Callose deposition in the phloem plasmodesmata and inhibition of phloem transport in citrus leaves infected with “Candidatus Liberibacter asiaticus”

Huanglongbing (HLB) is a difficult-to-control and highly destructive citrus disease that, in Brazil, is associated mainly with the bacterium ‘Candidatus Liberibacter asiaticus’ transmitted by the psyllid Diaphorina citri. The aim of this study was to improve our understanding of the ‘Ca. L. asiaticus’ infection process by exposing excised, fully expanded, immature citrus leaves in 50-ml Falcon tubes to one, four, or eight adults from a ‘Ca. L. asiaticus’-exposed colony for 1-, 3-, 7-, or 15-day periods for access to inoculation (IAP). The leaves were incubated at 26°C for 1, 3, 7, 15, and 21 days (incubation period [IP]). Infection frequencies and ‘Ca. L. asiaticus’ titers were assessed by quantitative polymerase chain reaction (qPCR). ‘Ca. L. asiaticus’ infection was a function of leaf age, number of insects, IAP, and IP. In general, higher infection rates were observed on younger leaves inoculated with higher numbers of insects and after longer IAP and IP. The immature excised leaf method allowed determination of 3 to 7 days as the range of time required by ‘Ca. L. asiaticus’ to reach qPCR detectable levels. Even though leaf survival could be prolonged by the maintenance of a branch segment at the base of the leaf petiole, leaf degradation, visible after about 15 days IP, did not allow observation of the entire infection process which, in the intact plant, culminates with the appearance of the blotch mottling symptom on leaf blades.

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Comparative Transcriptional and Anatomical Analyses of Tolerant Rough Lemon and Susceptible Sweet Orange in Response to ‘Candidatus Liberibacter asiaticus’ Infection

Molecular Plant-Microbe Interactions ◽

10.1094/mpmi-06-12-0150-r ◽

2012 ◽

Vol 25 (11) ◽

pp. 1396-1407 ◽

Cited By ~ 45

Author(s):

Jing Fan ◽

Chunxian Chen ◽

Qibin Yu ◽

Abeer Khalaf ◽

Diann S. Achor ◽

...

Keyword(s):

Sweet Orange ◽

Phloem Transport ◽

Differential Sensitivity ◽

Transcriptional Analysis ◽

Candidatus Liberibacter Asiaticus ◽

Transport Activity ◽

Phloem Cell ◽

Rough Lemon ◽

Candidatus Liberibacter ◽

Liberibacter Asiaticus

Although there are no known sources of genetic resistance, some Citrus spp. are reportedly tolerant to huanglongbing (HLB), presumably caused by ‘Candidatus Liberibacter asiaticus’. Time-course transcriptional analysis of tolerant rough lemon (Citrus jambhiri) and susceptible sweet orange (C. sinensis) in response to ‘Ca. L. asiaticus’ infection showed more genes differentially expressed in HLB-affected rough lemon than sweet orange at early stages but substantially fewer at late time points, possibly a critical factor underlying differences in sensitivity to ‘Ca. L. asiaticus’. Pathway analysis revealed that stress responses were distinctively modulated in rough lemon and sweet orange. Although microscopic changes (e.g., callose deposition in sieve elements and phloem cell collapse) were found in both infected species, remarkably, phloem transport activity in midribs of source leaves in rough lemon was much less affected by HLB than in sweet orange. The difference in phloem cell transport activities is also implicated in the differential sensitivity to HLB between the two species. The results potentially lead to identification of key genes and the genetic mechanism in rough lemon to restrain disease development and maintain (or recover) phloem transport activity. These potential candidate genes may be used for improving citrus tolerance (or even resistance) to HLB by genetic engineering.

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Detection of Liberibacter asiaticus causing Citrus Vein Phloem Degeneration from Siam Citrus leaves (Citrus nobilis var. microcarpa) in Singkawang City plantation, Pontianak, West Kalimantan

Microbiology Indonesia ◽

10.5454/mi.14.3.2 ◽

2020 ◽

Vol 14 (3) ◽

pp. 2

Author(s):

RAHMAWATI RAHMAWATI ◽

ILIANA ILIANA ◽

AGUS RACHMAT ◽

LATIFFAH ZAKARIA ◽

MUKARLINA MUKARLINA

Keyword(s):

16S Rrna ◽

Protein Gene ◽

Ribosomal Protein Gene ◽

Rrna Gene ◽

Candidatus Liberibacter Asiaticus ◽

Specific Primers ◽

West Kalimantan ◽

Candidatus Liberibacter ◽

Liberibacter Asiaticus ◽

Citrus Leaves

The objective of the present study was to detect presence of pathogenic fastidious bacterium, Candidatus Liberibacter asiaticus using PCR from leaves of Siam citrus showing Citrus Vein Phloem Degeneration (CVPD) symptoms in Singkawang City plantation, Pontianak, West Kalimantan, Indonesia. Citrus leaf samples were collected based on visual observation of symptoms showing CVPD infection. Typical symptoms of CVPD include leaf yellowing (chlorosis), vein banding, leaves become stiff, thicker and smaller in size. The pathogenic bacterium, Candidatus Liberibacter asiaticus was detected using two specific primers, namely, OI1/ OI2c amplified 16S rRNA gene and A2/J5, amplified ribosomal protein gene of the rplKAJL-rpoBC operon (β-operon). PCR amplification detected the presence of 1100 bp band using OI1/ OI2c primers, and 703 bp band using A2/J5 primers from symptomatic Siam citrus leaves. PCR products were not detected from healthy plants serve as control. By using two sets of specific primers to amplify 16S rRNA gene and ribosomal protein gene, Candidatus Liberibacter asiaticus was detected in symptomatic Siam citrus leaves in Singkawang City, Pontianak, Indonesia. Detection of the bacterial pathogen causing CVPD is important to prevent spreading of the disease which could affect production of citrus fruits.

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Physiologic, Anatomic, and Gene Expression Changes in Citrus sunki, Poncirus trifoliata, and Their Hybrids After ‘Candidatus Liberibacter asiaticus’ Infection

Phytopathology ◽

10.1094/phyto-02-16-0077-r ◽

2017 ◽

Vol 107 (5) ◽

pp. 590-599 ◽

Cited By ~ 15

Author(s):

Leonardo Pires Boava ◽

Mariângela Cristofani-Yaly ◽

Marcos Antonio Machado

Keyword(s):

Poncirus Trifoliata ◽

Starch Accumulation ◽

Candidatus Liberibacter Asiaticus ◽

Callose Deposition ◽

Expression Of Genes ◽

Hybrid Plants ◽

Distinct Disease ◽

Candidatus Liberibacter ◽

Liberibacter Asiaticus ◽

Lower Titer

Huanglongbing (HLB) is a destructive disease of citrus caused by phloem-limited bacteria, namely ‘Candidatus Liberibacter asiaticus’ (Las), ‘Candidatus Liberibacter africanus’, and ‘Candidatus Liberibacter americanus’. Although there are no known HLB-resistant citrus species, studies have reported Poncirus trifoliata as being more tolerant. Assuming that callose deposition in the phloem of infected plants can inhibit translocation of photosynthetic products and cause starch accumulation, we compared callose deposition in petioles and starch accumulation in infected leaves of three genotypes (Citrus sinensis, C. sunki, and P. trifoliata) and 15 hybrids (C. sunki × P. trifoliata). Compared with the mock-inoculated plants, higher bacterial counts and greater accumulation of callose and starch were found in C. sinensis, C. sunki, and 10 of the hybrid plants. Lower titer and fewer metabolic changes due to Las infection were observed in P. trifoliata and in two Las-positive hybrids while three hybrids were Las-negative. Callose accumulation was linked to and correlated with genes involved in phloem functionality and starch accumulation was linked to up-regulation of genes involved in starch biosynthesis and repression of those related to starch breakdown. Lower expression of genes involved in phloem functionality in resistant and tolerant plants can partially explain the absence of distinct disease symptoms associated with starch accumulation that are usually observed in HLB-susceptible genotypes.

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Transmission and Propagation of ‘Candidatus Liberibacter asiaticus’ by Grafting with Individual Citrus Leaves

Phytopathology ◽

10.1094/phyto-09-15-0221-r ◽

2016 ◽

Vol 106 (5) ◽

pp. 452-458 ◽

Cited By ~ 6

Author(s):

Mark E. Hilf ◽

Reid S. Lewis

Keyword(s):

Infected Plant ◽

Systemic Infection ◽

Scale Model ◽

Small Scale ◽

Candidatus Liberibacter Asiaticus ◽

Leaf Petiole ◽

Candidatus Liberibacter ◽

Decline Disease ◽

Liberibacter Asiaticus ◽

Citrus Leaves

Huanglongbing (HLB) is a chronic, progressive decline disease in citrus associated with a systemic infection by the bacterium ‘Candidatus Liberibacter asiaticus’. Transmission of the bacterium in the field is by the Asian citrus psyllid, Diaphorina citri Kuwayama. Experimental propagation of ‘Ca. L. asiaticus’ is done primarily by grafting pieces of bud wood from an infected plant. To produce a small-scale model system for investigation of pathogen biology, we investigated grafting single leaves from infected citrus plants as sources of inoculum for propagation of the bacterium. In total, 162 plants ranging in age from 3 to 18 months were grafted. Grafting with intact asymptomatic and HLB-symptomatic leaves resulted in 61 of 78 (78%) and 35 of 41 (85%) of the plants infected with ‘Ca. L. asiaticus’, respectively. Inoculum consisting of the leaf petiole only or only an inoculum tissue remnant under the bark of the receptor tree resulted in 6 of 12 (50%) and 7 of 31 (23%) infected trees, respectively. Real-time polymerase chain reaction (qPCR) assays verified the infection in plants, a majority of which developed the foliar blotchy mottle symptom considered diagnostic for HLB, while some plants also displayed the stunted, chlorotic shoots for which the disease is named. The qPCR data together with the symptoms displayed demonstrated that individual leaves from infected trees can serve as effective inoculum sources for transmission and propagation of ‘Ca. L. asiaticus’ via grafting.

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‘Candidatus Liberibacter asiaticus’ putative effectors: in silico analysis and gene expression in citrus leaves displaying distinct huanglongbing symptoms

Tropical Plant Pathology ◽

10.1007/s40858-020-00382-5 ◽

2020 ◽

Vol 45 (6) ◽

pp. 646-657 ◽

Cited By ~ 1

Author(s):

Laís Moreira Granato ◽

Tiago S. Oliveira ◽

Raquel L. Boscariol-Camargo ◽

Diogo M. Galdeano ◽

Nicholas V. Silva ◽

...

Keyword(s):

Gene Expression ◽

In Silico ◽

In Silico Analysis ◽

Candidatus Liberibacter Asiaticus ◽

Candidatus Liberibacter ◽

Liberibacter Asiaticus ◽

Citrus Leaves ◽

Silico Analysis

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Deteksi dan Evaluasi Keragaman Genetika Candidatus Liberibacter asiaticus sebagai Penyebab Penyakit Huanglongbing di Indonesia Berdasarkan Gen β-operon

Jurnal Fitopatologi Indonesia ◽

10.14692/jfi.12.5.168 ◽

2017 ◽

Vol 12 (5) ◽

pp. 168

Author(s):

Muhammad Rizal ◽

Kikin Hamzah Mutaqin ◽

Gede Suastika

Keyword(s):

Dna Amplification ◽

Amino Acid Sequences ◽

Nucleotide Polymorphisms ◽

Candidatus Liberibacter Asiaticus ◽

Single Nucleotide ◽

Central Java ◽

Ctab Method ◽

Candidatus Liberibacter ◽

Liberibacter Asiaticus ◽

Citrus Leaves

Huanglongbing also known in Indonesia as citrus vein phloem degeneration (CVPD) is a devastating disease in citrus plantation worldwide, especially in Asia, Africa, and America. In Asian countries including Indonesia, Candidatus Liberibacter asiaticus (CLas) has been confirmed as the causal agent of huanglongbing disease on citrus. Distribution of CLas in Indonesia has been reported in West Borneo, East Nusa Tenggara, Bali, Yogyakarta, Central Java and East Java. The purpose of this study was to detect CLas in several Indonesia’s citrus plantations that has not and has been reported previously and to study its genetic diversity and their relationship. DNA of plant samples, i.e. citrus leaves, was extracted using CTAB method and CLas was amplified using PCR with the A2/J5 primer pair. DNA amplification results showed that infection of CLas was positively detected from samples from Bogor and Cibodas (West Java), Tuban and Jember (East Java), as well as Katung, Bayung Gede, Kerta, and Pancasari (Bali). Alignment of nucleotide sequences from positive samples showed that their ribosomal protein β-operon has high similiarity to that of CLas OK901 originated from Okinawa (Japan). Isolates of CLas originated from Bogor, Cibodas, Tuban, Jember, and Katung have been known to be identical to other CLas of Indonesian origins reported earlier. Isolates of CLas originated from Bayung Gede, Pancasari, and Kerta have single nucleotide polymorphisms at 6 points of bases of the 539 total bases compared in their conservative regions, although only 3 of the 6 bases could affect their amino acid sequences.

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Effect of Adjuvants on Oxytetracycline Uptake upon Foliar Application in Citrus

Antibiotics ◽

10.3390/antibiotics9100677 ◽

2020 ◽

Vol 9 (10) ◽

pp. 677

Author(s):

Nabil Killiny ◽

Faraj Hijaz ◽

Pedro Gonzalez-Blanco ◽

Shelley E. Jones ◽

Myrtho O. Pierre ◽

...

Keyword(s):

Foliar Application ◽

Foliar Spray ◽

Bacterial Pathogen ◽

Water Addition ◽

Candidatus Liberibacter ◽

Citrus Huanglongbing ◽

Liberibacter Asiaticus ◽

Trunk Injection ◽

Citrus Leaves ◽

Target Effects

Recently in Florida, foliar treatments using products with the antibiotics oxytetracycline and streptomycin have been approved for the treatment of citrus Huanglongbing (HLB), which is caused by the putative bacterial pathogen ‘Candidatus Liberibacter asiaticus’. Herein, we assessed the levels of oxytetracycline and ‘Ca. L. asiaticus’ titers in citrus trees upon foliar applications with and without a variety of commercial penetrant adjuvants and upon trunk injection. The level of oxytetracycline in citrus leaves was measured using an oxytetracycline ELISA kit and ‘Ca. L. asiaticus’ titer was measured using quantitative PCR. Low levels of oxytetracycline were taken up by citrus leaves after foliar sprays of oxytetracycline in water. Addition of various adjuvants to the oxytetracycline solution showed minimal effects on its uptake by citrus leaves. The level of oxytetracycline in leaves from trunk-injected trees was higher than those treated with all foliar applications. The titer of ‘Ca. L. asiaticus’ in the midrib of leaves from trees receiving oxytetracycline by foliar application was not affected after four days and thirty days of application, whereas the titer was significantly reduced in oxytetracycline-injected trees thirty days after treatment. Investigation of citrus leaves using microscopy showed that they are covered by a thick lipidized cuticle. Perforation of citrus leaf cuticle with a laser significantly increased the uptake of oxytetracycline, decreasing the titer of ‘Ca. L. asiaticus’ in citrus leaves upon foliar application. Taken together, our findings indicate that trunk injection is more efficient than foliar spray even after the use of adjuvants. Our conclusion could help in setting useful recommendations for the application of oxytetracycline in citrus to improve tree health, minimize the amount of applied antibiotic, reduce environmental exposure, and limit off-target effects.

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