Candidatus Liberibacter Americanus Research Articles

Oral delivery of double-stranded RNAs induces mortality in nymphs and adults of the Asian citrus psyllid, Diaphorina citri.

The Asian citrus psyllid (ACP), Diaphorina citri Kuwayama, is one of the most important citrus pests. ACP is the vector of the phloem-limited bacteria Candidatus Liberibacter americanus and Candidatus Liberibacter asiaticus, the causal agents of the devastating citrus disease huanglongbing (HLB). The management of HLB is based on the use of healthy young plants, eradication of infected plants and chemical control of the vector. RNA interference (RNAi) has proven to be a promising tool to control pests and explore gene functions. Recently, studies have reported that target mRNA knockdown in many insects can be induced through feeding with double-stranded RNA (dsRNA). In the current study, we targeted the cathepsin D, chitin synthase and inhibitor of apoptosis genes of adult and nymph ACP by feeding artificial diets mixed with dsRNAs and Murraya paniculata leaves placed in dsRNAs solutions, respectively. Adult ACP mortality was positively correlated with the amount of dsRNA used. Both nymphs and adult ACP fed dsRNAs exhibited significantly increased mortality over time compared with that of the controls. Moreover, qRT-PCR analysis confirmed the dsRNA-mediated RNAi effects on target mRNAs. These results showed that RNAi can be a powerful tool for gene function studies in ACP and perhaps for HLB control.

Technological Advances in Huanglongbing (HLB) or Citrus Greening Disease Management

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

Normes OEPP ‐ Diagnostic/EPPO Standards ‐ Diagnostics

Normes OEPP ‐ Diagnostic/EPPO Standards ‐ Diagnostics

PM 7/121 (1) ‘Candidatus Liberibacter africanus’, ‘Candidatus Liberibacter americanus’ and ‘Candidatus Liberibacter asiaticus’

Specific scopeThis Standard describes a diagnostic protocol for ‘Candidatus Liberibacter africanus’, ‘Candidatus Liberibacter americanus’ and ‘Candidatus Liberibacter asiaticus’ and for their detection in their psyllid vectors Diaphorina citri and Trioza erytreae.Specific approval and amendmentFirst approved in 2014‐09.

Candidatus Liberibacter americanus induces significant reprogramming of the transcriptome of the susceptible citrus genotype

Author(s): Mafra, V.; Martins, P. K.; Franscisco, C. S.; Ribeiro-Alves, M.; Freitas-Astua, J.; Machado, M. A. | Abstract: In Brazil, Huanglongbing (HLB) is caused by Candidatus Liberibacter americanus (CaLam) and Ca. L. asiaticus (CaLas). Both species are vectored by the Asian citrus psyllid and are restricted to the phloem of infected citrus, where they promote a severe imbalance in the translocation of nutrients and other important substances along the plant. Several studies of the transcriptional response of citrus to HLB have been reported, but only for infection caused by CaLas. This study evaluated the transcriptional reprogramming of a susceptible genotype (Pera sweet orange) challenged with CaLam, using a customized 385K microarray chip. The analyses showed that a large number of genes and biological processes were significantly altered upon CaLam infection. Among the changes we highlight induction of zinc transporters, modulation of enzymes related to sugar metabolism, decreased photosynthesis, induction of several defense-related genes and modulation of enzymes regulating ROS production. Several biological processes reported as differentially modulated upon infection with CaLas responded similarly to CaLam. The large number of receptor-like proteins, PR genes, NBS-LRR and transcription factors (such as WRKY and MYB) found showed that even a susceptible citrus genotype is able to actively respond to infection by CaLam, as reported for CaLas. Twenty candidate genes were selected for validation in symptomatic and asymptomatic PCR-positive leaves of Hamlin sweet orange infected with CaLas or CaLam. Finally, using in silico approaches, we compared our results with all published studies using CaLas to hypothesize a global feature of the defense/susceptibility mechanisms of citrus in response to the bacteria. These results have been explored in selection of target genes for genetic engineering to control HLB. Also, further transcriptome (RNAseq) experiments using tolerant and susceptible citrus genotypes infected with CaLam or CaLas using different time points are in progress to investigate the dynamics of expression of these genes during early stages of infection.

Clues into the metagenome of Huanglongbing infected Citrus by analysis of ancillary sequences from Ion Torrent whole genome Candidatus Liberibacter asiaticus sequencing

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.

Nutritional Analysis of Flowers from ‘Valencia’ Orange Trees Infected with Huanglongbing

Author(s): Saccini, V. A.V.; Dos Santos, D. M.M.; Medina, C. L.; Machado, R. S.; Cruz, F. J.R. | Abstract: In the mid2000s, Brazil has reported the disease citrus greening or huanglongbing (HLB), considered one of the most destructive diseases in plants by its complexity. As an agent associated with HLB, we have bacteria: Candidatus Liberibacter americanus, Liberibacter asiaticus , Ca. Liberibacter africanus. Symptoms of greening may be masked by other symptoms generated from some diseases, besides such symptoms are similar to those caused by various mineral deficiencies. The objective of this study was to verify if the HLB affects the accumulation of nutrients in citrus flowers. The experiment was in Valencia oranges trees (C. sinensis) on Rangpur lime (C. limonia) with 12 years of age. Treatments consisted of: 1) symptomatic branches of flowers (PCR+), 2) asymptomatic branches of flowers (PCR+) and 3) flowers of healthy plants (PCR-). The levels of macronutrients and micronutrients were measuredn in December of 2011. The results showed lower levels of N, P, K, Ca, Mg, B, Fe, Mn in the flowers of diseased plants (asymptomatic and symptomatic) compared with the levels of certain nutrients in healthy plants. The reduction in nutrient concentration mobile (N, P and K) as not mobile in the phloem (Ca and B) shows that the absorption and distribution of nutrients were reduced in young tissues and justifies the diversity of symptoms found. The nutrients are involved in the activation / inactivation of enzymes related to metabolism and biosynthesis of plant hormone, auxin, gibberellins and cytokinins, which regulate the abortion of plant organs.

Candidatus Liberibacter americanus induces significant reprogramming of the transcriptome of the susceptible citrus genotype

Author(s): Mafra, V.; Martins, P. K.; Franscisco, C. S.; Ribeiro-Alves, M.; Freitas-Astua, J.; Machado, M. A. | Abstract: In Brazil, Huanglongbing (HLB) is caused by Candidatus Liberibacter americanus (CaLam) and Ca. L. asiaticus (CaLas). Both species are vectored by the Asian citrus psyllid and are restricted to the phloem of infected citrus, where they promote a severe imbalance in the translocation of nutrients and other important substances along the plant. Several studies of the transcriptional response of citrus to HLB have been reported, but only for infection caused by CaLas. This study evaluated the transcriptional reprogramming of a susceptible genotype (Pera sweet orange) challenged with CaLam, using a customized 385K microarray chip. The analyses showed that a large number of genes and biological processes were significantly altered upon CaLam infection. Among the changes we highlight induction of zinc transporters, modulation of enzymes related to sugar metabolism, decreased photosynthesis, induction of several defense-related genes and modulation of enzymes regulating ROS production. Several biological processes reported as differentially modulated upon infection with CaLas responded similarly to CaLam. The large number of receptor-like proteins, PR genes, NBS-LRR and transcription factors (such as WRKY and MYB) found showed that even a susceptible citrus genotype is able to actively respond to infection by CaLam, as reported for CaLas. Twenty candidate genes were selected for validation in symptomatic and asymptomatic PCR-positive leaves of Hamlin sweet orange infected with CaLas or CaLam. Finally, using in silico approaches, we compared our results with all published studies using CaLas to hypothesize a global feature of the defense/susceptibility mechanisms of citrus in response to the bacteria. These results have been explored in selection of target genes for genetic engineering to control HLB. Also, further transcriptome (RNAseq) experiments using tolerant and susceptible citrus genotypes infected with CaLam or CaLas using different time points are in progress to investigate the dynamics of expression of these genes during early stages of infection.

Detection of 16Sr IX phytoplasma (HLB phytoplasma) in Sunn Hemp (Crotalaria juncea) in São Paulo State, Brazil

In São Paulo State, besides the occurrence of Candidatus Liberibacter americanus and Ca. L. asiaticus, a 16Sr IX group phytoplasma was associated with HLB symptoms, indistinguishable from those caused by liberibacters. This phytoplasma is called HLB phytoplasma and was found widespread in citrus orchards, although at low incidence. The same phytoplasma was found in Sunn Hemp (Crotalaria juncea) in 2008 and witches’-broom was commonly found associated with 16Sr group IX detection. The aim of this work was to assess the phytoplasma diversity in Sunn Hemp with emphasis at the detection of group 16Sr IX phytoplasma and to establish an association between the occurrence of HLB phytoplasma and symptoms. Sunn Hemp samples were harvested close to the blooming period. Plants were selected in the field when showing symptoms common to phytoplasma infection. We employed universal primers do amplify phytoplasmas in general and group specific primers for 16Sr group IX. PCR products were sequenced to allow grouping of phytoplasmas. We identified five phytoplasmas groups in 48 out of 99 Sunn Hemp plants, belonging to phytoplasma groups 16Sr I, III, VII, IX and XV. The most abundant phytoplasma was the group 16Sr IX, present in 70% of the samples, found in central and north São Paulo State. The occurrence of HLB phytoplasma in Sunn Hemp samples, showing 100% of similarity to the citrus phytoplasma, was highly related to virescence and the second most conspicuous symptom for this infection was witches’-broom.

Variability of direction of tree-to-tree spread of HLB over time

Candidatus Liberibacter americanus and Candidatus Liberibacter asiaticus are two bacterial species that cause huanglongbing (HLB) disease in citrus-growing regions of Brazil. A concentrated sampling plan of a grove in Matao, Brazil was initiated to evaluate the spatial and temporal spread of these bacteria. The exact location of each of 8697 trees was recorded, and each symptomatic tree was assessed by PCR for the presence of Ca. L. americanus and Ca. L. asiaticus during 17 different months from April 2006 to May 2008 (Fig. 1). In the first month, only five trees were confirmed to have Ca. L. asiaticus. The first trees with confirmed cases of Ca. L. americanus were not found until February 2007. By the end of the study, 43 trees were confirmed to have Ca. L. americanus, 1164 trees were confirmed to have Ca. L. asiaticus, and three trees were coinfected.

The complete genome sequence of Candidatus Liberibacter americanus, a bacterium associated with Citrus Huanglongbing in Brazil

We used PFGE followed by CsCl bisbenzymide centrifugation to obtain sufficient DNA for pyrosequencing of the Ca. Liberibacter americanus (Lam) strain “Sao Paulo” genome. The complete circular genomic DNA sequence of Lam is 1,195,201 bp, with an average GC content of 31.12%, somewhat lower than other Liberibacters. There are 1,056 predicted Lam genes, with 1,002 encoding proteins, 9 encoding rRNA genes and 45 encoding tRNAs. The overall gene organization and structure of the Lam genome is more similar to Lso than to Las. There are 951 genes common to Lam, Lso and Las, 27 genes found in Lam and Lso but not Las, and only 8 genes common to Lam and Las but not found in Lso. Many pseudogenes or truncated genes were found among the unique genes of all 3 species. As with Las, two prophage were confirmed in Lam, with SP2 being 39,941 bp and SP1 being 16,398 bp in size; as in Las, the one that appears to replicate as an excision plasmid prophage carries putative lysogenic conversion genes, specifically peroxidases and a Type Vc secreted adhesin. These predicted peroxidases and adhesin were found in both Las and Lam, but appeared fragmentary or degenerated in Lso, indicating their potential for citrus host range determination. Liberibacter genomes seem to be under selective pressure to reduce GC% content and to lose unimportant genes. Although Lam have an outer membrane, most of the genes required for biosynthesis of lipopolysaccharide, which can trigger ROS production, are missing from the Lam genome.

Citrus huanglongbing: validation of Real-Time PCR (qPCR) for the detection of Candidatus Liberibacter asiaticus and Candidatus Liberibacter americanus in Colombia

Citrus huanglongbing (HLB) is the most destructive citrus disease. Two of the three known HLB-associated CandidatusLiberibacter species were recently found to be present in the Americas. In this study, eggs, nymphs and adults of Diaphorinacitri Kuwayama (Hemiptera: Liviidae) and suspect citrus plant materials were collected in 25 municipalities in the departments of Cundinamarca, Santander, Valle del Cauca, Meta and Quindio (Colombia). The detection sensitivity, specificity and assay performance of the 16S rDNA-based real-time PCR (qPCR) were validated for the field survey of the disease in Colombia. The validation confirmed the reliability and robustness of the real-time PCR method for the detection of HLB bacteria in host citrus plant tissues and the vector D. citri. The diagnosis was performed for Candidatus Liberibacter asiaticus (Ca. L. asiaticus) and for Candidatus Liberibacter americanus (Ca. L. americanus) on 168 citrus plant material samples and 239 insect samples. Neither Ca. L. asiaticus nor Ca. L. americanus were detected in the host plants or insects vector, confirming the absence of the disease in the citrus-producing areas of Colombia.

Tissue‐print and squash real‐time PCR for direct detection of ‘Candidatus Liberibacter’ species in citrus plants and psyllid vectors

Huanglongbing (HLB) disease is seriously threatening and/or damaging the citrus industry worldwide. Accurate detection of the three species associated with HLB disease, ‘Candidatus Liberibacter asiaticus’, ‘Candidatus Liberibacter africanus’ and ‘Candidatus Liberibacter americanus’, is essential for the preventive control of the disease. Real‐time PCR is a useful tool for bacterial detection. However, nucleic acid purification steps limit the number of samples that can be processed by PCR. Universal detection of ‘Ca. Liberibacter’ species was achieved by direct tissue‐printing and spotting of plant leaf petiole extracts or squashing of individual psyllids onto paper or nylon membranes. Primers were designed and used with TaqMan chemistry for accurate detection of the bacterium in immobilized targets (prints of 10 overlapping leaf pedicels per tree, or squashed single vectors), by extraction with water and direct use for real‐time PCR. This simplified method was validated and could detect HLB‐liberibacters in 100% of leaves with symptoms and 59% of symptomless leaves collected from HLB‐infected trees. The use of direct assays as template showed good agreement with use of purified DNA (κ = 0·76 ± 0·052). The squash assay allowed detection of the bacterium in 40% of mature Diaphorina citri that fed on leaves of HLB‐infected trees with or without symptoms. A commercial ready‐made kit based on this technology showed 96% accuracy in intra‐laboratory performance studies. The simplified direct methods of sample preparation presented herein can be effectively adopted for use in rapid screening of HLB agents in extensive surveys, certification schemes or for epidemiological and research studies.

Draft Genome Sequence of "Candidatus Liberibacter americanus" Bacterium Associated with Citrus Huanglongbing in Brazil.

We report here the draft genome sequence of “Candidatus Liberibacter americanus” strain PW_SP. The 1,176,071-bp genome, with 31.6% G+C content, comprises 948 open reading frames, 38 tRNAs, and three complete rRNAs.

Widespread occurrence of “Candidatus liberibacter africanus subspecies capensis” in Calodendrum capense in South Africa

Recent studies in citrus orchards confirmed that Citrus Greening, a heat sensitive citrus disease, similar to Huanglongbing (HLB), is associated with the presence of “Candidatus Liberibacter africanus” (Laf) in South Africa. Neither “Candidatus Liberibacter asiaticus” (Las), associated with HLB, “Candidatus Liberibacter americanus”, nor “Candidatus Liberibacter africanus ssp. capensis” (LafC), previously detected in the Western Cape, South Africa on an indigenous Rutaceous species, Calodendrum capense (L. f.) Thunb. (Cape Chestnut), were detected in citrus. The current study aims to determine the potential role of C. capense in the epidemiology of Citrus Greening in South Africa and whether LafC poses a risk to citriculture. A total of 278 C. capense samples were collected throughout South Africa and tested for Liberibacters using real-time PCR. While LafC was found in 100 samples, distributed from all areas where collected, no evidence of Laf infection in any sample was found . The identity of the LafC present was confirmed by sequencing the amplicon derived from conventional PCR of the ß-operon of the ribosomal protein gene region of the first 17 infected trees found and of a representative sample from each district. The Liberibacter status of 44 C. capense and 272 citrus (Midnight Valencia) trees growing in close proximity to each other for over 15 years was determined. Out of 44 C. capense specimens, 43 were infected with LafC, but none of the citrus trees were infected with LafC. Based on the results of this it appears that natural spread of LafC to citrus does not occur.

Influence of temperature on infection and establishment of ‘Candidatus Liberibacter americanus’ and ‘Candidatus Liberibacter asiaticus’ in citrus plants

The objectives of this work were (i) to determine the influence of temperature on infection of citrus by ‘Candidatus Liberibacter asiaticus’ and ‘Candidatus Liberibacter americanus’, the two bacterial species associated with citrus huanglongbing (HLB) in Brazil, and (ii) to determine the influence of temperature on citrus colonization by ‘Ca. L. asiaticus’, which has taken over from ‘Ca. L. americanus’ as the predominant species in Brazil since 2008. Two experiments were carried out with graft‐inoculated Valencia oranges on Rangpur lime rootstocks. Immediately after inoculation the plants were maintained for 423 days in growth chambers under the following night/day temperature conditions: 17/22, 22/27 or 27/32°C, with a dark/light photoperiod of 8/16 h. Infection and colonization of plants were determined using quantitative PCR (qPCR). ‘Candidatus Liberibacter americanus’ did not infect the plants maintained at 27/32°C; however, infection by ‘Ca. L. asiaticus’ occurred at all studied temperatures. Two months after inoculation, ‘Ca. L. asiaticus’ was distributed throughout the inoculated plants, with mean Ct values in the range of 30–31 for leaves and 25–28 for roots. Over time, ‘Ca. L. asiaticus’ reached the highest titres in mature leaves (mean Ct value = 26·7) of citrus plants maintained at 22/27°C. ‘Candidatus Liberibacter asiaticus’ colonization of citrus plants was negatively affected by the daily temperature regime of 27/32°C (mean Ct value in mature leaves = 33·6).

Liberibacters associated with orange jasmine in Brazil: incidence in urban areas and relatedness to citrus liberibacters

Two surveys (2005/2006 and 2009) were conducted in the state of São Paulo, Brazil, to investigate the incidence of ‘Candidatus Liberibacter asiaticus’ and ‘Ca. L. americanus’, two liberibacters associated with citrus huanglongbing (HLB) disease and both transmitted by Diaphorina citri, in orange jasmine (Murraya exotica), a widespread ornamental tree in cities and villages. The graft‐transmissibility of the two species, and their DNA relatedness to citrus‐associated liberibacters, were also investigated. Quantitative PCR was applied to PCR‐positive orange jasmine and HLB‐positive citrus growing in backyards and orchards to assess their inoculum source potentials. Liberibacters were detected in 91 of 786 sampled orange jasmine plants in 10 of 76 sampled locations. PCR‐positive trees exhibited yellow shoots and/or dieback symptoms indistinguishable from those on PCR‐negative trees. ‘Candidatus Liberibacter americanus’ was more common in 2005/2006 (96·6%) and ‘Ca. L. asiaticus’ in 2009 (84·8%). rplJ nucleotide sequences were identical within all populations of either species. Graft transmission succeeded only in homologous host combinations, including ‘Ca. L. americanus’ (2/10) from/to orange jasmine and ‘Ca. L. americanus’ (5/18) and ‘Ca. L. asiaticus’ (5/9) from/to citrus. Symptoms were mild and developed less rapidly in orange jasmine than in citrus, probably as a result of lower liberibacter multiplication rates. Respective titres of ‘Ca. L. americanus’ and ‘Ca. L. asiaticus’ in orange jasmine averaged 4·3 and 3·0 log cells g−1 tissue, compared with 5·5 and 7·3 in citrus. The results indicate that orange jasmine does not favour liberibacter multiplication as much as citrus. However, its importance in HLB epidemics should not be underestimated as it is a preferred host of D. citri and is not under any strict tree‐eradication programme or measures for insect control.

Sulfur volatiles from Allium spp. affect Asian citrus psyllid, Diaphorina citri Kuwayama (Hemiptera: Psyllidae), response to citrus volatiles

The Asian citrus psyllid, Diaphorina citri Kuwayama, vectors Candidatus Liberibacter asiaticus (Las) and Candidatus Liberibacter americanus (Lam), the presumed causal agents of huanglongbing. D. citri generally rely on olfaction and vision for detection of host cues. Plant volatiles from Allium spp. (Alliaceae) are known to repel several arthropod species. We examined the effect of garlic chive (A. tuberosum Rottl.) and wild onion (A. canadense L.) volatiles on D. citri behaviour in a two-port divided T-olfactometer. Citrus leaf volatiles attracted significantly more D. citri adults than clean air. Volatiles from crushed garlic chive leaves, garlic chive essential oil, garlic chive plants, wild onion plants and crushed wild onion leaves all repelled D. citri adults when compared with clean air, with the first two being significantly more repellent than the others. However, when tested with citrus volatiles, only crushed garlic chive leaves and garlic chive essential oil were repellent, and crushed wild onions leaves were not. Analysis of the headspace components of crushed garlic chive leaves and garlic chive essential oil by gas chromatography-mass spectrometry revealed that monosulfides, disulfides and trisulfides were the primary sulfur volatiles present. In general, trisulfides (dimethyl trisulfide) inhibited the response of D. citri to citrus volatiles more than disulfides (dimethyl disulfide, allyl methyl disulfide, allyl disulfide). Monosulfides did not affect the behaviour of D. citri adults. A blend of dimethyl trisulfide and dimethyl disulfide in 1:1 ratio showed an additive effect on inhibition of D. citri response to citrus volatiles. The plant volatiles from Allium spp. did not affect the behaviour of the D. citri ecto-parasitoid Tamarixia radiata (Waterston). Thus, Allium spp. or the tri- and di-sulphides could be integrated into management programmes for D. citri without affecting natural enemies.

Control of Huanglongbing (HLB) disease with reference to its occurrence in Malaysia

The center of diversity for citrus was originally found on the northeastern India, eastward through the Malay Archipelago and south to Australia. Today citrus is produced in 140 countries, mainly between the north and south 40o latitudes. Citrus ranked first among fruit crops in the international trade based on value. Citrus production of the world is around 105 million tons per year. Orange ( Citrus sinensis ) accounts for almost two thirds of the total citrus production (65%), followed by tangerine ( Citrus reticulata ) (21%), lemon ( C. limon ) (6%) and grapefruit ( C. paradisi ) (5.5%). Other significant commercially grown species are lime ( C. aurantifolia ), pummelo ( C. grandis ) and citron ( C. medica ). The largest citrus producers are Brazil (20%), United States (14%), China (12%), Mexico (6%) and the countries of the mediterranean basin (15%). Humidity and day-to-night temperature fluctuations influence which varieties are best adapted to an area. Most citrus fruits are produced for fresh market consumption and only around 30% is processed. Fresh fruits are rich in vitamin C which plays a vital role in prevention of scurvy. After extraction of the juice, the skin and fruit pulp can be used as livestock feed or making compost. The rind acid (oil) of the citrus is considered an expensive commodity in international trade (F.A.O 2003). Unfortunately, the citrus industry is threatened by 2 destructive diseases namely Witches’ Broom disease of lime (WBDL) and Huanglongbing (HLB) disease. WBDL has been known to be caused by Candidatus phytoplasma aurantifolia . It is one of the destructive diseases on citrus industry in the Middle East, India and Pakistan. It was reported in 1970 for the first time from lime orchard of Oman, united Arabian emirate (UAE) (1988), Iran (1998) and India and Pakistan (1999). WBDL is a phloem limited phytoplasma disease of lime. HLB disease causing citrus greening ( Candidatus liberibacter spp.) is the second most severe disease on citrus industry all over the world. HLB has destroyed an estimated 60 million trees in Africa and Asia. More than 40 countries were infected by HLB in Africa, Asia and USA (Chau et al., 1996; Bove, 2006; Roux et al., 2006; Batool et al., 2007). The HLB pathogens are highly fastidious phloem-inhabiting bacteria in the genus Candidatus liberibacter . The bacteria have not been cultured yet in laboratory media and do not survive outside the host cells. Three types of phloem limited bacteria causing HLB disease have been described and identified (Bove, 2006). The isolate from South Africa ( Candidatus liberibacter africanus ) is considered heat-sensitive and found in Africa. It is vectored by African citrus psyllid ( Trioza erytreae) and was described by Guercio in 1918. The isolate from Asia ( Candidatus liberibacter asiaticus ) is more severe and widespread; it is vectored by Asian psyllid (Diaphorina citri Kuwayama) (Garnier et al., 2000). This type of HLB is heat-tolerant (Garnier et al., 2000). It can show the symptom on humid, cool and hot temperature, up to 35oC (Garnier et al., 2000; Bove 2006; Le Roux et al., 2006). The isolate from America has been named Candidatus liberibacter americanus ; it was detected in Brazil and Florida (Coletta-Filho et al., 2005; Texeira et al., 2005).

RRNA operons and genome size of 'Candidatus Liberibacter americanus', a bacterium associated with citrus huanglongbing in Brazil

Huanglongbing is one of the most severe diseases of citrus worldwide and is associated with 'Candidatus (Ca.) Liberibacter africanus' in Africa, 'Ca. Liberibacter asiaticus' in Asia and the Americas (Brazil, USA and Cuba) and 'Ca. Liberibacter americanus' (Lam) in Brazil. In the absence of axenic cultures, genetic information on liberibacters is scarce. The sequences of the entire 23S rRNA and 5S rRNA genes from Lam have now been obtained, using a consensus primer designed on known tRNAMet sequences of rhizobia. The size of the Lam genome was determined by PFGE, using Lam-infected periwinkle plants for bacterial enrichment, and was found to be close to 1.31 Mbp. In order to determine the number of ribosomal operons on the Lam genome, probes designed to detect the 16S rRNA gene and the 3' end of the 23S rRNA gene were developed and used for Southern hybridization with I-CeuI-treated genomic DNA. Our results suggest that there are three ribosomal operons in a circular genome. Lam is the first liberibacter species for which such data are available.