scholarly journals Influence of Photoperiod Duration and Phloem Disruption through Scoring on Growth, Disease Symptoms, and Bacterial Titer in Citrus Graft Inoculated with Candidatus Liberibacter asiaticus

HortScience ◽  
2016 ◽  
Vol 51 (10) ◽  
pp. 1215-1219 ◽  
Author(s):  
Ed Stover ◽  
Robert G. Shatters ◽  
Barrett Gruber ◽  
Prem Kumar ◽  
Gloria A. Moore
Keyword(s):  
Quantitative Polymerase Chain Reaction ◽  
Continuous Light ◽  
Disease Symptoms ◽  
Rough Lemon ◽  
Bacterial Titer ◽  
Accelerate Development ◽  
Dark Regime ◽  
Candidatus Liberibacter ◽  
Liberibacter Asiaticus

Plants inoculated with the huanglongbing (HLB)-associated bacterium, Candidatus Liberibacter asiaticus (CLas) typically must be monitored for 8–10 months to identify differences in susceptibility between genotypes. Continuous light is reported to accelerate development of HLB symptoms and field observations suggest that trees girdled by tags or tree ties showed greater symptoms. Therefore, an experiment was conducted assessing HLB susceptibility as influenced by light/dark periods of 12 hours: 12 hours and 24 hours: 0 hours, in combination with scoring tree trunks to disrupt phloem. Sixty trees of each of three citrus genotypes (‘Kuharske’, previously shown to be HLB resistant; rough lemon, previously shown to be HLB tolerant; and ‘Valencia’, highly HLB susceptible) were bud grafted using two CLas-infected buds (rough lemon and citron) per tree on 26 Mar. 2012, and were placed in controlled growth rooms (one 12 hour light: 12 hour dark and one constant light) on 4 June 2012. Ten trees of each genotype in each growth room were scored 10 cm above the soil (cutting through the bark but not the wood) with a knife on 18 July 2012 and the scoring was repeated at the same scoring wounds on 30 Aug. 2012. Trees were removed from growth rooms on 12 Dec. 2012 and subsequently maintained in a greenhouse. At two to three month intervals between June 2012 and May 2013, HLB symptoms and stem diameter at 5 cm above the soil were assessed, and three leaves per tree were collected for quantitative polymerase chain reaction (qPCR) determination of CLas titer. Six months after inoculation and 3 months following imposition of treatments, the ‘Valencia’ scored in the 12 hour light: 12 hour dark regime, the ‘Valencia’ non scored trees in 24 hours of light and the ‘Kuharske’ scored trees in 24 hours of light displayed higher CLas titers than most other trees. After an additional two months, both scored and non-scored trees of all three genotypes in 24 hours of light had significantly elevated CLas titers compared with trees in 12 hour light: 12 hour dark regime, but within most treatments all three genotypes had titers which were not statistically different from each other. Growth of ‘Kuharske’ and rough lemon was enhanced; whereas ‘Valencia’ growth was reduced when graft-inoculated plants were maintained in continuous light. Scoring enhanced early CLas development in ‘Kuharske’ when combined with continuous light, had no effect in rough lemon, and showed inconsistent effects in ‘Valencia’. Although continuous lighting enhanced disease progression, it did not reveal differences in HLB susceptibility.

scholarly journals Examination of the Responses of Different Genotypes of Citrus to Huanglongbing (Citrus Greening) Under Different Conditions

2009 ◽  
Vol 99 (12) ◽  
pp. 1346-1354 ◽  
Author(s):  
Svetlana Y. Folimonova ◽  
Cecile J. Robertson ◽  
Stephen M. Garnsey ◽  
Siddarame Gowda ◽  
William O. Dawson
Keyword(s):  
Continuous Light ◽  
Bacterial Titer ◽  
Wide Range ◽  
Growth Room ◽  
Candidatus Liberibacter ◽  
Citrus Huanglongbing ◽  
Phloem Translocation ◽  
Continue Growth ◽  
Liberibacter Asiaticus ◽  
Tristeza Virus

Citrus Huanglongbing (HLB) is one of the most devastating diseases of citrus worldwide. The causal agent of HLB in Florida is thought to be ‘Candidatus Liberibacter asiaticus’. In this work, we examined the responses of 30 different genotypes of citrus to Florida isolates of ‘Ca. L. asiaticus’ under controlled conditions in the greenhouse or growth room. Although ‘Ca. L. asiaticus’ was able to multiply in all of the plants, a wide range of responses was observed among different hosts. Based on the symptoms developed and the ability of plants to continue growth, the different genotypes were grouped into four categories: sensitive, which exhibited severe chlorosis on leaves, greatly reduced growth, and eventual death; moderately tolerant, which exhibited some scattered distinct symptoms but little or no growth reduction and no plant death; tolerant, which exhibited very minimal symptoms; and genotypes, which exhibited variable reactions. Interestingly, although ‘Ca. L. asiaticus’ was unevenly distributed within each particular plant, comparison of titers of the bacterium in different citrus genotypes revealed that most accumulated similar levels of ‘Ca. L. asiaticus’, demonstrating that there is no strict correlation between bacterial titer and severity of disease. Incubation of infected plants in the growth room with continuous light greatly affected symptoms production by reducing the time before distinctive symptoms developed and significantly increasing severity of chlorosis of leaves of all citrus genotypes. These results provide additional evidence of the correlation between disruption of phloem translocation of carbohydrates during HLB infection and the appearance of chlorotic symptoms in leaves of infected trees. We also examined interaction between ‘Ca. L. asiaticus’ and Citrus tristeza virus, which usually occurs in trees that become infected with HLB, and found no synergistic effect of the two pathogens. We trust that observations reported here will provide reagents for further examination of the ‘Ca. L. asiaticus’–citrus interaction to advance the understanding of how ‘Ca. L. asiaticus’ causes disease and to develop methods or trees to overcome the disease.

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 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 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 Resistance to ‘Candidatus Liberibacter asiaticus,’ the Huanglongbing Associated Bacterium, in Sexually and/or Graft-Compatible Citrus Relatives

2021 ◽  
Vol 11 ◽  
Author(s):  
Mônica N. Alves ◽  
Silvio A. Lopes ◽  
Laudecir L. Raiol-Junior ◽  
Nelson A. Wulff ◽  
Eduardo A. Girardi ◽  
...  
Keyword(s):  
Genetic Resistance ◽  
Evaluation Procedure ◽  
Challenge Inoculation ◽  
Bacterial Titer ◽  
Incurable Disease ◽  
The Family ◽  
Candidatus Liberibacter ◽  
Liberibacter Asiaticus ◽  
Crop Sustainability ◽  
First Time

Huanglongbing (HLB) is the most destructive, yet incurable disease of citrus. Finding sources of genetic resistance to HLB-associated ‘Candidatus Liberibacter asiaticus’ (Las) becomes strategic to warrant crop sustainability, but no resistant Citrus genotypes exist. Some Citrus relatives of the family Rutaceae, subfamily Aurantioideae, were described as full-resistant to Las, but they are phylogenetically far, thus incompatible with Citrus. Partial resistance was indicated for certain cross-compatible types. Moreover, other genotypes from subtribe Citrinae, sexually incompatible but graft-compatible with Citrus, may provide new rootstocks able to restrict bacterial titer in the canopy. Use of seedlings from monoembryonic species and inconsistencies in previous reports likely due to Las recalcitrance encouraged us to evaluate more accurately these Citrus relatives. We tested for Las resistance a diverse collection of graft-compatible Citrinae species using an aggressive and consistent challenge-inoculation and evaluation procedure. Most Citrinae species examined were either susceptible or partially resistant to Las. However, Eremocitrus glauca and Papua/New Guinea Microcitrus species as well as their hybrids and those with Citrus arose here for the first time as full-resistant, opening the way for using these underutilized genotypes as Las resistance sources in breeding programs or attempting using them directly as possible new Las-resistant Citrus rootstocks or interstocks.

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 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 Murraya paniculata and Related Species as Potential Hosts and Inoculum Reservoirs of ‘Candidatus Liberibacter asiaticus’, Causal Agent of Huanglongbing

Plant Disease ◽  
2010 ◽  
Vol 94 (5) ◽  
pp. 528-533 ◽  
Author(s):  
V. D. Damsteegt ◽  
E. N. Postnikova ◽  
A. L. Stone ◽  
M. Kuhlmann ◽  
C. Wilson ◽  
...  
Keyword(s):  
Bacterial Disease ◽  
Diaphorina Citri ◽  
Candidatus Liberibacter Asiaticus ◽  
Infection Rates ◽  
Disease Symptoms ◽  
Murraya Paniculata ◽  
Candidatus Liberibacter ◽  
Liberibacter Asiaticus ◽  
Polymerase Chain ◽  
Southern Florida

Huanglongbing (HLB), considered to be the most serious insect-vectored bacterial disease of citrus, is transmitted in nature by the Asian citrus psyllid Diaphorina citri and the African citrus psyllid Trioza erytreae. D. citri was discovered in southern Florida in 1998 and the HLB disease in 2005. Both have become established throughout citrus-producing areas of Florida. Murraya species are widely grown in southern Florida as ornamental hedges and are readily colonized by D. citri vectors. Colonies of D. citri, isolates of ‘Candidatus Liberibacter asiaticus’ from Taiwan and Florida, and the Murraya species were established in the BSL-3 biosecurity facility at Fort Detrick. In controlled inoculation experiments, D. citri transmitted ‘Ca. L. asiaticus’ into M. paniculata (34/36 plants) and M. exotica (22/23 plants), but not into Bergera (Murraya) koenigii. Disease symptoms rarely developed in Murraya plants; however, positive infections were determined by conventional and real-time polymerase chain reaction (PCR). Back-inoculations of ‘Ca. L. asiaticus’ from M. paniculata to Madam Vinous sweet orange resulted in disease development in 25% of the inoculated plants. Considerable variability was observed in infection rates, titer, and persistence of ‘Ca. L. asiaticus’ in infected Murraya.

scholarly journals Comparative Transcriptional and Anatomical Analyses of Tolerant Rough Lemon and Susceptible Sweet Orange in Response to ‘Candidatus Liberibacter asiaticus’ Infection

2012 ◽  
Vol 25 (11) ◽  
pp. 1396-1407 ◽  
Author(s):  
Jing Fan ◽  
Chunxian Chen ◽  
Qibin Yu ◽  
Abeer Khalaf ◽  
Diann S. Achor ◽  
...  
Keyword(s):  
Sweet Orange ◽  
Phloem Transport ◽  
Differential Sensitivity ◽  
Transcriptional Analysis ◽  
Candidatus Liberibacter Asiaticus ◽  
Transport Activity ◽  
Phloem Cell ◽  
Rough Lemon ◽  
Candidatus Liberibacter ◽  
Liberibacter Asiaticus

Although there are no known sources of genetic resistance, some Citrus spp. are reportedly tolerant to huanglongbing (HLB), presumably caused by ‘Candidatus Liberibacter asiaticus’. Time-course transcriptional analysis of tolerant rough lemon (Citrus jambhiri) and susceptible sweet orange (C. sinensis) in response to ‘Ca. L. asiaticus’ infection showed more genes differentially expressed in HLB-affected rough lemon than sweet orange at early stages but substantially fewer at late time points, possibly a critical factor underlying differences in sensitivity to ‘Ca. L. asiaticus’. Pathway analysis revealed that stress responses were distinctively modulated in rough lemon and sweet orange. Although microscopic changes (e.g., callose deposition in sieve elements and phloem cell collapse) were found in both infected species, remarkably, phloem transport activity in midribs of source leaves in rough lemon was much less affected by HLB than in sweet orange. The difference in phloem cell transport activities is also implicated in the differential sensitivity to HLB between the two species. The results potentially lead to identification of key genes and the genetic mechanism in rough lemon to restrain disease development and maintain (or recover) phloem transport activity. These potential candidate genes may be used for improving citrus tolerance (or even resistance) to HLB by genetic engineering.

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