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 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.

A protocol for successful transmission of ‘Candidatus Liberibacter asiaticus’ from citrus to citrus using Diaphorina citri

2021 ◽  
Author(s):  
Silvio Aparecido Lopes ◽  
Juan Camilo Cifuentes-Arenas
Keyword(s):  
Ambient Air ◽  
Diaphorina Citri ◽  
Candidatus Liberibacter Asiaticus ◽  
Mature Leaves ◽  
Air Temperatures ◽  
Tree Canopies ◽  
Candidatus Liberibacter ◽  
Liberibacter Asiaticus ◽  
Time Required ◽  
Transmission Studies

A protocol to successfully transmit the huanglongbing (HLB) pathogen, ‘Candidatus Liberibacter asiaticus’ (Las), from citrus-to-citrus plants using the Asian citrus psyllid (ACP), and an alternative way to help growers control ACP are proposed. Best results were obtained when pathogen acquisition by adults reared on fully symptomatic Las-positive plants, latency, and inoculation, occurred at ambient air temperatures ranging from 24 to 28°C, and by confining single infective adult ACP for seven days on soft newly developing vegetative shoots (stages v2 to v4). No infection resulted from confinement of infective ACP adults on mature leaves (stage v6). Under the described conditions, single ACP adults could successfully transmit Las to an average of 56.5% (35 to 83%) of plantlets with v2 to v4 shoots growing in 0.3 L tubes, and to 80.5% (76 to 86%) of plants with v2 to v4 shoots growing in 4.7 L pots. The use of single insects and plantlets reduces labor, space and other resources required to undertake transmission tests. It also reduces time required for transmission studies and should help accelerate research on HLB. The results were used to develop an index for favorability to infection (IFI) to determine orchard vulnerabilities to Las. The IFI is based on the heterogeneous population of new shoots that occurs on tree canopies and may offer alternative or complementary alternatives to the laborious and costly insect surveys currently used in most instances to determine threshold levels for insecticide applications.

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 ‘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 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 Detection of ‘Candidatus Liberibacter asiaticus’ in Diaphorina citri and Its Importance in the Management of Citrus Huanglongbing in Florida

2008 ◽  
Vol 98 (4) ◽  
pp. 387-396 ◽  
Author(s):  
K. L. Manjunath ◽  
S. E. Halbert ◽  
C. Ramadugu ◽  
S. Webb ◽  
R. F. Lee
Keyword(s):  
Quantitative Polymerase Chain Reaction ◽  
Diagnostic Methods ◽  
Diaphorina Citri ◽  
Candidatus Liberibacter Asiaticus ◽  
Citrus Production ◽  
Candidatus Liberibacter ◽  
Citrus Huanglongbing ◽  
Liberibacter Asiaticus ◽  
Polymerase Chain ◽  
Polymerase Chain Reaction Methodology

Citrus huanglongbing (HLB or citrus greening), is a highly destructive disease that has been spreading in both Florida and Brazil. Its psyllid vector, Diaphorina citri Kuwayama, has spread to Texas and Mexico, thus threatening the future of citrus production elsewhere in mainland North America. Even though sensitive diagnostic methods have been developed for detection of the causal organisms, Candidatus Liberibacter spp., the pathogen cannot be detected consistently in plants until symptoms develop, presumably because of low titer and uneven distribution of the causal bacteria in nonsymptomatic tissues. In the present study, TaqMan based real-time quantitative polymerase chain reaction methodology was developed for detection of ‘Ca. L. asiaticus’ in D. citri. Over 1,200 samples of psyllid adults and nymphs, collected from various locations in Florida, from visually healthy and HLB symptomatic trees at different times of the year were analyzed to monitor the incidence and spread of HLB. The results showed that spread of ‘Ca. L. asiaticus’ in an area may be detected one to several years before the development of HLB symptoms in plants. The study suggests that discount garden centers and retail nurseries may have played a significant role in the widespread distribution of psyllids and plants carrying HLB pathogens in Florida.

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’.

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