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Four pathogenic bacterial species of the genus ‘Candidatus Liberibacter’, transmitted by psyllid vectors, have been associated with serious diseases affecting economically important crops of Rutaceae, Apiaceae and Solanaceae families. The most severe disease of citrus plants, huanglongbing (HLB), is associated with ‘Ca. Liberibacter asiaticus’ (CaLas), ‘Ca. Liberibacter americanus’ (CaLam) and ‘Ca. Liberibacter africanus’ (CaLaf), while ‘Ca. Liberibacter solanacearum’ (CaLsol) is associated with zebra chip disease in potatoes and vegetative disorders in apiaceous plants. Since these bacteria remain non-culturable and their symptoms are non-specific, their detection and identification are done by molecular methods, mainly based on PCR protocols. In this study, a new quantitative real-time PCR protocol based on TaqMan probe, which can also be performed in a conventional PCR version, has been developed to detect the four known phytopathogenic species of the genus Liberibacter. The new protocol has been validated according to European Plant Protection Organization (EPPO) guidelines and is able to detect CaLas, CaLam, CaLaf and CaLsol in both plants and vectors, not only using purified DNA but also using crude extracts of potato and citrus or psyllids. A comparative analysis with other previously described qPCR protocols revealed that this new one developed in this study is more specific and equally or more sensitive. Thus, other genus-specific qPCR protocols have important drawbacks regarding the lack of specificity, while with the new protocol there was no cross-reactions in 250 samples from 24 different plant and insect species from eight different geographical origins. Therefore, it can be used as a rapid and time-saving screening test, as it allows simultaneous detection of all plant pathogenic species of ‘Ca. Liberibacter’ in a one-step assay.

El Psílido de los cítricos Diaphorina citri kuwayama (Hemiptera: liviidae) es el vector más importante de la bacteria asociada a la enfermedad más grave y destructiva de la citricultura mundial, el Huanglongbing o HLB. En julio de 2021, D. citri se detectó en la región de Emek Jefer (Israel) en una zona donde existen muchas plantaciones de cítricos. La llegada de este Psílido a la cuenca mediterránea, junto con la expansión del otro vector del HLB Trioza erytreae (Del Guercio) (Hemiptera: Triozidae), ya presente en la España peninsular desde 2014, supone la mayor amenaza para la citricultura mediterránea, por tratarse de una enfermedad incurable que podría causar daños catastróficos en los cítricos españoles. En particular, la detección de D. citri en el Mediterráneo hace vislumbra un escenario futuro nada alentador. El potencial biótico de D. citri es mucho mayor que T. erytreae y es, además, capaz de multiplicarse rápidamente sobre las principales variedades y especies de cítricos cultivadas en el Mediterráneo. Por todo ello, es de vital importancia impedir la llegada de D. citri a nuestro territorio y, con el fin de evitar su propagación y conseguir su erradicación, permanecer alerta para actuar con rapidez y eficacia en caso de detección.

En una de las líneas de Félix Morán y Ester Marco-Noales. investigación del equipo de Centro de Protección Vegetal y Biotecnología, Instituto Valenciano de Investigaciones Agrarias (IVIA), Moncada, Valencia, España. Bacteriología del IVIA, y enmarcado dentro del proyecto LIFE Vida For Citrus en el que participan, se está llevando a cabo el diseño y desarrollo de una nueva herramienta de detección molecular, basada en la amplificación isotérmica de ADN mediante el uso de enzimas de recombinación y polimerización, con el objetivo de que pueda llegar a ser un kit de diagnóstico rápido in situ de las tres bacterias asociadas al Huanglongbing.

El HLB es la enfermedad más devastadora de los cítricos que se conoce hoy en día. Se asocia a tres especies bacterianas del género Liberibacter: `Candidatus Liberibacter asiaticus´, `Ca. L. africanus´ y `Ca. L. americanus´. Estas bacterias son transmitidas por insectos vectores de la familia Psyllidae, y no se han podido cultivar hasta el momento en el laboratorio, lo que dificulta en gran medida su estudio y control. La prevención de la diseminación de estas tres especies y de su entrada en áreas libres de la enfermedad, como el continente europeo y, en particular, la cuenca mediterránea, requiere el uso de técnicas de detección específicas y sensibles.

Inability to culture the phloem-restricted alpha-proteobacterium “Candidatus Liberibacter asiaticus” (“Ca. L. asiaticus”) or the closely related species (“Candidatus Liberibacter americanus” and “Candidatus Liberibacter africanus”) that are associated with Huanglongbing (HLB) hampers the development of effective long-term control strategies for this devastating disease. Here we report successful establishment and long-term maintenance of host-free “Ca. L. asiaticus” cultures, with the bacterium growing within cultured biofilms derived from infected citrus tissue. The biofilms were grown in a newly designed growth medium under specific conditions. The initial biofilm-based culture has been successfully maintained for over two years and has undergone over a dozen subcultures. Multiple independent cultures have been established and maintained in a biofilm reactor system, opening the door to the development of pure culture of “Ca. L. asiaticus” and the use of genetics-based methods to understand and mitigate the spread of HLB.

Huanglongbing (ex-greening) disease is one of the most serious diseases of citrus. It is caused by the phloem-limited, gram-negative bacterium “Candidatus Liberibacter spp.”. This bacterium is not well characterized mainly because it is still uncultured. There are two known strains, Asian (“Candidatus Liberibacter asiaticus”) and African (“Candidatus Liberibacter africanus”) that cause severe damage to citrus plants including twig dieback, decline, and death. Symptoms first appear as leaf mottling and chlorosis occurring in one shoot or sector of trees. Later, leaf symptoms resemble nutritional deficiencies (Zn, Ca, and N) that vary depending on the strains, with more severe symptoms caused by “Ca. L. asiaticus”. The Asian strains are transmitted by the Asian citrus psyllid (AsCP), Diaphorina citri, which is present in Brazil. The bacterium has been detected in citrus plants in many geographic locations including China, Japan, Thailand, India, the Philippines, the Arabian Peninsula, and Africa. In 2004, plants showing Huanglongbing symptoms were observed in the Araraquara County, a central region of the State of Sao Paulo, the largest citrus-producing area in Brazil. To verify the presence of “Ca. L. spp.” in these plants, leaf samples of sweet orange cvs. Hamlin and Valencia were used for DNA extraction and polymerase chain reaction amplification using the specific OI1 and Oi2c primers (1). Amplification of the 16S rDNA was positive for 2 (cvs. Hamlin and Valencia) of 10 analyzed plants. The amplified fragments were cloned and sequenced. The amplicons obtained from both plants showed the same sequence, which differed from “Ca. L. africanus”, utilized as the positive control in the amplification experiment (27 divergent bases in 1,160). The sequences were used for BLAST searches, and the results showed identities ranging from 94.71 to 100% with “Ca. L. spp.” sequences available at the National Center for Biotechnology Information database (on-line publication). The highest scores were obtained with “Ca. L. asiaticus sequences. These analyses confirmed the presence of such agent in the State of Sao Paulo. To our knowledge, this is the first report of “Ca. L. asiaticus” in Brazil as well as elsewhere in the Americas. The significance of this report relates to the potential damage that this pathogen could cause to the citrus industry in the largest citrus-producing country in the world. It remains unclear how and when the pathogen entered Brazil. Reference: (1) S. Jagoueix et al. Mol. Cell Probes 10:43, 1996.

Maladie du huanglongbing. Analyse du risque phytosanitaire pour l’Union européenne

Maladie du huanglongbing. Analyse du risque phytosanitaire pour l’Union européenne

Huanglongbing (HLB), previously citrus greening disease, is the most destructive of citrus species causing major threat to the world citrus industry. The disease was reported from China in 1919 and now known to occur in more than 40 different countries of Asia, Africa, South and North America. Three species of gram negative bacterium namely Candidatus Liberibacter asiaticus, Candidatus Liberibacter africanus and Candidatus Liberibacter americanus are the casual organisms of HLB, respectively prevailing in the continent of Asia, Africa and South America. It is one of the most extensively researched subjects in citriculture world. HLB was detected in 2004 and 2005, respectively in San Paulo of Brazil and Florida of USA: the two leading citrus production hub of the world causing huge economic loss within 5 years of first detection. Since then research on HLB detection and management was further accelerated in American continents. This paper presents the scientific advancement made on detection, spread, economic losses caused by HLB in different parts of the world and controlling management strategies. Remarkable achievements have been made on HLB detection techniques including iodine test, qPCR and more recently in spectroscopy. While efforts are being made to develop resistance varieties using conventional and biotechnological tools management strategy which includes reduction of inoculums source, vector control and replant with disease-free planting materials still remains major option for HLB control. Citrus intercropping with guava have shown promising results for vector reduction.Journal of Nepal Agricultural Research Council Vol.1 2015, pp.41-50

Citrus Huanglongbing (HLB) is one of the most devastating citrus diseases worldwide. Sensitive and accurate assays are vital for efficient prevention of the spread of HLB-associated "Candidatus Liberibacter spp". "Candidatus Liberibacter spp" that infect Citrus includes "Candidatus Liberibacter asiaticus" (Las), "Candidatus Liberibacter africanus" (Laf) and "Candidatus Liberibacter americanus" (Lam). Of them, Las is the most widespread species. In this study, a set of nested PCR primer pairs were screened to diagnose Las, and the nested PCR method greatly enhanced the sensitivity to detect Las up to 10 times and 100 times compared to qPCR and conventional PCR, respectively. Totally, 1112 samples from 5 different citrus cultivars in 39 different counties and cities were assayed by nested PCR. The results show that 384 samples were HLB-infected; the highest positive detection rate was 79.7% from the lopsided fruit samples, and the lowest positive detection rate was 16.3% from the apical dieback samples. The results indicate that the designed nested PCR primer pairs can detect Las from different symptomatic tissues, different citrus cultivars and different geographic regions. The set of nested PCR primers designed in the present study will provide a very useful supplementation to the current approaches for Las detection.

Huanglongbing (HLB) is a globally devastating disease of citrus. Presently, three etiological agents are associated with HLB and include; Candidatus Liberibacter asiaticus (CLas), Candidatus Liberibacter americanus; and Candidatus Liberibacter africanus. Attempts to determine alternate (non-Liberibacter) associated etiological agents of HLB have been performed, namely by metagenomic analyses with HLB phenotypic citrus of phloem tissue isolated from bark [1] and whole leaf midribs [2]. These reports indicated a strong correlation for Liberibacter species associated with HLB etiology, but they do not indicate the presence of other significant associated etiological agents. Utilizing both PCR and non-PCR based metagenomic strategies; these previous reports present an undersized view (relating to inherent technique limits, restricted sample scope, and/or bacterial bias) into the etiology of HLB. Here we report ancillary contiguous metagenomic sequences contained amongst whole CLas genome amplification contiguous sequences that were sequenced on the next generation Ion Torrent PGM sequencing system. Within these ancillary sequences, a diverse metagenomic community is present, indicating a rich diversity of both prokaryotic and eukaryotic organisms that are unique and in common within these globally isolated HLB citrus samples. Although this report does not represent a complete metagenomic study of HLB diseased citrus, it does suggest that the use of Ion Torrent PGM sequencing system can be employed for metagenomic analysis. These findings justify a more complete analysis of the metagenome of HLB etiology, which may help further elucidate the HLB disease complex.

Author(s): Bove, Josy | Abstract: It will be recalled how the notions of “heat-sensitive/heat-tolerant HLB” and “African HLB/Asian HLB” were developed. These notions benefited from the possibility to confirm, for the first time, the non-specific HLB symptoms by reliable laboratory techniques: detection of the HLB-associated bacterium by transmission electron microscopy from 1970 onwards to DNA-hybridization and PCR of the liberibacters in the 1990s. With these tools, the early history of citrus HLB in the many countries surveyed could be more precisely described. This presentation also shows or proposes why: (i) African HLB is heat-sensitive and Asian HLB, heat-tolerant; (ii) only one of the seven known liberibacters, namely Candidatus Liberibacter asiaticus (Las), is heat-tolerant, Candidatus Liberibacter africanus (Laf) being a heat-sensitive liberibacter; (iii) African HLB is native to Africa, and Asian HLB is native to Asia; (iv)n “Continental Drift” supports astonishingly well the presence of Laf in Africa andn that of Las in Asia; presence of Las in the Americas is the result of incursions; (v) HLBn is not native to the Arabian Peninsula, but is the result of African and Asian HLB incursions into the peninsula; vi) recent presence of Las in Ethiopia is also the result of an incursion; vii) Candidatus Liberibacter americanus (Lam) in South America is the result of an incursion too; viii) while Laf and Las are of Gondwanan origin, Candidatus Liberibacter europaeus

Background: Citrus Huanglongbing (HLB) is the most devastating bacterial citrus disease worldwide. Three Candidatus Liberibacter species are associated with different forms of the disease: Candidatus Liberibacter asiaticus, Candidatus Liberibacter americanus and Candidatus Liberibacter africanus. Amongst them, Candidatus Liberibacter asiaticus is the most widespread and economically important. These Gram-negative bacterial plant pathogens are phloem-limited and vectored by citrus psyllids. The current management strategy of HLB is based on early and accurate detection of Candidatus Liberibacter asiaticus in both citrus plants and vector insects. Nowadays, real time PCR is the method of choice for this task, mainly because of its sensitivity and reliability. However, this methodology has several drawbacks, namely high equipment costs, the need for highly trained personnel, the time required to conduct the whole process, and the difficulty in carrying out the detection reactions in field conditions. Results: A recent DNA amplification technique known as Loop Mediated Isothermal Amplification (LAMP) was adapted for the detection of Candidatus Liberibacter asiaticus. This methodology was combined with a Lateral Flow Dipstick (LFD) device for visual detection of the resulting amplicons, eliminating the need for gel electrophoresis. The assay was highly specific for the targeted bacterium. No cross-reaction was observed with DNA from any of the other phytopathogenic bacteria or fungi assayed. By serially diluting purified DNA from an infected plant, the sensitivity of the assay was found to be 10 picograms. This sensitivity level was proven to be similar to the values obtained running a real time PCR in parallel. This methodology was able to detect Candidatus Liberibacter asiaticus from different kinds of samples including infected citrus plants and psyllids. Conclusions: Our results indicate that the methodology here reported constitutes a step forward in the development of new tools for the management, control and eradication of this destructive citrus disease. This system constitutes a potentially field-capable approach for the detection of the most relevant HLB-associated bacteria in plant material and psyllid vectors. Fil: Rigano, Luciano Ariel. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Parque Centenario. Instituto de Ciencias y Tecnología "Dr. Cesar Milstein"; Argentina. University of Otago. Department of Microbiology and Immunology; Nueva Zelanda Fil: Malamud, Florencia. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Parque Centenario. Instituto de Ciencias y Tecnología "Dr. Cesar Milstein"; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Parque Centenario. Instituto de Investigaciones Fisiológicas y Ecológicas Vinculadas a la Agricultura; Argentina Fil: Orce, Ingrid Georgina. Instituto de Tecnología Agroindustrial del Noroeste Argentino. Estación Experimental Agroindustrial Obispo Colombres; Argentina Fil: Filippone, María Paula. Instituto de Tecnología Agroindustrial del Noroeste Argentino. Estación Experimental Agroindustrial Obispo Colombres; Argentina Fil: Marano, Maria Rosa. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Rosario. Instituto de Biología Molecular y Celular de Rosario; Argentina Fil: Do Amaral, Alexandre Morais. Ministerio Da Agricultura Pecuaria E Abastecimento de Brasil. Empresa Brasileira de Pesquisa Agropecuaria; Brasil Fil: Castagnaro, Atilio Pedro. Instituto de Tecnología Agroindustrial del Noroeste Argentino. Estación Experimental Agroindustrial Obispo Colombres; Argentina Fil: Vojnov, Adrian Alberto. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Parque Centenario. Instituto de Ciencias y Tecnología "Dr. Cesar Milstein"; Argentina

Maladie du huanglongbing. Analyse du risque phytosanitaire pour l’Union européenne