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

2016 ◽  
Vol 106 (5) ◽  
pp. 452-458 ◽  
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.

scholarly journals Improvement in the Excised Citrus Leaf Assay to Investigate Inoculation of ‘Candidatus Liberibacter asiaticus’ by the Asian Citrus Psyllid Diaphorina citri

Plant Disease ◽  
2017 ◽  
Vol 101 (3) ◽  
pp. 409-413 ◽  
Author(s):  
Laudecir L. Raiol-Junior ◽  
Ana D. B. Baia ◽  
Fernanda Q. B. F. Luiz ◽  
Camila G. Fassini ◽  
Viviani V. Marques ◽  
...  
Keyword(s):  
Quantitative Polymerase Chain Reaction ◽  
Leaf Age ◽  
Infection Process ◽  
Diaphorina Citri ◽  
Candidatus Liberibacter Asiaticus ◽  
Leaf Petiole ◽  
Candidatus Liberibacter ◽  
Liberibacter Asiaticus ◽  
Citrus Leaves ◽  
Time Required

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.

scholarly journals Inoculation Period and Citrus Host Effect Establishment of New Infections of ‘Candidatus Liberibacter asiaticus’ Transmitted via Vegetative Grafting

Plant Disease ◽  
2020 ◽  
Vol 104 (7) ◽  
pp. 1894-1899
Author(s):  
Mark E. Hilf ◽  
Weiqi Luo
Keyword(s):  
Survival Analysis ◽  
Bacterial Species ◽  
Systemic Infection ◽  
Candidatus Liberibacter Asiaticus ◽  
Susceptibility To Infection ◽  
Successful Infection ◽  
Significant Difference ◽  
Candidatus Liberibacter ◽  
Liberibacter Asiaticus ◽  
Pathogen Dna

Vegetative grafting is a common method of transmitting and propagating ‘Candidatus Liberibacter asiaticus’, the bacterial species accepted as the causal agent of the citrus disease huanglongbing (HLB). Generally, infected tissue that is grafted to a receptor tree remains in place indefinitely to ensure transmission. In this study, individual HLB-symptomatic leaves were grafted as ‘Ca. L. asiaticus’ inoculum sources to receptor trees of six citrus types and removed after an inoculation period (IP) of 21, 51, or 81 days. The goal was to assess the effect of varying IPs on transmission of bacteria to the receptor tree and on the successful establishment of a new infection. Survival analysis of data from three trials showed a significantly reduced proportion of infected trees with an IP of 21 days compared with IPs of 51 and 81 days but that there was no significant difference in the proportion of infected trees between IPs of 51 and 81 days. In addition, the time to first detection of pathogen DNA in an infected tree was delayed significantly for the 21-day IP when compared with the 51- and 81-day IPs. Survival analysis showed that the probability of infection of sweet orange trees was significantly higher than for trees of five other citrus types throughout the experiment. There was no significant difference between the infection probabilities of these latter five citrus types. The data from this study show that successful infection by grafting is dependent upon the time of exposure to the inoculum, that shorter IPs increase the time needed to establish a systemic infection, and that citrus types vary in their overall susceptibility to infection by ‘Ca. L. asiaticus’.

Maternal Contribution of Candidatus Liberibacter asiaticus to Asian Citrus Psyllid (Hemiptera: Liviidae) Nymphs Through Oviposition Site Inoculation and Transovarial Transmission

2019 ◽  
Author(s):  
Alicia J Kelley ◽  
Kirsten S Pelz-Stelinski
Keyword(s):  
Vertical Transmission ◽  
Horizontal Transmission ◽  
Infected Plant ◽  
Bacterial Pathogen ◽  
Oviposition Site ◽  
Asian Citrus Psyllid ◽  
Candidatus Liberibacter Asiaticus ◽  
Infected Adult ◽  
Candidatus Liberibacter ◽  
Liberibacter Asiaticus

Abstract Asian citrus psyllid, Diaphorina citri Kuwayama (Hemiptera: Liviidae), transmits Candidatus Liberibacter asiaticus (Las), the bacterial pathogen putatively responsible for citrus huanglongbing. Multiple studies have shown psyllids acquire Las more frequently, and are more likely to inoculate susceptible plants, when they acquire Las as nymphs. Understanding the transmission of Las to nymphs is critical to the Las lifecycle. The objective of this study was to determine the transmission Las by female D. citri to their offspring. Two transmission pathways were quantified: horizontal transmission (acquisition of Las via feeding at the oviposition site) and vertical transmission (transovarial). Eggs of individual, infected females were transferred to an uninfected seedling to assess vertical transmission. In a second experiment, horizontal transmission was evaluated by replacing eggs laid by infected females with uninfected nymphs. Nymphs exposed to Las via horizontal transmission of the oviposition site were more likely to acquire Las than from vertical transmission. Las deposited in flush by an infected adult female feeding during oviposition was sufficient for infecting nymphs. Combined results of both experiments suggest that vertical transmission allows Las to spread in low amounts even when infected plant hosts are not available and that inoculation of the oviposition site provides a source of Las to developing nymphs via the plant phloem. These data support the hypothesis that transmission through infected plant material via maternal inoculation is a primary pathway of Las transmission between vector and host.

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

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.

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

2020 ◽  
Vol 45 (6) ◽  
pp. 646-657 ◽  
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

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

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. 

scholarly journals The Asian Citrus Psyllid Host Murraya koenigii Is Immune to Citrus Huanglongbing Pathogen ‘Candidatus Liberibacter asiaticus’

2018 ◽  
Vol 108 (9) ◽  
pp. 1089-1094 ◽  
Author(s):  
Vitor H. Beloti ◽  
Gustavo R. Alves ◽  
Helvécio D. Coletta-Filho ◽  
Pedro T. Yamamoto
Keyword(s):  
Infected Plant ◽  
Asian Citrus Psyllid ◽  
Test Plant ◽  
Insect Vector ◽  
Candidatus Liberibacter Asiaticus ◽  
Murraya Koenigii ◽  
Trap Crop ◽  
Candidatus Liberibacter ◽  
Citrus Huanglongbing ◽  
Liberibacter Asiaticus

The Asian citrus psyllid (ACP) Diaphorina citri, vector of ‘Candidatus Liberibacter asiaticus’ (CLas), the putative causal agent of citrus Huanglongbing (HLB), is controlled by application of insecticides, which, although effective, has resulted in serious biological imbalances. New management tools are needed, and the technique known as “trap crop” has been attracting attention. A potential plant for use as a trap crop in the management of the ACP is Murraya koenigii (curry leaf). However, for this plant to be used in the field, it needs to be attractive for the vector and must not harbor CLas. To verify the potential of curry leaf as trap crop for the management of HLB, we investigated the ability of D. citri to transmit CLas to M. koenigii, and to other test plants, including M. paniculata (orange jasmine) and cultivar Valencia sweet-orange seedlings. For the tests, the insects were reared on a symptomatic CLas-infected plant and allowed to feed on the three test plant species. The overall maximum transmission rate for the citrus seedlings was 83.3%, and for orange jasmine was 33.3%. Successful transmission of CLas by ACP to the curry-leaf seedlings was not observed, and it was treated as immune to CLas. Supported by the previous results that M. koenigii is attractive for ACP, these results indicate that curry leaf is an excellent candidate for use as a trap crop, to improve the management of the insect vector and consequently of HLB.

scholarly journals Effect of Adjuvants on Oxytetracycline Uptake upon Foliar Application in Citrus

Antibiotics ◽  
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.

Sign in / Sign up

Export Citation Format

Share Document