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.

scholarly journals Viability of ‘Candidatus Liberibacter asiaticus’ Prolonged by Addition of Citrus Juice to Culture Medium

2014 ◽  
Vol 104 (1) ◽  
pp. 15-26 ◽  
Author(s):  
Jennifer K. Parker ◽  
Sarah R. Wisotsky ◽  
Evan G. Johnson ◽  
Faraj M. Hijaz ◽  
Nabil Killiny ◽  
...  
Keyword(s):  
Grapefruit Juice ◽  
Culture Medium ◽  
Quantitative Polymerase Chain Reaction ◽  
Chemical Characterization ◽  
Candidatus Liberibacter Asiaticus ◽  
Citrus Greening Disease ◽  
Candidatus Liberibacter ◽  
The Media ◽  
Liberibacter Asiaticus

Huanglongbing, or citrus greening disease, is associated with infection by the phloem-limited bacterium ‘Candidatus Liberibacter asiaticus’. Infection with ‘Ca. L. asiaticus’ is incurable; therefore, knowledge regarding ‘Ca. L. asiaticus’ biology and pathogenesis is essential to develop a treatment. However, ‘Ca. L. asiaticus’ cannot currently be successfully cultured, limiting its study. To gain insight into the conditions conducive for growth of ‘Ca. L. asiaticus’ in vitro, ‘Ca. L. asiaticus’ inoculum obtained from seed of fruit from infected pomelo trees (Citrus maxima ‘Mato Buntan’) was added to different media, and cell viability was monitored for up to 2 months using quantitative polymerase chain reaction in conjunction with ethidium monoazide. Media tested included one-third King's B (K), K with 50% juice from the infected fruit, K with 50% commercially available grapefruit juice, and 100% commercially available grapefruit juice. Results show that juice-containing media dramatically prolong viability compared with K in experiments reproduced during 2 years using different juice sources. Furthermore, biofilm formed at the air–liquid interface of juice cultures contained ‘Ca. L. asiaticus’ cells, though next-generation sequencing indicated that other bacterial genera were predominant. Chemical characterization of the media was conducted to discuss possible factors sustaining ‘Ca. L. asiaticus’ viability in vitro, which will contribute to future development of a culture medium for ‘Ca. L. asiaticus’.

scholarly journals Tracing Penicillin Movement in Citrus Plants Using Fluorescence-Labeled Penicillin

Antibiotics ◽  
2019 ◽  
Vol 8 (4) ◽  
pp. 262
Author(s):  
Nabil Killiny ◽  
Pedro Gonzalez-Blanco ◽  
Yulica Santos-Ortega ◽  
Fuad Al-Rimawi ◽  
Amit Levy ◽  
...  
Keyword(s):  
Vascular Tissue ◽  
Vascular System ◽  
Citrus Greening ◽  
Application Site ◽  
Asian Citrus Psyllid ◽  
Diaphorina Citri ◽  
Candidatus Liberibacter Asiaticus ◽  
Citrus Industry ◽  
Candidatus Liberibacter ◽  
Liberibacter Asiaticus

Huánglóngbìng (HLB), citrus greening, is one of the most destructive diseases of citrus plants worldwide. In North America, HLB is caused by the phloem-limited bacterium Candidatus Liberibacter asiaticus and is transmitted by the Asian citrus psyllid, Diaphorina citri. No cure exists at present, and the use of antibiotics for the control of HLB has gained interest due to the significant losses to the citrus industry. Because of unsatisfactory results when using foliar applications of antibiotics, concerns were raised regarding the uptake and translocation of these materials within trees. We, therefore, investigated a method that allows us to study the movement of antibiotic materials in citrus plants. Herein, we utilized a fluorescence-labeled penicillin, BOCILLIN™ FL-Penicillin (FL-penicillin), to study the uptake and translocation of penicillin in citrus plants. FL-penicillin was applied by puncture to the stem of young citrus seedlings and was traced by using fluorescence microscopy. After application, we detected FL-penicillin in the leaves and in the stem xylem and phloem tissues above and below the application site in both intact and partially bark-girdled citrus seedlings, indicating that it is easily taken up and transported through the plant vascular system. In addition, we detected FL-penicillin in the gut of D. citri, which were allowed to feed on the treated plants, suggesting translocation of this molecule into the vascular tissue. We propose that the use of fluorescent-labeled molecules could be an effective tool for understanding the uptake and translocation of antibiotics and other macromolecules in plants and insects.

scholarly journals Low Incidence of ‘Candidatus Liberibacter asiaticus’ in Murraya paniculata and Associated Diaphorina citri

Plant Disease ◽  
2012 ◽  
Vol 96 (6) ◽  
pp. 827-832 ◽  
Author(s):  
Abigail J. Walter ◽  
David G. Hall ◽  
Yong Ping Duan
Keyword(s):  
Quantitative Polymerase Chain Reaction ◽  
Diaphorina Citri ◽  
Candidatus Liberibacter Asiaticus ◽  
Disease Epidemiology ◽  
Citrus Groves ◽  
Murraya Paniculata ◽  
Candidatus Liberibacter ◽  
Liberibacter Asiaticus ◽  
Low Incidence ◽  
A Minor

Huanglongbing (HLB) is one of the most devastating diseases of citrus worldwide. ‘Candidatus Liberibacter asiaticus’ is the prevalent species of three HLB-associated Liberibacter spp., which is vectored by the psyllid Diaphorina citri. The vector and the bacteria have host plants outside the genus Citrus, and these plants have the potential to affect disease epidemiology within citrus groves. Murraya paniculata could be especially problematic because it is a popular ornamental plant and a host of both psyllid and bacteria. We conducted a year-long survey of eight urban plantings of M. paniculata in east-central Florida to characterize ‘Ca. L. asiaticus’ infection rates in plants and associated psyllids. Using sensitive quantitative polymerase chain reaction (qPCR) primers targeting two prophage genes of ‘Ca. L. asiaticus’, we found infection to be extremely low: less than 1% of psyllids and 1.8% of plants. With qPCR primers targeting ‘Ca. L. asiaticus’ 16S rDNA, none of the plants and only one psyllid were ‘Ca. L. asiaticus’-positive. Therefore, the titer of ‘Ca. L. asiaticus’ is low in M. paniculata and associated psyllids. These results suggest that urban plantings of M. paniculata may serve as a minor source of ‘Ca. L. asiaticus’ inoculum.

scholarly journals Seasonal Variation of ‘Candidatus Liberibacter asiaticus’ Titers in New Shoots of Citrus in Distinct Environments

Plant Disease ◽  
2017 ◽  
Vol 101 (4) ◽  
pp. 583-590 ◽  
Author(s):  
Silvio A. Lopes ◽  
Fernanda Q. B. F. Luiz ◽  
Hermes T. Oliveira ◽  
Juan C. Cifuentes-Arenas ◽  
Laudecir L. Raiol-Junior
Keyword(s):  
Sweet Orange ◽  
Quantitative Polymerase Chain Reaction ◽  
Diaphorina Citri ◽  
Candidatus Liberibacter Asiaticus ◽  
Cumulative Rainfall ◽  
Data Loggers ◽  
Minas Gerais State ◽  
Candidatus Liberibacter ◽  
Liberibacter Asiaticus ◽  
Polymerase Chain

The major citrus area of Brazil occupies near 450,000 ha between the Triângulo Mineiro (TM) region of Minas Gerais State and the south of São Paulo State (SPS). Significant climatic variation occurs between regions which could affect huanglongbing (HLB) progress, which is lower in TM. To investigate this possibility, young sweet orange shoots were sampled periodically over 2 years to determine ‘Candidatus Liberibacter asiaticus’ titers in naturally infected trees in orchards in Analândia, central SPS, and Frutal and Comendador Gomes, within TM. Data-loggers recorded local temperature and relative humidity hourly. In the laboratory, five ‘Ca. L. asiaticus’-free Diaphorina citri adults were placed on each sampled shoot for 48 h to feed and acquire the pathogen. Shoots and insects were individually analyzed by quantitative polymerase chain reaction to determine ‘Ca. L. asiaticus’ titers. The incidence of ‘Ca. L. asiaticus’-positive shoots, ‘Ca. L. asiaticus’ titers, and acquisition rates were lower for shoots from Comendador Gomes than those from Frutal or Analândia. Stronger association was observed between ‘Ca. L. asiaticus’ titers and the number of hours below 15°C (h < 15°C) or above 30°C (h > 30°C), and cumulative rainfall registered during the 15 days prior to sampling of shoots on each occasion. ‘Ca. L. asiaticus’ titers associated positively with h < 15°C and rainfall and negatively with h > 30°C. The slower spread and lower incidence of HLB in TM may be related to lower incidences of ‘Ca. L. asiaticus’-positive young shoots and lower titers of ‘Ca. L. asiaticus’ in the same shoots as a consequence of the warmer and drier conditions.

scholarly journals Heat Treatment Eliminates ‘Candidatus Liberibacter asiaticus’ from Infected Citrus Trees Under Controlled Conditions

2013 ◽  
Vol 103 (1) ◽  
pp. 15-22 ◽  
Author(s):  
Michele T. Hoffman ◽  
Melissa S. Doud ◽  
Lisa Williams ◽  
Mu-Qing Zhang ◽  
Fang Ding ◽  
...  
Keyword(s):  
Quantitative Polymerase Chain Reaction ◽  
Thermal Exposure ◽  
Candidatus Liberibacter Asiaticus ◽  
Time Duration ◽  
Bacterial Titer ◽  
Heat Treated ◽  
Candidatus Liberibacter ◽  
Vigorous Growth ◽  
Liberibacter Asiaticus ◽  
Polymerase Chain

Huanglongbing (HLB) is one of the most destructive diseases of citrus worldwide. The three known causal agents of HLB are species of α-proteobacteria: ‘Candidatus Liberibacter asiaticus’, ‘Ca. L. africanus’, and ‘Ca. L. americanus’. Previous studies have found distinct variations in temperature sensitivity and tolerance among these species. Here, we describe the use of controlled heat treatments to cure HLB caused by ‘Ca. L. asiaticus’, the most prevalent and heat-tolerant species. Using temperature-controlled growth chambers, we evaluated the time duration and temperature required to suppress or eliminate the ‘Ca. L. asiaticus’ bacterium in citrus, using various temperature treatments for time periods ranging from 2 days to 4 months. Results of quantitative polymerase chain reaction (qPCR) after treatment illustrate significant decreases in the ‘Ca. L. asiaticus’ bacterial titer, combined with healthy vigorous growth by all surviving trees. Repeated qPCR testing confirmed that previously infected, heat-treated plants showed no detectable levels of ‘Ca. L. asiaticus’, while untreated control plants remained highly infected. Continuous thermal exposure to 40 to 42°C for a minimum of 48 h was sufficient to significantly reduce titer or eliminate ‘Ca. L. asiaticus’ bacteria entirely in HLB-affected citrus seedlings. This method may be useful for the control of ‘Ca. Liberibacter’-infected plants in nursery and greenhouse settings.

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 ‘Candidatus Liberibacter asiaticus’ Titers in Citrus and Acquisition Rates by Diaphorina citri Are Decreased by Higher Temperature

Plant Disease ◽  
2013 ◽  
Vol 97 (12) ◽  
pp. 1563-1570 ◽  
Author(s):  
S. A. Lopes ◽  
F. Q. B. F. Luiz ◽  
E. C. Martins ◽  
C. G. Fassini ◽  
M. C. Sousa ◽  
...  
Keyword(s):  
Quantitative Polymerase Chain Reaction ◽  
Disease Spread ◽  
Diaphorina Citri ◽  
Candidatus Liberibacter Asiaticus ◽  
Ambient Temperatures ◽  
Air Temperatures ◽  
Temperature Regimes ◽  
Candidatus Liberibacter ◽  
Liberibacter Asiaticus ◽  
Higher Temperature

‘Candidatus Liberibacter asiaticus’ is the most prevalent Liberibacter sp. associated with huanglongbing (HLB) in Brazil. Within São Paulo state (SP), HLB has spread more rapidly to and reached higher incidence in regions with relatively mild (cooler) summer temperatures. This suggests that climate can influence disease spread and severity. ‘Ca. L. asiaticus’ titers on soft, immature leaves from infected ‘Valencia’ sweet orange plants exposed to different temperature regimes and adult Diaphorina citri fed for 48 h on these plants for ‘Ca. L. asiaticus’ acquisition were determined by quantitative polymerase chain reaction in two experiments. The first experiment included plants with three levels of infection, three incubation periods (IPs), and air temperatures favorable (14.6 to 28°C) and unfavorable (24 to 38°C) to ‘Ca. L. asiaticus’. The second included plants with severe late-stage infections, 10 IPs (based on 3-day intervals over 27 days), and three air temperature regimes (12 to 24, 18 to 30, and 24 to 38°C). Overall, ‘Ca. L. asiaticus’ titers and the percentages of ‘Ca. L. asiaticus’-positive psyllids were lower in plants maintained at the warmer temperature regime (24 to 38°C) than in plants maintained in the cooler regimes. The results suggest that the lower incidence and slower spread of ‘Ca. L. asiaticus’ to warmer regions of SP are related to the influence of ambient temperatures on titers of ‘Ca. L. asiaticus’ in leaves.

scholarly journals Incidence of Diaphorina citri Carrying Candidatus Liberibacter asiaticus in Brazil’s Citrus Belt

Insects ◽  
2020 ◽  
Vol 11 (10) ◽  
pp. 672
Author(s):  
Nelson Wulff ◽  
Bruno Daniel ◽  
Rodrigo Sassi ◽  
Alécio Moreira ◽  
Renato Bassanezi ◽  
...  
Keyword(s):  
Spectral Density ◽  
Management Practices ◽  
Quantitative Polymerase Chain Reaction ◽  
Diaphorina Citri ◽  
Candidatus Liberibacter Asiaticus ◽  
Good Management ◽  
Northern Regions ◽  
Candidatus Liberibacter ◽  
Density Analysis ◽  
Liberibacter Asiaticus

Huanglongbing (HLB) is a citrus disease of worldwide importance, associated with the presence of Candidatus Liberibacter asiaticus (Las) and vectored by the psyllid Diaphorina citri in Asia and the Americas. To properly manage HLB, removal of inoculum sources and control of the psyllid are undertaken. We evaluated the percentage of the psyllid population with Las, sampled from yellow sticky traps over a three-year period and its relationship with insect population, regions, season of the year, and HLB management in citrus areas in the southwestern, central, and northern regions of São Paulo (SP) and southwestern region of Minas Gerais states, Brazil. In each reading, up to 50 psyllids per region were collected and detection of Las in individual psyllids were made by quantitative polymerase chain reaction. The percentage of psyllids with Las—an average of 65.3%—was constant throughout the year in the southwestern region of SP state, while showing an increase from spring to autumn when sampled from central to northern regions. The proportion of psyllids carrying Las from each region and year period were compared by a proportion test and spectral density analysis. The proportion of psyllids carrying Las evaluated in the same region in different seasons presented statistical differences in central (Araraquara) and southwestern (Santa Cruz do Rio Pardo) regions in 2015, with higher values in the first semester (summer and autumn) than in the second semester (winter and spring). Orchards with poor HLB management had higher incidence of psyllids with Las. Spectral density analysis indicated that good management areas had 50% less relevant peaks of psyllids with Las than in areas with poor HLB management practices. The relationship between the percentage of psyllids carrying Las and the number of captured psyllids in the region in a given time denotes the most critical intake time for HLB spread in citrus orchards. The reduction in the population of psyllids carrying Las is a direct benefit from the use of good management practices.

scholarly journals Genome Sequence Resource of ‘Candidatus Liberibacter asiaticus’ from Thailand

Plant Disease ◽  
2020 ◽  
Vol 104 (3) ◽  
pp. 624-626
Author(s):  
Tao Li ◽  
Narit Thaochan ◽  
Jiaquan Huang ◽  
Jianchi Chen ◽  
Xiaoling Deng ◽  
...  
Keyword(s):  
Genome Sequence ◽  
Candidatus Liberibacter Asiaticus ◽  
Citrus Industry ◽  
Candidatus Liberibacter ◽  
Citrus Huanglongbing ◽  
Liberibacter Asiaticus

‘Candidatus Liberibacter asiaticus’ (Las) is an unculturable α-proteobacterium associated with citrus huanglongbing (HLB), a devastating disease currently threatening the citrus industry worldwide. Here, we present the genome sequence of Las strain TaiYZ2 from an HLB-affected pomelo tree in Hat Yai district, Songkhla Province, Thailand. The TaiYZ2 genome is composed of 1,230,623 bp with G+C content of 36.4%. This is the first Las genome sequence from Thailand, which will enrich current Las genome resource and facilitate HLB research and management.

scholarly journals Identification of Gene Candidates Associated with Huanglongbing Tolerance, Using ‘Candidatus Liberibacter asiaticus’ Flagellin 22 as a Proxy to Challenge Citrus

2018 ◽  
Vol 31 (2) ◽  
pp. 200-211 ◽  
Author(s):  
Qingchun Shi ◽  
Vicente J. Febres ◽  
Shujian Zhang ◽  
Fahong Yu ◽  
Greg McCollum ◽  
...  
Keyword(s):  
Quantitative Polymerase Chain Reaction ◽  
Citrus Canker ◽  
Defense Responses ◽  
Candidatus Liberibacter Asiaticus ◽  
Tolerance Mechanisms ◽  
Molecular Pattern ◽  
Number Of Genes ◽  
Candidatus Liberibacter ◽  
Liberibacter Asiaticus ◽  
Polymerase Chain

The 22–amino acid (flg22) pathogen-associated molecular pattern from the flagellin of Xanthomonas citri subsp. citri has been shown to induce defense responses correlated with citrus canker resistance. Here, flg22 of ‘Candidatus Liberibacter asiaticus’, the putative causal agent of Huanglongbing (HLB), elicited differential defense responses that were weaker than those from Xcc-flg22, between those of the HLB-tolerant mandarin cultivar Sun Chu Sha and susceptible grapefruit cultivar Duncan. Transcriptomics was used to compare the effect of CLas-flg22 and Xcc-flg22 between the citrus genotypes and identified 86 genes induced only by CLas-flg22 in the tolerant mandarin. Expression of 16 selected genes was validated, by reverse transcription-quantitative polymerase chain reaction, and was evaluated in citrus during ‘Ca. L. asiaticus’ infection. Differential expression of a number of genes occurred between tolerant and susceptible citrus infected with ‘Ca. L. asiaticus’, suggesting their involvement in HLB tolerance. In addition, several genes were similarly regulated by CLas-flg22 and ‘Ca. L. asiaticus’ treatments, while others were oppositely regulated in the tolerant mandarin, suggesting similarity and interplay between CLas-flg22 and ‘Ca. L. asiaticus’–triggered defenses. Genes identified are valuable in furthering the study of HLB tolerance mechanisms and, potentially, for screening for HLB-tolerant citrus using CLas-flg22 as a pathogen proxy.

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