)

Candidatus Liberibacter solanacearum
Candidatus Liberibacter psyllaurous
Pathogen detection System for Integrated disease management

Table 2
Genetic diversity in vector population of huanglongbing disease.
References
Markers
Population
Diversity estimate/Seasonal abundance
Tsai et al. (2002)
Psyllid population levels were positively

correlated with new shoot ushes, weekly
minimum temperature and rainfall. Natural
enemies did not regulate the population
Boykin et al. (2007)
12 SSR
Diaphorina citri Kuwayama,

population was studied weekly in
two orange jasmine
[Murraya paniculata (L.) Jack] plots
in southern Florida
288 individuals from Florida,
Texas, and Brazil
Young irrigated grapefruit trees and
mature no irrigated orange trees
Heterozygosities ranged from 0.014 to 0.569

and from 0.052 to 0.653
Hall et al. (2008)
26.5, 16.8, and 0.27 eggs, nymphs, and adults

per ush shoot, respectively, in the young
grapefruit trees while 16.0, 12.7, and 0.31 for
mature orange trees
D. citri eggs, nymphs, and adults were
Setamou et al. (2008)
34 grapefruit trees (Citrus paradisi Macfad.)
signicantly higher on sweet orange than on
and 6 sweet orange trees (Citrus sinensis (L.)
grapefruit
Hunter et al. (2009) 636 cDNA sequences
Gene data will provide information regarding
physiological processes of vector
Boykin et al. (2012) 821 bp portion of the 212 individuals from 52 collections representing Eight haplotype DCit1-Dcit8 belonging to
mtCOI gene
15 countries
south eastern asia to south western asia
De Leon and
Cytochrome oxidase
22 populations of Diaphorina citri Kuwayama Twenty-three haplotypes (hp) were identied
Setamou (2010)
subunit I gene (433 bp)
which were grouped into two: hp18 were
identied in South America (group 1) and
hp923 were identied in North America and
Hawaii (group 2). Hp1 and 9 were the most
frequent
Two separate introductions of citrus psyllid in
American continent
live in the host for many years under masked conditions.

Therefore, the presence of sophisticated technology that allows
early and rapid identication of the pathogen is a pre-requisite.
A summary of the advantages and disadvantages of various
techniques is presented in Table 3. Sdoodee and Garnett
(1994) developed polyclonal antibodies against African strains
of the bacterium which could detect infection in infected
branches but were unable to do so in apparently healthylooking branches. Alternatively, a PCR-based detection system has provided a powerful solution for early and quick
detection of the pathogen. Freedom of its dependence from
morphological symptoms, concentration of bacteria within
host and cheaper to apply are further advantages over traditional technologies. Das (2004) compared the PCR method
with the conventional method of biological indexing of HLB.
The PCR-based method provided several advantages such as
rapid detection while the whole procedure could be completed
within 6 h and could overcome the problem of low and uneven
distribution of pathogen within the host.
Several PCR methods and protocols have been employed
for the detection of HLB. Comparative performance of two
PCR protocols i.e. long and standard showed that the long
PCR protocol was more consistent and sensitive than the standard protocol. The long PCR protocol included another DNA
polymerase for proof reading. A number of studies have indicated that multiplex PCR faithfully detected HLB in host tissues (Baranwal et al., 2005). Loop-mediated isothermal
amplication (LAMP) was also found to be effective for detection of HLB (Okuda et al., 2005). Real time PCR is another
powerful tool for the detection of HLB. It can identify and
quantify the concentration of HLB with a high degree of sensitivity. In comparison with other traditional PCR methods,
real time PCR is the most effective for determination of pathogen concentration within the host because of its ability to
indicate pathogenicity in the sample at early stages. It is based
on the principle of the TaqMan probe, which relies on orescence resonance energy transfer (FRET). Xiaolan et al. (2004)
noted many benets of this technique including high speed,
sensitivity, and specicity and stable reproduction of the
results. The pathogen detection system can enhance the
efciency of the disease control system for the production of
disease-free citrus plants. Nageswara-Rao et al. (2013a,b)
designed digoxigenin labeled probe specic to the Ca. L. asiaticusa. L. asiaticuso for the simple, fast, sensitive and nonradioactive detection of the pathogen.
Use of imaging techniques for the detection of disease was
reported by Sankaran et al. (2013). They measured the reectance of diseased and healthy foliage and found a signicant
difference in values at 560 and 710 nm. Similarly, Pourreza
et al. (2013) obtained images at 591 nm and identied diseased
leaves with 100% accuracy. Li et al. (2013) have proposed
extended spectral angle mapping (ESAM) for the detection
and translocation of disease.
3.1. Variability of results due to primer, probes and enzymes
Gene specic primer pairs for polymerase chain reaction based
detection of disease have been investigated (Nageswara-Rao
et al., 2013a). Primers OII and O12c designed from 16S rDNA
sequences are used globally to detect pathogen in the host tissue (Teixeira et al., 2005; Gouda et al., 2006). Das (2004) used
these primers to amplify the band of 1160 bp. Several other
primers have also been used in various studies giving variable
Y. Iftikhar et al.
Table 3
Properties of various systems for the detection of pathogens.
Detection system
Properties
References
Symptomology
Able to score disease on the

basis of visible sample
Unable to do so in apparently healthy
looking plant material
Time consuming but requires low
bacterial concentration. Requires highly skilled labor
Bos (1999);
Batool et al. (2007)
Requires high and even concentration

of pathogen in infected material
Rapid and Qualitative detection
Low concentration of Pathogen may be detected
Detection of low copies of bacterial genome
Nylon membrane for resolution instead
of gel-electrophoresis
High speed, sensitivity, specicity and
reproducibility of the results
Quantitative detection
Two step PCR using the specic-specic
outer and inner primer TaqMan
Average reectance of diseased and healthy
plant was signicantly dierent at 560 and 710 nm infrared
Las bacterial cells were extracted from mid rib
of the infested plant which were suitable for the highly sensitive direct PCR
Ratana and Garnett (1994)
Biological indexing
(propagation and
insect transmission)
ELISA*/Electron-microscopy
Traditional PCR
Loop-mediated isothermal
amplication (LAMP)
Real-time PCR*
Ultrasensitive detection system

Visible, Infrared and thermal
imaging techniques
Direct sensitive PCR
*
McClean and Oberholzer

(1965), Graca (1991),
Das (2004),
Baranwal et al. (2005)
Okuda et al. (2005)
Xiaolan et al. (2004)
Lin et al. (2010)

Sankaran et al. (2013)
Fujikawa et al. (2013)
Polymerase chain reaction, enzyme linked immunosorbent assay.
amplication products. For instance Gouda et al. (2006) compared the results of three primers (A, B and C). These primers
produced an amplication band of 1160, 703 and 451 bp with
two enzymes (Taq and Klen Taq Polymerase). It was concluded that Primer C and Klen Taq polymerase enzyme were
more effective in detecting HLB pathogen. Nageswara-Rao
et al. (2013b) successfully developed 32 gene specic primers
across the genome of Ca. Liberibacter asiaticus for the detection of disease. Das et al. (2013) used primerprobe combination of HLBas-HLBr-HLBp for the detection of pathogen
associated specically with HLB.
3.2. Bacterial gene sequences/genetic diversity
Various pathogen genes have been extensively used to detect
the presence of pathogen in host plant (Tables 4 and 5). Occasional mutation result changes in the base sequences of these
genes. These variations have been detected in various studies
Table 4
and were used to identify new strains of bacterium. On the

basis of nucleotide diversity in conserved sequences of various
genes dendograms were constructed which identied new
bacterial strains (Table 5). For instance, new bacterial strain
now known as Brazilian (L. americanus) was identied and
proposed as new species. On the basis of serological studies
and sequence analysis of 16S rDNA, intergenic 16/23S r
DNA ribosomal protein gene it was found that sub-species
(L. africanus sub. capensis) existed in South Africa. Samples
of bacterium collected from various regions of Japan, Philippine, Indonesia and Thailand showed 100% sequence homology for gene 16S rRNA to that Nepalese bacterium and
98.8% of Indian and 97.5% of African races (Subandiyah
et al., 2000). It was concluded that these isolates were similar
to Indian and Nepalese strain but distinct from African.
Uncharacterized regions of the nusG-rp/KAS-rpo B gene
cluster of causal organism obtained from Japan and Indonesia
were sequenced which showed only differences for 3
Summary of base homology for gene 16S rRNA and 23S rRNA in various bacterial strains.
Gene
Comparison
Homology %
References
16S rRNA
23S rRNA
L. americanus vs. asiaticus or africanus

L. americanus vs. asiaticus
L. americanus vs. africanus
L. africanus vs. asiaticus
Chinese vs. L. asiaticus
Chinese vs. L. africanus
L. africanus subspecies capensis vs. L. africanus
Far East vs. Nepalese
Far East vs. India
Far East vs. African
African vs. Kenya
Asian vs. Kenya
96
66
79.5
98.4
99.8
98.7
98.2
100
98.8
97.5
84
50
Teixeira et al. (2005)
16S rRNA
16SrRNA
16Sr RNA/23 sRNA
16Sr RNA
L10 and L12 rDNA
Teixeira et al. (2005)

Subandiyah et al. (2000)
Magomere et al. (2009)

Table 5
Genetic diversity in pathogen as revealed through molecular markers.
Pathogen
Polymorphism
Conclusion
References
Candidatus
Liberibacter asiaticus
16S ribosomal DNA

(rDNA),16S/23S intergenic
spacer regions; the outer
membrane protein (omp)
gene region; the trmU-tufB-secEnusG-rplKAJL-rpoB
Omp-based PCR (RFLP)
Pathogen diversity was not correlated

with hosts (mandarin or pomelo). Indonesian
isolates clustered together
Tomimura et al. (2009)
Pathogen contains several dierent variants

within particular regions
Japanese isolates comprise at least two
distinct genotypes
Bastianel et al. (2005)
One unique genetic group is dominant

around Okinawa Main Island, whereas several
dierent are commonly distributed
around islands near Taiwan
Genetic diversity with asiaticus populations
was higher in Asia than America. Indian
and Florida isolates were genetically
distinct while east southern asia
and Brazilian isolates were similar.
There were three founder haplotypes which
were progenitor of the population worldwide
Presence of new genetic linage
of Ca. Liberibacter
asiaticus in Indian subcontinent
Furuya et al. (2010)
Ca. Liberibacter
asiaticus
Candidatus
Liberibacter
asiaticus
Candidatus
Liberibacter asiaticus
Bacteriophagetype DNA
polymerase region (DNA pol)
1168-nucleotide sequence of the
wserA-trmU-tufB-secE-nusGrplKAJL-rpoB gene cluster
Ca. L. asiaticus
7 micro-satellite
Ca. Liberibacter
asiaticus
SNPS based on 16S rRNA

and b-operon ribosomal
protein (b-rp) gene
nucleotides when they were compared. Diversity analysis is

important to detect the hot spot for the evolution of new races
of pathogen, summary of the results is presented in Table 5.
4. Management practices to control the pathogen
Huanglongbing requires integrated management program for
the control with the combination of plant pathologist, horticulturist and breeders as shown in Fig. 1 and 2.
Tomimura et al. (2009)
Islam et al. (2012)
Adkar-Purushothama
et al. (2009)
Binh and Lam (2004) introduced the term intensive farming, which includes pruning, insect protection, and fertilizer
application to improve growth and production in infected
plants. These tree husbandry practices increased crop production by 57.6%. The continuous foliar application of micronutrients such as (ZnSO4 + MnSO4 (3: 1)) for 7 weeks was also
effective to induce growth and production in infected trees
(Nguyen and Nguyen, 2004).
4.2. Biological control of the vector population
4.1. Cultural Practices

Horticultural and agronomic practices have also been shown
to regulate the intensity of symptoms. Hand selective pruning
during summer (January) reduced the incidence of the disease,
along with a positive and signicant impact on yield and fruit
size (Joubert and Stassen, 2000). Shoot tip grafting along with
pre-heat treatment also reduced the disease with 100% efciency (Ruilin et al., 1990).
Vector populations have also been known to be regulated by

natural enemies such as wasp (Tamarixia radiate), and fungal
mycelia (Paecilomyces fumosoroseus and Hirsutella citriformis)
(Qureshi et al., 2009). In France, vector populations were controlled through discharge of 4600 parasites (T. radiata) in an
island on an experimental basis. The presence of a pathogen
(Liberbacter asiaticus) within a vector parasite (Tamarixia radiata) showed that this parasite was free from pathogens (Hoy
et al., 2001). Similarly, in Indonesia, possible use of fungal
mycelia to control the disease was also investigated
(Subandiyah et al., 2000). Result summary of various biological controls of the vector has been given in Table 6.
4.3. Chemotherapy
Figure 1
Phyto-sanitory strategies against Huanglongbing.
Use of various types of antibiotics has also been recommended

as part of disease management even though the chemotherapy
is not new. Martinez et al. (1970) indicated the suppression of
disease symptoms through various antibiotics. Nariana et al.
(1975) exposed tree branches by various antibiotics i.e. achromycin and ledermycin at the concentration of 500 PPM which
effectively suppressed the disease symptoms. Shokrollah et al.
(2011) compared various treatment effect on control of
Y. Iftikhar et al.
Figure 2
Table 6
Disease resistance strategies against Huanglongbing (HLB).
Biological control of targeted vector population.
Predator/Parasite
Results
Tamarixia radiata; Coccinellid species,

Olla v-nigrum; Harmonia axyridis;
hunting spiders (Aranae: Anyphaenidae,
Clubionidae, Oxyopidae, and Salticidae),
lacewings (Neuroptera: Chrysopidae, Hemerobiidae),
hoveries (Diptera: Syrphidae), and
predatory bugs (Hemiptera: Anthocoridae)
Harmonia axyridis (Pallas), Olla v-nigrum
(Mulsant), Cycloneda sanguine L.,
and Exochomus childreni (Mulsant)
Lacewings, Ceraeochrysa sp. Chrysoperla
rulabris (Burmeister), a spider,
Hibana velox (Becker),
parasitoid Tamarixia radiata (Waterston)
Isaria fumosorosea
Olla v-nigrum; Harmonia axyridis

Asian citrus psyllid Michaud (2002)
caused the highest mortality of the
vector and completed their life cycle
exclusively on the diet of Asian citrus psyllid
Tamarixia triozae (Burks)

Tamarixia radiata
Tamarixia radiata
Targeted vector
References
All the predator and parasites

contributed to the mortality of the vector
Asian citrus psyllid Michaud (2004)
Fungus caused disease in vector population
Diaphorina citri
Meyer et al. (2008)
Kuwayama
Asian citrus psyllid De Leon and
Setamou (2010)
No genetic dierentiation in both

species as observed through inter
genomic sequence repeats
Vietnam and Pakistani colonies were
distinct from other Asian colonies
diseases and it was noted that antibiotic (Oxi-tetracycline)

+2 g/L) + GA3 (15 mg/L) and antibiotic (2 g/L) + GA3
(15 mg/L) + foliar fertilizer (20 ml/4 L) were most effective
in improving fruit yield and quality. Zhang et al. (2008) used
integrated approaches i.e. micro-propagation and antibiotics
(pencillin @50mgL 1) to reduce the incidences of disease.
Zhang et al. (2010) identied two chemical agents, penicillin
G sodium and 2,2-dibromo-3-nitrilopropionamide (DBNPA),
to be effective at eliminating or suppressing the Ca. L. asiaticus bacterium. Zhang et al. (2011, 2012) also demonstrated
that the combination of penicillin and streptomycin (PS) was
effective in eliminating or suppressing the Ca. L. asiaticus.
Application of the PS via trunk injection or root soaking also
eliminated or suppressed the Ca. L. asiaticus bacterium in the
HLB-affected citrus plants. Wang and Valkonen (2009) used a
novel cryo-preservation method in liquid nitrogen for the
eradication of pathogens which eliminated the virus, phytoplasmas and bacteria. Some research highlight for the
Asian citrus psyllid Barr et al. (2009)
utilization of nano-particle technology to cure the disease

has been shown in Table 7. These nano-particles were intended
to block the production of essential amino-acids within
pathogen.
4.4. Breeding for huanglongbing disease resistance
4.4.1. Genetic variation for resistance
Presence of adequate amount of genetic variation within citrus
cultivated or wild germplasm is pre-requisite while breeding for
disease tolerance or resistance. This genetic variation may be
uncovered by exposing the species to natural or articial
inoculums of disease. Identied sources of resistance may be
incorporated by conventional means of breeding or advanced
molecular techniques (Table 8). Cultivated germplasm usually
has a lower degree of genetic diversity due to continued articial
selection on few economically important traits such as yield and

Table 7
Research highlights of the utilization of Nano-particles to cure the huanglongbing diseases.
Nano particle
Treatment
References
Multifunctional silica
based nano particle gel
Eective for treating and preventing the spread of

citrus canker, citrus greening and vector Asian citrus
psyllid. The chemical incorporates Dimethyl disulde
(DMDS) with copper in a multifunctional silica nanogel
Screening showed that 17 and 4 compounds inhibit the
protein production by 50% and 65%, respectively
Santra (2011)
5 compounds were found to inhibit the SecA protein

of pathogens at nano-level
Akula et al. (2011)
Trade mark is special formulation of benecial and

essential nutrient that suppresses bacterial plant diseases
Humber (2011)
Novel inhibitory compounds

against SecA protein of
Ca. L. asiaticus
In-silico screening of
compounds from ZINC
database
Peak formulation
quality (Coletta et al., 1998; Breto et al., 2001). It may happen

that some valuable sources of resistance may be washed out
during this selection. Self-pollination in some species used as
rootstocks explains their low heterozygosity values. However,
occasional variation due to mutations in the allelic base
sequence alleles may result in evolution of new alleles having
economic benets. Breto et al. (2001) indicated the substitution
of older alleles because of the origin of new alleles in citrus population. They also showed that current citrus varieties aroused
through selection for spontaneous mutation rather than
through hybridization or direct breeding program.
4.5. Molecular breeding
4.5.1. Reverse genetics: a strategy for disease resistance
breeding
Conventionally in eld crops, identied source of resistance is
incorporated in the economically desirable germplasm through
backcrosses. Back cross is a tedious technique in which F1
(Resistance susceptible) is backcrossed to susceptible species
Table 8
Akula et al. (2011)
many times i.e. up to six times. This type of strategy may not
be applicable in the long-lived species such as citrus (Talon and
Gmitter, 2008). Reverse genetics provide an opportunity to
identify a specic transcript that is involved in the resistance
against disease through their expression (Dixon, 2001;
Forment et al., 2005). Resistance alleles may be forced to
express by exposing the species to heavy bacterial inoculums
(Kiedrowski et al., 1992). Afterward, RNA prole of a species
produced under disease inoculums and stress free environment
may be compared and any RNA molecule produced specically under the disease stress environment may be picked
and converted to cDNA molecule. Summary of the studies carried out to determine the differential expression of gene has
been shown in Table 9.
4.5.2. Single nucleotide polymorphism (SNP): a way to screen
germplasm and segregating generations
Under eld conditions, screening citrus germplasm for disease
resistance is a tedious job. Citrus breeders have to expose the
material to inoculum through articial means and wait for
Inter and Intra species variation for huanglongbing resistance.
References
Conclusion
Koizumi et al. (1993)
Sweet orange were most susceptible. Many mandarins including Fairchild, Murcott, Kinnow, Clementine,
Fremont, Ponkan, King, and Som-keo-wan were moderately tolerant. Queen mandarin, Avon Ever Bearing
(calamondin) and rough lemon grew well with mild symptoms. Ladu mandarin and Som-pan mandarin
showed the most resistance, with good growth, few symptoms and yielding healthy fruit
Persian lime (C. aurantifolia (Christm.) Swingle) Tolerantlittle or no symptoms
Carrizo citrange (X Citroncirus webberi J. Ingram & H. E. Moore)
+ Tolerantlittle or no chlorosis; Severinia buxifolia (Poiret) Ten.
+ Tolerantno distinct symptoms
US-897 (hybrid of trifoliate orange and Cleopatra mandarin
(C. reticulata Blanco) seedlings declared (PCR)-positive for the pathogen but exhibited a superior
performance compared with Cleopatra mandarin seedlings, which displayed severe disease symptoms soon
after inoculation. The superior performance of US-897 plants in greenhouse and eld locations suggests
tolerance of this genotype to Ca. L. asiaticus
Temple tangor showed the most consistently low incidence of HLB symptoms and CLas titer; in contrast,
Murcott tangor and Minneola tangelo had the highest incidence of HLB symptoms and highest CLas titer
Seedlings of M. paniculata, and C. grandis showed no HLB symptoms six months after inoculation period.
They also showed negative results by polymerase chain reaction (PCR) test
Embryo rescue of seed from healthy chimera sections of fruits. Two resistant plants were isolated
Sweet orange, grapefruit and rootstock cultivars were genetically transformed using several natural and
synthetic antibacterial genes as well as SAR inducing genes
Poncirus trifoliata hybrids (most resistant to HLB), Citrus maxima and hybrids (susceptible to both diseases)
No symptoms of HLB when C. grandis was used as rootstock with
C. hystrix as the interstock. high rate of disease severity was observed when C. aurantium was used as
rootstock and C. aurantifolia as the interstock
Folimonova et al. (2009)
Albercht and Bowman (2008)
Stover and Mccollum (2011)

Ahmad et al. (2011)
van Vuuren and Manicom (2009)
Dutt et al. (2011)
Stover and Mccollum (2011)
Shokrollah et al. (2011)
Y. Iftikhar et al.
Table 9
Micro array and microchip analyses to identify relevant genes with huanglongbing.
Array/Gene
Results
References
Aymetrix GeneChip
Tolerant US-897 (Citrus reticulata Blanco Poncirus trifoliata L. Raf.)

and susceptible Cleopatra mandarin (C. reticulata) transcript analysis
after infection at seedling level was carried out through microarray
analysis which identied 326 genes which were 4-fold upregulated in the
susceptible genotype, while 17 genes were upregulated in tolerant cultivar
More than eight hundred genes were expressed at much higher levels in
US-897 independent of infection with Las. Among these, genes for a
constitutive disease resistance protein (CDR1) were notable
33,000 probe sets on the microarray detected 21,067 genes which expressed
in the leaves. 794 were dierentially expressed
14 selected genes were found to be relevant with huanglongbing
HLB infection signicantly aected expression of 624 genes. The genes
were associated with sugar metabolism, plant defense, phytohormone, and
cell wall metabolism
A microarray experiment was designed to compare gene expression
response of two citrus cultivars to Liberibacter infection, sweet orange (C.
sinensis) that is extremely susceptible and rough lemon (C. jambhiri), a
more tolerant variety
Genetic transformation with spinach defensin protein provided broad
spectrum resistance against bacteria and fungus
Aymetrix GeneChip
Micro array
Agilent GeneChip Array
Spinch Defensin
symptoms, which may take many weeks. Alternatively, molecular markers such as SNPs provide a powerful technique to
discriminate species on the basis of disease resistance without
exposing them to natural environment (Hayashi et al., 2004).
It has been noted that SNPs are the most abundant sequence
variations in plants (Jiang et al., 2010). They used cleaved
amplied polymorphic sequences to discover SNPs in 30 accessions. A total of 3348 SNPs were identied. The basis of the
polymorphism was transition, transversion and indels which
occurred at the frequency of 47.9%, 36% and 16%, respectively. Possibility of using SNPs to discriminate accessions
for molecular marker was also undertaken which showed that
SNPs could be applied in citrus genetic research and breeding.
4.5.2.1. Probe designing. Identied and sequenced molecules of
resistance and susceptible alleles may also be converted into
probes and can be used to screen genotypes. Probes may be
designed by using the conserved regions having single nucleotide polymorphism so that designed probes may differ for each
other at one or few bases only. In all cases probes are labeled
with a radioactive source. Similar strategy was also used by Bo
and Yong (2010).
4.5.3. Genetic transformation
Genetic transformation has also been proposed as a tool to
induce resistance against the diseases. In order to provide resistance against the huanglongbing disease, transgenes are incorporated with objective to reduce survival, growth and virulence
of the pathogen as well as they are detrimental for the vector
citrus psyllid. Anti microbial peptides have been the focus of
the studies to induce resistance against the disease. For
instance spinach defensins have also been reported to be active
at 20 lM against gram negative and gram positive bacterial
pathogens as well as against fungi (Segura et al., 1998). Protein
defensins are small cystein rich molecules and none of the protein belonging to this group has been shown to be toxic or
allergenic. Genes transcribing the spinach defensins have been
Kim et al. (2009)
Khalaf et al. (2010)
Mirkov et al. (2010)
incorporated in the citrus species to induce resistance against

bacteria and fungi (Mirkov et al., 2010). Transgenes under
evaluation for the induction of resistance against citrus greening can be broadly described as antimicrobial genes (i.e. Attacin E, LIMA) or genes conferring systemic acquired resistance
(SAR). This type of gene activates immune system of the plant.
SAR genes include SABP2 (Salicylic acid binding protein 2)
and NRP2 (non-expresser of PR genes from Arabidopsis).
NRP1 genes mediate the salicylic acid induced expression of
pathogenesis related genes. Grosser et al. (2010) transformed
the citrus species with anti-microbial genes (LIMA and ATTE)
and showed that HLB pathogen was undetermined through qPCR when transgenic plants were grafted with sweet orange
infected budwood.
5. Conclusion
A comprehensive review of the literature showed that control
of the disease may be carried out at three steps. Firstly, control
of the vector population to reduce the spread of pathogen
inoculums in the eld and biological control of the vector
may be preferred to control the vector population cheaply
and effectively. Secondly, Management practices would
include the identication of infected plants, removal of
infected branches, and development of disease free citrus propagating material through utilization of suitable pathogen
detection system. Furthermore, infected plants may be cured
through chemotherapies, nano-particles that have the properties to block the important amino-acid translation within pathogen or supply of better nutrition package that may enhance
growth and production. Third step is to control the disease
including the development of disease resistant propagating
material. Inter species and intra species variation for disease
resistance has been shown among the citrus species and either
tolerant species may be chosen for propagation or they may be
the source of resistance genes. These resistance genes may be
identied by utilization of micro-array technology.

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Huanglongbing - Pathogen Detection System For Integrated Disease Management

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Journal of the Saudi Society of Agricultural Sciences (2014) xxx, xxxxxx

King Saud University

Journal of the Saudi Society of Agricultural Sciences

Huanglongbing: Pathogen detection system

, Saeed Rauf b, Umbreen Shahzad c, Muhammad Awais Zahid

Department of Plant Pathology, University College of Agriculture, University of Sargodha, Pakistan

Received 15 January 2014; revised 14 April 2014; accepted 22 April 2014

Production and hosting by Elsevier

4.4. Breeding for huanglongbing disease resistance . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

disease. Two species, Diaphorina citri and Trioza erytreae, are

2. Disease vector: population dynamics

Traditionally, HLB is scored on the basis of variable disease

List of alternative host of Candidatus Liberibacter.

Candidatus Liberibacter asiaticus

Cleome rutidosperma, Pisonia aculeate,

Brown et al. (2011)

Damsteegt et al. (2010)

Candidatus Liberibacter asiaticus

Pathogen detection System for Integrated disease management

Genetic diversity in vector population of huanglongbing disease.

Diversity estimate/Seasonal abundance

Tsai et al. (2002)

Psyllid population levels were positively

Boykin et al. (2007)

Diaphorina citri Kuwayama,

Heterozygosities ranged from 0.014 to 0.569

26.5, 16.8, and 0.27 eggs, nymphs, and adults

live in the host for many years under masked conditions.

Properties of various systems for the detection of pathogens.

Able to score disease on the

Requires high and even concentration

Ratana and Garnett (1994)

Ultrasensitive detection system

McClean and Oberholzer

Xiaolan et al. (2004)

Lin et al. (2010)

Polymerase chain reaction, enzyme linked immunosorbent assay.

and were used to identify new strains of bacterium. On the

L. americanus vs. asiaticus or africanus

Teixeira et al. (2005)

L10 and L12 rDNA

Teixeira et al. (2005)

Magomere et al. (2009)

Pathogen detection System for Integrated disease management

Genetic diversity in pathogen as revealed through molecular markers.

16S ribosomal DNA

Pathogen diversity was not correlated

Tomimura et al. (2009)

Pathogen contains several dierent variants

Bastianel et al. (2005)

One unique genetic group is dominant

Furuya et al. (2010)

SNPS based on 16S rRNA

nucleotides when they were compared. Diversity analysis is

Tomimura et al. (2009)

Islam et al. (2012)

4.1. Cultural Practices

Vector populations have also been known to be regulated by

Phyto-sanitory strategies against Huanglongbing.

Use of various types of antibiotics has also been recommended

Disease resistance strategies against Huanglongbing (HLB).

Biological control of targeted vector population.