Huanglongbing: Pathogen detection system for ...

Journal of the Saudi Society of Agricultural Sciences (2016) 15, 1–11

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

a

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 Available online 13 May 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 field which are based on several approaches such as the presence of a reliable pathogen detection system, control over vector populations, cultural practices, chemotherapy and finally 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. ª 2014 King Saud University. Production and hosting by Elsevier B.V. All rights reserved.

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: [email protected] (Y. Iftikhar). Peer review under responsibility of King Saud University.

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Production and hosting by Elsevier 1658-077X ª 2014 King Saud University. Production and hosting by Elsevier B.V. All rights reserved. http://dx.doi.org/10.1016/j.jssas.2014.04.006

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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 30–100% in yield losses depending upon the severity of the disease. It takes 2–5 years for a tree to become unproductive from the first appearance of the symptoms and the total life span of the tree is reduced to 7–10 years, although significant 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 deficiency 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 identified (Table 1).

6 6 7 7 7 8 8 9

disease. Two species, Diaphorina citri and Trioza erytreae, are known as vectors of specific 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 specific 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 October–January. 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 identified 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 asiaticus’ ‘Candidatus Liberibacter solanacearum’ Candidatus Liberibacter psyllaurous Candidatus Liberibacter asiaticus

Pathogen detection System for Integrated disease management Table 2

3

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 flushes, 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 flush 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 Se´tamou et al. (2008) – 34 grapefruit trees (Citrus paradisi Macfad.) significantly 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 identified Setamou (2010) subunit I gene (433 bp) which were grouped into two: hp1–8 were identified in South America (group 1) and hp9–23 were identified 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 identification 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 amplification (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 florescence resonance energy transfer (FRET). Xiaolan et al. (2004) noted many benefits of this technique including high speed, sensitivity, and specificity and stable reproduction of the results. The pathogen detection system can enhance the efficiency of the disease control system for the production of disease-free citrus plants. Nageswara-Rao et al. (2013a,b) designed digoxigenin labeled probe specific to the Ca. L. asiaticus’a. 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 reflectance of diseased and healthy foliage and found a significant difference in values at 560 and 710 nm. Similarly, Pourreza et al. (2013) obtained images at 591 nm and identified 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 specific 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

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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, specificity and reproducibility of the results Quantitative detection Two step PCR using the specific-specific outer and inner primer TaqMan Average reflectance of diseased and healthy plant was significantly different 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 amplification (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.

amplification products. For instance Gouda et al. (2006) compared the results of three primers (A, B and C). These primers produced an amplification 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 specific primers across the genome of Ca. Liberibacter asiaticus for the detection of disease. Das et al. (2013) used primer–probe combination of HLBas-HLBr-HLBp for the detection of pathogen associated specifically 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 identified new bacterial strains (Table 5). For instance, new bacterial strain now known as Brazilian (L. americanus) was identified 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) Xiaolan et al. (2004) Xiaolan et al. (2004) Subandiyah et al. (2000)

Magomere et al. (2009)

Pathogen detection System for Integrated disease management Table 5

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 different 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 different 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 significant impact on yield and fruit size (Joubert and Stassen, 2000). Shoot tip grafting along with pre-heat treatment also reduced the disease with 100% efficiency (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

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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), hoverflies (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 rufilabris (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 differentiation 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) identified 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 artificial inoculums of disease. Identified 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 artificial selection on few economically important traits such as yield and

Pathogen detection System for Integrated disease management Table 7

7

Research highlights of the utilization of Nano-particles to cure the huanglongbing diseases.

Nano particle

Treatment

References

Multifunctional silica based nano particle gel

Effective for treating and preventing the spread of citrus canker, citrus greening and vector Asian citrus psyllid. The chemical incorporates Dimethyl disulfide (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 beneficial 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 benefits. 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 field crops, identified 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 specific 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 profile of a species produced under disease inoculums and stress free environment may be compared and any RNA molecule produced specifically 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 field conditions, screening citrus germplasm for disease resistance is a tedious job. Citrus breeders have to expose the material to inoculum through artificial 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) Tolerant––little or no symptoms Carrizo citrange (X Citroncirus webberi J. Ingram & H. E. Moore) + Tolerant––little or no chlorosis; Severinia buxifolia (Poiret) Ten. + Tolerant––no 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 field 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)

8

Y. Iftikhar et al. Table 9

Micro array and microchip analyses to identify relevant genes with huanglongbing.

Array/Gene

Results

References

Affymetrix 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 identified 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 differentially expressed 14 selected genes were found to be relevant with huanglongbing HLB infection significantly affected 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

Albercht and Bowman (2008)

Affymetrix GeneChip

Micro array

Agilent GeneChip Array

Spinch Defensin

symptoms, which may take many weeks. Alternatively, molecular markers such as SNP’s 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 amplified polymorphic sequences to discover SNPs in 30 accessions. A total of 3348 SNPs were identified. 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. Identified 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

Albercht and Bowman (2008)

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 field and biological control of the vector may be preferred to control the vector population cheaply and effectively. Secondly, Management practices would include the identification 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 identified by utilization of micro-array technology.

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