scholarly journals Visualization of ‘Candidatus Liberibacter asiaticus’ Cells in the Vascular Bundle of Citrus Seed Coats with Fluorescence In Situ Hybridization and Transmission Electron Microscopy

2013 ◽  
Vol 103 (6) ◽  
pp. 545-554 ◽  
Author(s):  
Mark E. Hilf ◽  
Kenneth R. Sims ◽  
Svetlana Y. Folimonova ◽  
Diann S. Achor
Keyword(s):  
16S Rrna ◽  
Rrna Gene ◽  
Bacterial Cells ◽  
Candidatus Liberibacter Asiaticus ◽  
Transmission Electron ◽  
Candidatus Liberibacter ◽  
Liberibacter Asiaticus ◽  
Citrus Seed ◽  
Seed Coats

‘Candidatus Liberibacter asiaticus’ is the bacterium implicated as a causal agent of the economically damaging disease of citrus called huanglongbing (HLB). Vertical transmission of the organism through seed to the seedling has not been demonstrated. Previous studies using real-time polymerase chain reaction assays indicated abundant bacterial 16S rRNA sequences in seed coats of citrus seed but the presence of intact bacterial cells was not demonstrated. We used microscopy to verify that intact bacterial cells were present in citrus seed coats. Bacterial cells with the morphology and physical dimensions appropriate for ‘Ca. L. asiaticus’ were seen in phloem sieve elements in the vascular bundle of grapefruit seed coats using transmission electron microscopy (TEM). Fluorescence in situ hybridization (FISH) analyses utilizing probes complementary to the ‘Ca. L. asiaticus’ 16S rRNA gene revealed bacterial cells in the vascular tissue of intact seed coats of grapefruit and pummelo and in fragmented vascular bundles excised from grapefruit seed coats. The physical measurements and the morphology of individual bacterial cells were consistent with those ascribed in the literature to ‘Ca. L. asiaticus’. No bacterial cells were observed in preparations of seed from fruit from noninfected trees. A small library of clones amplified from seed coats from a noninfected tree using degenerate primers targeting prokaryote 16S rRNA gene sequences contained no ‘Ca. L. asiaticus’ sequences, whereas 95% of the sequences in a similar library from DNA from seed coats from an infected tree were identified as ‘Ca. L. asiaticus’, providing molecular genetic corroboration that the bacterial cells observed by TEM and FISH in seed coats from infected trees were ‘Ca. L. asiaticus’.

scholarly journals Co-cultivation of ‘Candidatus Liberibacter asiaticus’ with Actinobacteria from Citrus with Huanglongbing

Plant Disease ◽  
2008 ◽  
Vol 92 (11) ◽  
pp. 1547-1550 ◽  
Author(s):  
Michael J. Davis ◽  
Sachindra N. Mondal ◽  
Huiqin Chen ◽  
Michael E. Rogers ◽  
Ronald H. Brlansky
Keyword(s):  
16S Rrna ◽  
Rrna Gene ◽  
Insect Vector ◽  
Candidatus Liberibacter Asiaticus ◽  
Citrus Greening Disease ◽  
Transmission Electron ◽  
Pcr Products ◽  
Candidatus Liberibacter ◽  
Liberibacter Asiaticus ◽  
Polymerase Chain

Huanglongbing (HLB), also known as citrus greening disease, is a devastating disease of citrus caused by phloem-limited bacteria that have not been grown in culture. Three species, ‘Candidatus Liberibacter asiaticus’, ‘Ca. L. africanus’, and ‘Ca. L. americanus’, are known. ‘Ca. L. asiaticus’ and its insect vector, the psyllid Diaphorina citri, have been recently introduced into Florida. We attempted to isolate ‘Ca. L. asiaticus’ using media formulations developed in response to the growth of another bacterium that appears to be related to the liberibacters based on 16S rRNA gene identities. Cultures were obtained that were polymerase chain reaction (PCR) positive for ‘Ca. L. asiaticus’. However, transmission electron microscope examination of the culture, PCR using generic primers, and sequencing of the PCR products revealed the presence of other bacteria in the cultures. These were actinobacteria related to Propionibacterium acnes based on 16S rRNA identities. The co-cultures remained after attempts to purify the cultures by single-colony isolation, suggesting that the bacteria might be mutually beneficial to each other in culture. The co-cultures have survived more than 10 weekly passages to fresh medium. PCR using P. acnes-specific primers indicated that actinobacteria are common inhabitants of citrus and psyllids, whether or not ‘Ca. L. asiaticus’ is present.

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.

scholarly journals Colonization of Citrus Seed Coats by ‘Candidatus Liberibacter asiaticus’: Implications for Seed Transmission of the Bacterium

2011 ◽  
Vol 101 (10) ◽  
pp. 1242-1250 ◽  
Author(s):  
Mark E. Hilf
Keyword(s):  
Sweet Orange ◽  
Seed Transmission ◽  
Candidatus Liberibacter Asiaticus ◽  
Real Time Quantitative Pcr ◽  
Candidatus Liberibacter ◽  
Liberibacter Asiaticus ◽  
Citrus Varieties ◽  
Citrus Seed ◽  
Pathogen Dna ◽  
Seed Coats

Huanglongbing is an economically damaging disease of citrus associated with infection by ‘Candidatus Liberibacter asiaticus’. Transmission of the organism via infection of seeds has not been demonstrated but is a concern since some citrus varieties, particularly those used as rootstocks in commercial plantings are propagated from seed. We compared the incidence of detection of ‘Ca. Liberibacter asiaticus’ DNA in individual fruit peduncles, seed coats, seeds, and in germinated seedlings from ‘Sanguenelli’ sweet orange and ‘Conners’ grapefruit fruits sampled from infected trees. Using real-time quantitative PCR (qPCR) we detected pathogen DNA in nucleic acid extracts of 36 and 100% of peduncles from ‘Sanguenelli’ and from ‘Conners’ fruits, respectively. We also detected pathogen DNA in extracts of 37 and 98% of seed coats and in 1.6 and 4% of extracts from the corresponding seeds of ‘Sanguenelli’ and ‘Conners’, respectively. Small amounts of pathogen DNA were detected in 10% of ‘Sanguenelli’ seedlings grown in the greenhouse, but in none of 204 extracts from ‘Conners’ seedlings. Pathogen DNA was detected in 4.9% and in 89% of seed coats peeled from seeds of ‘Sanguenelli’ and ‘Conners’ which were germinated on agar, and in 5% of ‘Sanguenelli’ but in none of 164 ‘Conners’ seedlings which grew from these seeds on agar. No pathogen DNA was detected in ‘Ridge Pineapple’ tissue at 3 months post-grafting onto ‘Sanguenelli’ seedlings, even when pathogen DNA had been detected initially in the ‘Sanguenelli’ seedling. Though the apparent colonization of ‘Conners’ seeds was more extensive and nearly uniform compared with ‘Sanguenelli’ seeds, no pathogen DNA was detected in ‘Conners’ seedlings grown from these seeds. For either variety, no association was established between the presence of pathogen DNA in fruit peduncles and seed coats and in seedlings.

scholarly journals A New ‘Candidatus Liberibacter’ Species Associated with Diseases of Solanaceous Crops

Plant Disease ◽  
2009 ◽  
Vol 93 (3) ◽  
pp. 208-214 ◽  
Author(s):  
Lia W. Liefting ◽  
Paul W. Sutherland ◽  
Lisa I. Ward ◽  
Kerry L. Paice ◽  
Bevan S. Weir ◽  
...  
Keyword(s):  
16S Rrna ◽  
Pcr Primers ◽  
23S Rrna ◽  
Rrna Gene ◽  
High Identity ◽  
Specific Pcr ◽  
Transmission Electron ◽  
Candidatus Liberibacter ◽  
Polymerase Chain ◽  
Specific Pcr Primer

A new disease of glasshouse-grown tomato and pepper in New Zealand has resulted in plant decline and yield loss. Affected plants are characterized by spiky, chlorotic apical growth, curling or cupping of the leaves, and overall stunting. Transmission electron microscopy revealed the presence of phloem-limited bacterium-like organisms in symptomatic plants. The strategy used to identify the bacterium involved using specific prokaryote polymerase chain reaction (PCR) primers in combination with universal 16S rRNA primers. Sequence analysis of the 16S rRNA gene, the 16S/23S rRNA spacer region, and the rplKAJL-rpoBC operon revealed that the bacterium shared high identity with ‘Candidatus Liberibacter’ species. Phylogenetic analysis showed that the bacterium is distinct from the three citrus liberibacter species previously described and has been named ‘Candidatus Liberibacter solanacearum’. This is the first report of a liberibacter naturally infecting a host outside the Rutaceae family. A specific PCR primer pair was developed for its detection.

scholarly journals First Report of the Causal Agent of Huanglongbing (“Candidatus Liberibacter asiaticus”) Infecting Kumquat in Taiwan

Plant Disease ◽  
2006 ◽  
Vol 90 (10) ◽  
pp. 1360-1360 ◽  
Author(s):  
C.-H. Tsai ◽  
H.-J. Su ◽  
Y.-C. Liao ◽  
T.-H. Hung
Keyword(s):  
16S Rrna ◽  
Lateral Roots ◽  
Severe Disease ◽  
Intergenic Spacer ◽  
Sieve Tube ◽  
Causal Agent ◽  
Candidatus Liberibacter Asiaticus ◽  
Intergenic Spacer Region ◽  
Candidatus Liberibacter ◽  
Liberibacter Asiaticus

Huanglongbing (greening) disease caused by a nonculturable, phloem-limited bacterium is a severe disease of citrus. On the basis of the influence of temperature on host symptoms and the causal agent, this disease can be categorized as Asian caused by “Candidatus Liberibacter asiaticus”, African caused by “Ca. L. africanus”, and American caused by “Ca. L. americanus”. Kumquat (Fortunella margarita (Lour.) Swingle), a member of the Rutaceae is an economically important crop for export and local consumption in Taiwan. Recently, a Huanglongbing-like disease was found on kumquat in Yilan County, the largest kumquat-producing area in northeastern Taiwan. Even though the disease has been reported on Citrus spp. from Taiwan, it has never been reported on kumquat. Symptoms of infected kumquat were mottling, yellowing, hardening, and curling of leaves followed by premature defoliation, twig dieback, decay of feeder rootlets and lateral roots, and ultimately the death of the entire plant. Typical sieve-tube-restricted bacteria were observed in kumquat cells by electron microscopy (1). In addition, psyllid-transmission tests demonstrated that the Asian psyllid (Diaphorina citri) could transmit this bacterium to healthy kumquats. Positive bud graft transmissions were obtained to F. margarita, F. japonica (Thunb.) Swingle, F. obovata Hort. ex Tanaka, Luchen sweet orange (Citrus sinensis (L.) Osb.), and Wentan pummelo (C. maxima f. sp. butan Hay.). These inoculated plants showed symptoms in 3 to 8 months, and bacteria could be detected by polymerase chain reaction (PCR) using a common primer pair that amplified a 226-bp specific DNA fragment (2). For further molecular identification, the bacterial DNA was extracted from the inoculated plants and PCR was performed by using two sets of primers selected from the 16S rRNA region (GenBank Accession No. L22532) and 16S/23S intergenic spacer region (GenBank Accession No. AB019793). The expected DNA fragments of 1,389 bp and 862 bp were, respectively, amplified from symptomatic plants but not from healthy plants. The PCR products were cloned and sequenced (GenBank Accession Nos. DQ302750 and DQ207841). The 16S rRNA has 98 to 99% identity and 16S/23S intergenic spacer region has 99% identity to the corresponding region of “Ca. L. asiaticus” in GenBank. These molecular analyses confirm the presence of “Ca. L. asiaticus” in kumquat. Since Huanglongbing has been rarely reported naturally on kumquat, further analysis of this bacterium as a special strain of “Ca. L. asiaticus” is needed. References: (1) M. Garnier et al. Ann. Microbiol. 135A:169, 1984. (2) T. H. Hung et al. J. Phytopathol. 147:599, 1999.

scholarly journals Endosymbiotic Bacteroidales Bacteria of the Flagellated Protist Pseudotrichonympha grassii in the Gut of the Termite Coptotermes formosanus

2005 ◽  
Vol 71 (12) ◽  
pp. 8811-8817 ◽  
Author(s):  
Satoko Noda ◽  
Toshiya Iida ◽  
Osamu Kitade ◽  
Hideaki Nakajima ◽  
Toshiaki Kudo ◽  
...  
Keyword(s):  
16S Rrna ◽  
Elongation Factor ◽  
Sequence Divergence ◽  
Rnase H ◽  
Coptotermes Formosanus ◽  
Cell Counting ◽  
Fish Cell ◽  
Rrna Gene ◽  
Bacterial Cells ◽  
Transmission Electron

ABSTRACT A unique lineage of bacteria belonging to the order Bacteroidales was identified as an intracellular endosymbiont of the protist Pseudotrichonympha grassii (Parabasalia, Hypermastigea) in the gut of the termite Coptotermes formosanus. We identified the 16S rRNA, gyrB, elongation factor Tu, and groEL gene sequences in the endosymbiont and detected a very low level of sequence divergence (<0.9% of the nucleotides) in the endosymbiont population within and among protist cells. The Bacteroidales endosymbiont sequence was affiliated with a cluster comprising only sequences from termite gut bacteria and was not closely related to sequences identified for members of the Bacteroidales attached to the cell surfaces of other gut protists. Transmission electron microscopy showed that there were numerous rod-shaped bacteria in the cytoplasm of the host protist, and we detected the endosymbiont by fluorescence in situ hybridization (FISH) with an oligonucleotide probe specific for the 16S rRNA gene identified. Quantification of the abundance of the Bacteroidales endosymbiont by sequence-specific cleavage of rRNA with RNase H and FISH cell counting revealed, surprisingly, that the endosymbiont accounted for 82% of the total bacterial rRNA and 71% of the total bacterial cells in the gut community. The genetically nearly homogeneous endosymbionts of Pseudotrichonympha were very abundant in the gut symbiotic community of the termite.

scholarly journals Colonization of Dodder, Cuscuta indecora, by ‘Candidatus Liberibacter asiaticus’ and ‘Ca. L. americanus’

2010 ◽  
Vol 100 (8) ◽  
pp. 756-762 ◽  
Author(s):  
John S. Hartung ◽  
Cristina Paul ◽  
Diann Achor ◽  
R. H. Brlansky
Keyword(s):  
Cross Sections ◽  
Quantitative Polymerase Chain Reaction ◽  
Morphological Differentiation ◽  
Ultrastructural Level ◽  
Bacterial Cells ◽  
Candidatus Liberibacter Asiaticus ◽  
Citrus Industry ◽  
Vessel Elements ◽  
Candidatus Liberibacter ◽  
Liberibacter Asiaticus

Huanglongbing, or citrus greening, threatens the global citrus industry. The presumptive pathogens, ‘Candidatus Liberibacter asiaticus’ and ‘Ca. L. americanus’ can be transferred from citrus to more easily studied experimental hosts by using holoparasitic dodder plants. However, the interaction between ‘Candidatus Liberibacter’ spp. and the dodder has not been studied. We combined quantitative polymerase chain reaction with electron microscopy to show that only 65% of tendrils of Cuscuta indecora grown on ‘Ca. Liberibacter’ spp.-infected host plants had detectable levels of the pathogen. Among tendrils that were colonized by Liberibacter in at least one 2 cm segment, most were not colonized in all segments. Furthermore, the estimated population levels of the pathogen present in serial 2 cm segments of dodder tendrils varied widely and without any consistent pattern. Thus, there was generally not a concentration gradient of the pathogen from the source plant towards the recipient and populations of the pathogen were sometimes found in the distal segments of the dodder plant but not in the proximal or middle segments. Populations of the pathogens ranged from 2 × 102 to 3.0 × 108 cells per 2 cm segment. On a fresh weight basis, populations as high as 1.4 × 1010 cells per g of tissue were observed demonstrating that ‘Ca. Liberibacter’ spp. multiplies well in Cuscuta indecora. However, 55% of individual stem segments did not contain detectable levels of the pathogen, consistent with a pattern of nonuniform colonization similar to that observed in the much more anatomically complex citrus tree. Colonization of dodder by the pathogen is also nonuniform at the ultrastructural level, with adjacent phloem vessel elements being completely full of the pathogen or free of the pathogen. We also observed bacteria in the phloem vessels that belonged to two distinct size classes based on the diameters of cross sections of cells. In other sections from the same tendrils we observed single bacterial cells that were apparently in the process of differentiating between the large and round forms to the long and thin forms (or vice versa). The process controlling this morphological differentiation of the pathogen is not known. The highly reduced and simplified anatomy of the dodder plant as well as its rapid growth rate compared with citrus, and the ability of the plant to support multiplication of the pathogen to high levels, makes it an interesting host plant for further studies of host–pathogen interactions.

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