scholarly journals Nanopore Sequencing as a Surveillance Tool for Plant Pathogens in Plant and Insect Tissues

Plant Disease ◽  
2018 ◽  
Vol 102 (8) ◽  
pp. 1648-1652 ◽  
Author(s):  
Aline Bronzato Badial ◽  
Diana Sherman ◽  
Andrew Stone ◽  
Anagha Gopakumar ◽  
Victoria Wilson ◽  
...  
Keyword(s):  
Plant Pathogens ◽  
Massively Parallel Sequencing ◽  
High Titer ◽  
Treatment Options ◽  
Plum Pox Virus ◽  
Specific Strain ◽  
Candidatus Liberibacter ◽  
Liberibacter Asiaticus ◽  
Whole Transcriptome Amplification ◽  
Whole Transcriptome

Plant pathogens are constantly emerging and spreading into new areas and there are often limited postdiagnosis treatment options for infection, making surveillance key to their control. Here we present results from a study testing the efficacy of a portable nanopore-based massively parallel sequencing (MPS) technology for use in the detection of diverse plant pathogens in selected samples. The Oxford MinION device was coupled with whole transcriptome amplification (WTA) to sequence the metatranscriptome of plant and insect tissues infected with either Candidatus Liberibacter asiaticus or plum pox virus. Results showed that this methodology is useful for detecting unsuspected viral and bacterial pathogens in plant and insect tissues. The percentage of generated reads assigned to plum pox virus was 95% from infected tissue and 3% from the viruliferous insect, Myzus persicae. Diaphorina citri sequencing led to 22% of the reads mapping as Ca. L. asiaticus. Plum pox virus and Ca. L. asiaticus were detected in both tissue and insect samples near the beginning of each sequencing run, demonstrating the capability of this methodology to obtain results rapidly. This approach also proved the capability of this system to determine the major components of the insect vector’s microbiome and the specific strain of small-genome, high-titer pathogens.

scholarly journals A Phytoplasma Related to ‘Candidatus Phytoplasma asteris’ Detected in Citrus Showing Huanglongbing (Yellow Shoot Disease) Symptoms in Guangdong, P. R. China

2009 ◽  
Vol 99 (3) ◽  
pp. 236-242 ◽  
Author(s):  
J. Chen ◽  
X. Pu ◽  
X. Deng ◽  
S. Liu ◽  
H. Li ◽  
...  
Keyword(s):  
Plant Pathogens ◽  
23S Rrna ◽  
Tem Analysis ◽  
Transmission Electron ◽  
Citrus Production ◽  
Candidatus Liberibacter ◽  
Citrus Huanglongbing ◽  
Liberibacter Asiaticus ◽  
Sieve Tubes ◽  
Primer Sets

Citrus huanglongbing (HLB) or yellow shoot disease (i.e., greening disease) is highly destructive to citrus production worldwide. Understanding the etiology of HLB is critical for managing the disease. HLB is currently associated with infection by ‘Candidatus Liberibacter spp.’ around the world, including China. However, Koch's postulates have not been fulfilled. In addition, other plant pathogens also may be involved in HLB. In a survey performed in Guangdong Province, P. R. China in 2006 and 2007, 141 citrus samples showing typical symptoms of HLB from 11 different cities were collected. Polymerase chain reaction (PCR) using phytoplasma-specific primer sets fU5/rU3 nested with primer set P1/P7 identified 110 (78.0%) positive samples. A 1,785-bp amplicon was obtained with primer set P1/P7. Analysis showed a 100% identity of this sequence in the region of 16S rDNA and 16S-23S rRNA intergenic transcribed spacer to three strains of ‘Candidatus Phytoplasma asteris’ (onion yellows [Japan], aster yellows ‘watercress’ [Hawaii], and valeriana yellows [Lithuania]). Of the 141 samples, 89 (63.1%) samples were positive for ‘Ca. Liberibacter asiaticus’. When mixed infection was considered, 69 (48.9%) samples were positive for both ‘Ca. P. asteris’ and ‘Ca. L. asiaticus’. Transmission electron microscopy (TEM) showed low titers of both walled and wall-less bodies in the phloem sieve tubes of HLB citrus. When transmission from symptomatic citrus to periwinkle (Catharanthus roseus) via dodder (Cuscuta campestris) was conducted, both phytoplasma and ‘Ca. L. asiaticus’ were detected from the affected periwinkle. In addition to yellowing/mottling, the infected periwinkle showed symptoms of virescence and phyllody which are commonly associated with phytoplasmal diseases. TEM analysis of affected periwinkle revealed pleomorphic and wall-less organisms, characteristic of phytoplasmas, filling some phloem sieve tubes. In contrast, walled bacteria were at low titer. This study showed that in addition to ‘Ca. L. asiaticus’, a phytoplasma related to ‘Ca. P. asteris’ could also be detected in citrus showing HLB symptoms in Guangdong.

scholarly journals Progress and Obstacles in Culturing ‘Candidatus Liberibacter asiaticus’, the Bacterium Associated with Huanglongbing

2019 ◽  
Vol 109 (7) ◽  
pp. 1092-1101 ◽  
Author(s):  
Marcus V. Merfa ◽  
Edel Pérez-López ◽  
Eber Naranjo ◽  
Mukesh Jain ◽  
Dean W. Gabriel ◽  
...  
Keyword(s):  
Plant Pathogens ◽  
The State ◽  
Economic Consequences ◽  
Candidatus Liberibacter Asiaticus ◽  
Wide Range ◽  
Plant Hosts ◽  
Substantial Progress ◽  
Candidatus Liberibacter ◽  
Liberibacter Asiaticus ◽  
Scientific Challenge

In recent decades, ‘Candidatus Liberibacter spp.’ have emerged as a versatile group of psyllid-vectored plant pathogens and endophytes capable of infecting a wide range of economically important plant hosts. The most notable example is ‘Candidatus Liberibacter asiaticus’ (CLas) associated with Huanglongbing (HLB) in several major citrus-producing areas of the world. CLas is a phloem-limited α-proteobacterium that is primarily vectored and transmitted among citrus species by the Asian citrus psyllid (ACP) Diaphorina citri. HLB was first detected in North America in Florida (USA) in 2005, following introduction of the ACP to the State in 1998. HLB rapidly spread to all citrus growing regions of Florida within three years, with severe economic consequences to growers and considerable expense to taxpayers of the state and nation. Inability to establish CLas in culture (except transiently) remains a significant scientific challenge toward effective HLB management. Lack of axenic cultures has restricted functional genomic analyses, transfer of CLas to either insect or plant hosts for fulfillment of Koch’s postulates, characterization of host-pathogen interactions and effective screening of antibacterial compounds. In the last decade, substantial progress has been made toward CLas culturing: (i) three reports of transient CLas cultures were published, (ii) a new species of Liberibacter was identified and axenically cultured from diseased mountain papaya (Liberibacter crescens strain BT-1), (iii) psyllid hemolymph and citrus phloem sap were biochemically characterized, (iv) CLas phages were identified and lytic genes possibly affecting CLas growth were described, and (v) genomic sequences of 15 CLas strains were made available. In addition, development of L. crescens as a surrogate host for functional analyses of CLas genes, has provided valuable insights into CLas pathogenesis and its physiological dependence on the host cell. In this review we summarize the conclusions from these important studies.

scholarly journals Prophage-Encoded Peroxidase in ‘Candidatus Liberibacter asiaticus’ Is a Secreted Effector That Suppresses Plant Defenses

2015 ◽  
Vol 28 (12) ◽  
pp. 1330-1337 ◽  
Author(s):  
Mukesh Jain ◽  
Laura A. Fleites ◽  
Dean W. Gabriel
Keyword(s):  
Plant Pathogens ◽  
Conversion Factor ◽  
Shuttle Vector ◽  
Symptom Development ◽  
Candidatus Liberibacter Asiaticus ◽  
In Planta ◽  
Enhanced Resistance ◽  
Candidatus Liberibacter ◽  
Liberibacter Asiaticus ◽  
Lysogenic Conversion

‘Candidatus Liberibacter asiaticus’ is transmitted by psyllids and causes huanglongbing (HLB), a lethal disease of citrus. Most pathogenic ‘Ca. L. asiaticus’ strains carry two nearly identical prophages similar to SC1 and SC2 in strain UF506. SC2 was observed to replicate as a moderately high-copy excision plasmid encoding a reactive oxygen species–scavenging peroxidase (SC2_gp095), a predicted lysogenic conversion factor. SC2_gp095 was expressed at significantly higher levels in periwinkle than in citrus and was suppressed in psyllids. SC2_gp095 was cloned in a shuttle vector and transformed into Escherichia coli and Liberibacter crescens, a culturable proxy for ‘Ca. L. asiaticus’. Transformed L. crescens cells showed 20 to 25% enhanced resistance to H2O2 on agar plates, 47% greater enzymatic activity, and enhanced growth in liquid cultures. A nonclassical secretion potential was predicted for SC2_gp095 and secretion from L. crescens was confirmed by enzymatic and Western blot analyses. Transient expression of SC2_gp095 in planta resulted in strong transcriptional downregulation of RbohB, the key gatekeeper of the H2O2-mediated defense signaling in plants, helping explain the surprisingly long incubation period (years) before HLB symptoms appear in ‘Ca. L. asiaticus’–infected citrus. ‘Ca. L. asiaticus’ peroxidase is likely a secreted, horizontally acquired effector that suppresses host symptom development, a tactic used by most biotrophic plant pathogens.

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.

scholarly journals Two ‘Candidatus Liberibacter asiaticus’ Strains Recently Found in California Harbor Different Prophages

2017 ◽  
Vol 107 (6) ◽  
pp. 662-668 ◽  
Author(s):  
Z. Zheng ◽  
F. Wu ◽  
L. B. Kumagai ◽  
M. Polek ◽  
X. Deng ◽  
...  
Keyword(s):  
Los Angeles ◽  
Geographic Region ◽  
Mapping Technique ◽  
Polymorphism Analysis ◽  
Candidatus Liberibacter Asiaticus ◽  
Next Generation Sequencing Technology ◽  
Candidatus Liberibacter ◽  
Liberibacter Asiaticus

‘Candidatus Liberibacter asiaticus’ (CLas), an α-proteobacterium, is associated with citrus Huanglongbing (HLB; yellow shoot disease). In California, two cases of CLas have been detected in Los Angeles County, one in Hacienda Heights in 2012 and the other in San Gabriel in 2015. Although all infected trees were destroyed in compliance with a state mandate, citrus industry stakeholder concerns about HLB in California are high. Little is known about the biology of CLas, particularly the California strains, hindering effective HLB management efforts. In this study, next-generation sequencing technology (Illumina MiSeq) was employed to characterize the California CLas strains. Data sets containing >4 billion (Giga) bp of sequence were generated from each CLas sample. Two prophages (P-HHCA1-2 and P-SGCA5-1) were identified by the MiSeq read mapping technique referenced to two known Florida CLas prophage sequences, SC1 and SC2. P-HHCA1-2 was an SC2-like or Type 2 prophage of 38,989 bp in size. P-SGCA5-1 was an SC1-like or Type 1 prophage of 37,487 bp in size. Phylogenetic analysis revealed that P-HHCA1-2 was part of an Asiatic lineage within the Type 2 prophage group. Similarly, P-SGCA5-1 was part of an Asiatic lineage within Type 1 prophage group. The Asiatic relatedness of both P-HHCA1-2 and P-SGCA5-1 was further presented by single nucleotide polymorphism analysis at terL (encoding prophage terminase) that has been established for CLas strain differentiation. The presence of different prophages suggests that the two California CLas strains could have been introduced from different sources. An alternative explanation is that there was a mixed CLas population containing the two types of prophages, and limited sampling in a geographic region may not accurately depict the true CLas diversity. More accurate pathway analysis may be achieved by including more strains collected from the regions.

scholarly journals Susceptibility of Sixteen Citrus Genotypes to ‘Candidatus Liberibacter asiaticus’

Plant Disease ◽  
2016 ◽  
Vol 100 (6) ◽  
pp. 1080-1086 ◽  
Author(s):  
Greg McCollum ◽  
Mark Hilf ◽  
Mike Irey ◽  
Weiqi Luo ◽  
Tim Gottwald
Keyword(s):  
The United States ◽  
Insect Vector ◽  
Candidatus Liberibacter Asiaticus ◽  
Citrus Germplasm ◽  
Citrus Production ◽  
Candidatus Liberibacter ◽  
Liberibacter Asiaticus ◽  
Florida Citrus ◽  
Citrus Macrophylla ◽  
Free Ranging

Huanglongbing (HLB) disease is the most serious threat to citrus production worldwide and, in the last decade, has devastated the Florida citrus industry. In the United States, HLB is associated with the phloem-limited α-proteobacterium ‘Candidatus Liberibacter asiaticus’ and its insect vector, the Asian citrus psyllid (ACP; Diaphorina citri). Significant effort is being put forth to develop novel citrus germplasm that has a lower propensity to succumb to HLB than do currently available varieties. Effective methods of screening citrus germplasm for susceptibility to HLB are essential. In this study, we exposed small, grafted trees of 16 citrus types to free-ranging ACP vectors and ‘Ca. L. asiaticus’ inoculum in the greenhouse. During 45 weeks of exposure to ACP, the cumulative incidence of ‘Ca. L. asiaticus’ infection was 70%. Trees of Citrus macrophylla and C. medica were most susceptible to ‘Ca. L. asiaticus’, with 100% infection by the end of the test period in three trials, while the complex genetic hybrids ‘US 1-4-59’ and ‘Fallglo’ consistently were least susceptible, with approximately 30% infection. Results obtained in this greenhouse experiment showed good agreement with trends observed in the orchard, supporting the validity of our approach for screening citrus germplasm for susceptibility to HLB.

scholarly journals A New Diagnostic System for Detection of ‘Candidatus Liberibacter asiaticus’ Infection in Citrus

Plant Disease ◽  
2013 ◽  
Vol 97 (10) ◽  
pp. 1295-1300 ◽  
Author(s):  
Lianming Lu ◽  
Baoping Cheng ◽  
Jinai Yao ◽  
Aitian Peng ◽  
Danchao Du ◽  
...  
Keyword(s):  
Affinity Chromatography ◽  
Polyclonal Antibodies ◽  
Diagnostic System ◽  
Enzyme Linked Immunosorbent Assay ◽  
Candidatus Liberibacter Asiaticus ◽  
Negative Results ◽  
Candidatus Liberibacter ◽  
Liberibacter Asiaticus ◽  
Polymerase Chain

In this study, two polyclonal antibodies were produced against the Omp protein of ‘Candidatus Liberibacter asiaticus’. First, omp genes were sequenced to exhibit 99.9% identity among 137 isolates collected from different geographical origins. Then, two peptides containing the hydrophobic polypeptide-transport-associated (POTRA) domain and β-barrel domain, respectively, were identified on Omp protein. After that, these two peptides were overexpressed in Escherichia coli and purified by affinity chromatography to immunize the white rabbits. Finally, the antiserum was purified by affinity chromatography. The two Omp antibodies gave positive results (0.454 to 0.633, 1:1,600 dilution) in enzyme-linked immunosorbent assay against ‘Ca. L. asiaticus’-infected samples collected from different geographical origins but revealed negative results against other pathogen-infected, nutrient-deficient and healthy samples. The antibody against the POTRA domain of Omp protein could detect ‘Ca. L. asiaticus’ in 45.7% of the symptomatic samples compared with a 56.2% detection rate with a polymerase chain reaction assay. These new antibodies will provide a very useful supplement to the current approaches to ‘Ca. L. asiaticus’ detection and also provide powerful research tools for tracking distribution of this pathogen in vivo.

Sign in / Sign up

Export Citation Format

Share Document