Controlled replication of ‘Candidatus Liberibacter asiaticus‘ DNA in citrus leaf discs

‘Candidatus Liberibacter asiaticus’ is a fastidious bacterium and a putative agent of citrus greening disease (a.k.a., huanglongbing, HLB), a significant agricultural disease that affects citrus fruit quality and tree health. In citrus, ‘Ca. L. asiaticus’ is phloem limited. Lack of culture tools to study ‘Ca. L. asiaticus’ complicates analysis of this important organism. To improve understanding of ‘Ca. L. asiaticus’–host interactions including parameters that affect ‘Ca. L. asiaticus’ replication, methods suitable for screening pathogen responses to physicochemical and nutritional variables are needed. We describe a leaf disc-based culture assay that allows highly selective measurement of changes in ‘Ca. L. asiaticus’ DNA within plant tissue incubated under specific physicochemical and nutritional conditions. qPCR analysis targeting the hypothetical gene CD16-00155 (strain A4) allowed selective quantification of ‘Ca. L. asiaticus’ DNA content within infected tissue. ‘Ca. L. asiaticus’ DNA replication was observed in response to glucose exclusively under microaerobic conditions, and the antibiotic amikacin further enhanced ‘Ca. L. asiaticus’ DNA replication. Metabolite profiling revealed a moderate impact of ‘Ca. L. asiaticus’ on the ability of leaf tissue to metabolize and respond to glucose.     

Characterization of an ATP Translocase Identified in the Destructive Plant Pathogen “Candidatus Liberibacter asiaticus”

ATP/ADP translocases transport ATP across a lipid bilayer, which is normally impermeable to this molecule due to its size and charge. These transport proteins appear to be unique to mitochondria, plant plastids, and obligate intracellular bacteria. All bacterial ATP/ADP translocases characterized thus far have been found in endosymbionts of protozoa or pathogens of higher-order animals, including humans. A putative ATP/ADP translocase was uncovered during the genomic sequencing of the intracellular plant pathogen “Candidatus Liberibacter asiaticus,” the causal agent of citrus huanglongbing. Bioinformatic analysis of the protein revealed 12 transmembrane helices and predicted an isoelectric point of 9.4, both of which are characteristic of this family of proteins. The “Ca. Liberibacter asiaticus” gene (nttA) encoding the translocase was subsequently expressed in Escherichia coli and shown to enable E. coli to import ATP directly into the cell. Competition assays with the heterologous E. coli system demonstrated that the translocase was highly specific for ATP and ADP but that other nucleotides, if present in high concentrations, could also be taken up and/or block the ability of the translocase to import ATP. In addition, a protein homologous to NttA was identified in “Ca. Liberibacter solanacearum,” the bacterium associated with potato zebra chip disease. This is the first reported characterization of an ATP translocase from “Ca. Liberibacter asiaticus,” indicating that some intracellular bacteria of plants also have the potential to import ATP directly from their environment.Citrus huanglongbing (HLB), also known as citrus greening, is a disease of citrus that was first reported in China in the early 20th century (33) and identified in the United States in August 2005 in South Florida (22). As it spread rapidly across Florida, HLB has caused substantial economic losses to the citrus industry, and now other citrus-producing states may be in danger as well. The effects of this disease range from mild to severe and include symptoms such as yellow shoots, blotchy mottles on leaves, vein yellowing and corking, lopsided fruit with aborted seeds, early fruit dropping, and limb dieback, which can ultimately lead to the total loss of the infected tree. The disease has been associated with three species of bacteria known as “Candidatus Liberibacter” species. Each of the three “Ca. Liberibacter” species was discovered and named based on its presumptive origin, with “Ca. Liberibacter asiaticus” being found in Asia, “Ca. Liberibacter africanus” in Africa (13), and “Ca. Liberibacter americanus” in South America (24). A fourth species, known as “Ca. Liberibacter solanacearum,” is genetically related, although it is not naturally associated with HLB in citrus plants (16). “Ca. Liberibacter solanacearum” is associated with the emerging zebra chip disease of potatoes and tomatoes (15). “Ca. Liberibacter” species are Gram-negative, fastidious alphaproteobacteria (13) that reside in the sieve tube elements of infected plants (23). The same bacteria found in citrus plants have also been found in two phloem-feeding insects, the Asian citrus psyllid (Diaphorina citri) and the African citrus psyllid (Trioza erytreae), which act as vectors for the disease (for recent reviews, see references 3 and 9). Since insects that carry the pathogen do not have a shortened life span or other adverse effects (12), “Ca. Liberibacter” is thought to act more as an endosymbiont than as a pathogen in insects. There is no known cure for HLB, and current management strategies include elimination of infected trees and methods aimed at vector control. Because of the rapid spread and devastating consequences of infection with “Ca. Liberibacter,” understanding this obligate intracellular pathogen will be critical for the survival of the citrus industry.Recently, the complete genome sequence of “Ca. Liberibacter asiaticus” was obtained via metagenomics (5). Within this “Ca. Liberibacter asiaticus” genome, an open reading frame encoding a putative ATP/ADP translocase was found. Translocases are enzymes that aid in the transport of molecules, in this case adenosine phosphate, across a cell membrane. These adenylate transporters can be placed into one of three groups based upon where they reside. The first group was discovered in mitochondria and is involved in transporting the ATP synthesized in the mitochondrial matrix to the cytosol of the cell (28). The second type of transporter is found in plant plastids (19, 21, 31). In contrast to the mitochondrial transporters, which transport ATP to the cytosol, this set of transporters import ATP from the cytosol. Their function is to provide the stroma with a supply of cytosolic ATP in order to facilitate many of the anabolic reactions that take place there. The third set of transporters was originally discovered in the obligate intracellular bacterium Rickettsia prowazekii (30). Similar to their plastid counterparts, these transporters import ATP from the host cell''s cytosol and translocate it into the bacterial cell. Bacteria that posses this enzyme can act as “energy parasites” and import ATP directly from their hosts.Since its discovery in Rickettsia, the ATP/ADP translocase has been identified in other obligate intracellular parasites of animals, such as Chlamydia psittaci and Lawsonia intracellularis (11, 20), in addition to some protist endosymbionts, such as Caedibacter caryophilus and “Protochlamydia amoebophila” (4, 10). Analyses of the translocase proteins in these bacteria have demonstrated that certain translocase homologs can be used by the cell to import nucleotides other than ATP (2, 4, 10, 26), and thus, the family of proteins has come to be known more generally as nucleotide transporters. In spite of all of the previous research in this area, an ATP/ADP translocase from a bacterial plant pathogen has yet to be characterized. Here, we present the first characterization of a nucleotide transport protein (NttA) from the obligate intracellular plant pathogen “Ca. Liberibacter asiaticus.”     

Diversity of “Candidatus Liberibacter asiaticus,” Based on the omp Gene Sequence

Huanglongbing (yellow dragon disease) is a destructive disease of citrus. The etiological agent is a noncultured, phloem-restricted alpha-proteobacterium, “Candidatus Liberibacter africanus” in Africa and “Candidatus Liberibacter asiaticus” in Asia. In this study, we used an omp-based PCR-restriction fragment length polymorphism (RFLP) approach to analyze the genetic variability of “Ca. Liberibacter asiaticus” isolates. By using five different enzymes, each the 10 isolates tested could be associated with a specific combination of restriction profiles. The results indicate that the species “Ca. Liberibacter asiaticus,” even within a given region, may comprise several different variants. Thus, omp-based PCR-RFLP analysis is a simple method for detecting and differentiating “Ca. Liberibacter asiaticus” isolates.     

The flagella of ‘Candidatus Liberibacter asiaticus’ and its movement in planta

Citrus huanglongbing (HLB) is the most devastating citrus disease worldwide. ‘Candidatus Liberibacter asiaticus’ (Las) is the most prevalent HLB causal agent that is yet to be cultured. Here, we analysed the flagellar genes of Las and Rhizobiaceae and observed two characteristics unique to the flagellar proteins of Las: (i) a shorter primary structure of the rod capping protein FlgJ than other Rhizobiaceae bacteria and (ii) Las contains only one flagellin-encoding gene flaA (CLIBASIA_02090), whereas other Rhizobiaceae species carry at least three flagellin-encoding genes. Only flgJ_Atu but not flgJ_Las restored the swimming motility of Agrobacterium tumefaciens flgJ mutant. Pull-down assays demonstrated that FlgJ_Las interacts with FlgB but not with FliE. Ectopic expression of flaA_Las in A. tumefaciens mutants restored the swimming motility of ∆flaA mutant and ∆flaAD mutant, but not that of the null mutant ∆flaABCD. No flagellum was observed for Las in citrus and dodder. The expression of flagellar genes was higher in psyllids than in planta. In addition, western blotting using flagellin-specific antibody indicates that Las expresses flagellin protein in psyllids, but not in planta. The flagellar features of Las in planta suggest that Las movement in the phloem is not mediated by flagella. We also characterized the movement of Las after psyllid transmission into young flush. Our data support a model that Las remains inside young flush after psyllid transmission and before the flush matures. The delayed movement of Las out of young flush after psyllid transmission provides opportunities for targeted treatment of young flush for HLB control.     

A prophage-encoded effector from “Candidatus Liberibacter asiaticus” targets ASCORBATE PEROXIDASE6 in citrus to facilitate bacterial infection

Citrus huanglongbing (HLB), associated with the unculturable phloem-limited bacterium “Candidatus Liberibacter asiaticus” (CLas), is the most devastating disease in the citrus industry worldwide. However, the pathogenicity of CLas remains poorly understood. In this study, we show that AGH17488, a secreted protein encoded by the prophage region of the CLas genome, suppresses plant immunity via targeting the host ASCORBATE PEROXIDASE6 (APX6) protein in Nicotiana benthamiana and Citrus sinensis. The transient expression of AGH17488 reduced the chloroplast localization of APX6 and its enzyme activity, inhibited the accumulation of reactive oxygen species (H₂O₂ and O₂⁻) and the lipid oxidation endproduct malondialdehyde in plants, and promoted the proliferation of Pseudomonas syringae pv. tomato DC3000 and Xanthomonas citri subsp. citri. This study reveals a novel mechanism underlying how CLas uses a prophage-encoded effector, AGH17488, to target a reactive oxygen species accumulation-related gene, APX6, in the host to facilitate its infection.     

Deciphering the Bacterial Microbiome of Citrus Plants in Response to ‘Candidatus Liberibacter asiaticus’-Infection and Antibiotic Treatments

The bacterial microbiomes of citrus plants were characterized in response to ‘Candidatus Liberibacter asiaticus’ (Las)-infection and treatments with ampicillin (Amp) and gentamicin (Gm) by Phylochip-based metagenomics. The results revealed that 7,407 of over 50,000 known Operational Taxonomic Units (OTUs) in 53 phyla were detected in citrus leaf midribs using the PhyloChip™ G3 array, of which five phyla were dominant, Proteobacteria (38.7%), Firmicutes (29.0%), Actinobacteria (16.1%), Bacteroidetes (6.2%) and Cyanobacteria (2.3%). The {"type":"entrez-protein","attrs":{"text":"OTU62806","term_id":"1193547623","term_text":"OTU62806"}}OTU62806, representing ‘Candidatus Liberibacter’, was present with a high titer in the plants graft-inoculated with Las-infected scions treated with Gm at 100 mg/L and in the water-treated control (CK₁). However, the Las bacterium was not detected in the plants graft-inoculated with Las-infected scions treated with Amp at 1.0 g/L or in plants graft-inoculated with Las-free scions (CK₂). The PhyloChip array demonstrated that more OTUs, at a higher abundance, were detected in the Gm-treated plants than in the other treatment and the controls. Pairwise comparisons indicated that 23 OTUs from the Achromobacter spp. and 12 OTUs from the Methylobacterium spp. were more abundant in CK₂ and CK₁, respectively. Ten abundant OTUs from the Stenotrophomonas spp. were detected only in the Amp-treatment. These results provide new insights into microbial communities that may be associated with the progression of citrus huanglongbing (HLB) and the potential effects of antibiotics on the disease and microbial ecology.     

Predominance of Single Prophage Carrying a CRISPR/cas System in “Candidatus Liberibacter asiaticus” Strains in Southern China

“Candidatus Liberibacter asiaticus” (CLas) is an uncultureable α-proteobacterium associated with citrus Huanglongbing (HLB, yellow shoot disease), a highly destructive disease affecting citrus production worldwide. HLB was observed in Guangdong Province of China over a hundred years ago and remains endemic there. Little is known about CLas biology due to its uncultureable nature. This study began with the genome sequence analysis of CLas Strain A4 from Guangdong in the prophage region. Within the two currently known prophage types, Type 1 (SC1-like) and Type 2 (SC2-like), A4 genome contained only a Type 2 prophage, CGdP2, namely. An analysis on CLas strains collected in Guangdong showed that Type 2 prophage dominated the bacterial population (82.6%, 71/86). An extended survey covering five provinces in southern China also revealed the predominance of single prophage (Type 1 or Type 2) in the CLas population (90.4%, 169/187). CLas strains with two and no prophage types accounted for 7.2% and 2.8%, respectively. In silico analyses on CGdP2 identified a CRISPR (clustered regularly interspaced short palindromic repeats)/cas (CRISPR-associated protein genes) system, consisting of four 22 bp repeats, three 23 bp spacers and 9 predicted cas. Similar CRISPR/cas systems were detected in all 10 published CLas prophages as well as 13 CLas field strains in southern China. Both Type 1 and Type 2 prophages shared almost identical sequences in spacer 1 and 3 but not spacer 2. Considering that the function of a CRISPR/cas system was to destroy invading DNA, it was hypothesized that a pre-established CLas prophage could use its CRISPR/cas system guided by spacer 1 and/or 3 to defeat the invasion of the other phage/prophage. This hypothesis explained the predominance of single prophage type in the CLas population in southern China. This is the first report of CRISPR/cas system in the “Ca. Liberibacter” genera.     

Herbivory by the insect diaphorina citri?induces greater change in citrus plant volatile profile than does infection by the bacterium,Candidatus Liberibacter asiaticus

The volatile organic compound (VOC) profile in plant leaves often changes after biotic and abiotic stresses. Monitoring changes in VOCs in plant leaves could provide valuable information about multitrophic interactions. In the current study, we investigated the effect of Asian citrus psyllid (ACP) infestation, citrus greening pathogen (Candidatus Liberibacter asiaticus [CLas]) infection, and simultaneous attack by ACP and CLas on the VOC content of citrus leaves. Leaf volatiles were extracted using hexane and analyzed with gas chromatography-mass spectrometry (GC-MS). Although ACP is a phloem-sucking insect that causes minimal damage to plant tissues, the relative amount of 21 out of the 27 VOCs increased 2- to 10-fold in ACP-infested plants. The relative amount of d-limonene, β-phelandrene, citronellal, and undecanal were increased 4- to 20- fold in CLas-infected plants. A principle component analysis (PCA) and cluster analysis (CA) showed that VOC patterns of ACP-infested and CLas-infected plants were different from each other and were also different from the controls, while the VOC pattern of double-attacked plants was more like that of the controls than that of ACP-infested or CLas-infected plants. VOC amounts from leaves were compromised when plants were attacked by ACP and CLas. The results of this study showed that a simple direct extraction of citrus leaf volatiles could be successfully used to discriminate between healthy and CLas-infected plants. Information about the effects of insect and pathogen attack on the VOC content profile of plants might contribute to a better understanding of biotic stress.     

Changes in Variable Number of Tandem Repeats in ‘Candidatus Liberibacter asiaticus’ through Insect Transmission

Citrus greening (huanglongbing) is the most destructive citrus disease worldwide. The disease is associated with three species of ‘Candidatus Liberibacter’ among which ‘Ca. Liberibacter asiaticus’ has the widest distribution. ‘Ca. L. asiaticus’ is commonly transmitted by a phloem-feeding insect vector, the Asian citrus psyllid Diaphorina citri. A previous study showed that isolates of ‘Ca. L. asiaticus’ were clearly differentiated by variable number of tandem repeat (VNTR) profiles at four loci in the genome. In this study, the VNTR analysis was further validated by assessing the stability of these repeats after multiplication of the pathogen upon host-to-host transmission using a ‘Ca. L. asiaticus’ strain from Japan. The results showed that some tandem repeats showed detectable changes after insect transmission. To our knowledge, this is the first report to demonstrate that the repeat numbers VNTR 002 and 077 of ‘Ca. L. asiaticus’ change through psyllid transmission. VNTRs in the recipient plant were apparently unrelated to the growing phase of the vector. In contrast, changes in the number of tandem repeats increased with longer acquisition and inoculation access periods, whereas changes were not observed through psyllid transmission after relatively short acquisition and inoculation access periods, up to 20 and 19 days, respectively.     

Prophage-Mediated Dynamics of ‘Candidatus Liberibacter asiaticus’ Populations,the Destructive Bacterial Pathogens of Citrus Huanglongbing

Prophages are highly dynamic components in the bacterial genome and play an important role in intraspecies variations. There are at least two prophages in the chromosomes of Candidatus Liberibacter asiaticus’ (Las) Floridian isolates. Las is both unculturable and the most prevalent species of Liberibacter pathogens that cause huanglongbing (HLB), a worldwide destructive disease of citrus. In this study, seven new prophage variants resulting from two hyper-variable regions were identified by screening clone libraries of infected citrus, periwinkle and psyllids. Among them, Types A and B share highly conserved sequences and localize within the two prophages, FP1 and FP2, respectively. Although Types B and C were abundant in all three libraries, Type A was much more abundant in the libraries from the Las-infected psyllids than from the Las-infected plants, and Type D was only identified in libraries from the infected host plants but not from the infected psyllids. Sequence analysis of these variants revealed that the variations may result from recombination and rearrangement events. Conventional PCR results using type-specific molecular markers indicated that A, B, C and D are the four most abundant types in Las-infected citrus and periwinkle. However, only three types, A, B and C are abundant in Las-infected psyllids. Typing results for Las-infected citrus field samples indicated that mixed populations of Las bacteria present in Floridian isolates, but only the Type D population was correlated with the blotchy mottle symptom. Extended cloning and sequencing of the Type D region revealed a third prophage/phage in the Las genome, which may derive from the recombination of FP1 and FP2. Dramatic variations in these prophage regions were also found among the global Las isolates. These results are the first to demonstrate the prophage/phage-mediated dynamics of Las populations in plant and insect hosts, and their correlation with insect transmission and disease development.     

Effective Antibiotics against ‘Candidatus Liberibacter asiaticus’ in HLB-Affected Citrus Plants Identified via the Graft-Based Evaluation

Citrus huanglongbing (HLB), caused by three species of fastidious, phloem-limited ‘Candidatus Liberibacter’, is one of the most destructive diseases of citrus worldwide. To date, there is no established cure for this century-old and yet, newly emerging disease. As a potential control strategy for citrus HLB, 31 antibiotics were screened for effectiveness and phytotoxicity using the optimized graft-based screening system with ‘Candidatus Liberibacter asiaticus’ (Las)-infected citrus scions. Actidione and Oxytetracycline were the most phytotoxic to citrus with less than 10% of scions surviving and growing; therefore, this data was not used in additional analyses. Results of principal component (PCA) and hierarchical clustering analyses (HCA) demonstrated that 29 antibiotics were clustered into 3 groups: highly effective, partly effective, and not effective. In spite of different modes of actions, a number of antibiotics such as, Ampicillin, Carbenicillin, Penicillin, Cefalexin, Rifampicin and Sulfadimethoxine were all highly effective in eliminating or suppressing Candidatus Liberibacter asiaticus indicated by both the lowest Las infection rate and titers of the treated scions and inoculated rootstock. The non-effective group, including 11 antibiotics alone with three controls, such as Amikacin, Cinoxacin, Gentamicin, Kasugamycin, Lincomycin, Neomycin, Polymixin B and Tobramycin, did not eliminate or suppress Las in the tested concentrations, resulting in plants with increased titers of Las. The other 12 antibiotics partly eliminated or suppressed Las in the treated and graft-inoculated plants. The effective and non-phytotoxic antibiotics could be potential candidates for control of citrus HLB, either for the rescue of infected citrus germplasm or for restricted field application.     

Nicotianamine: mediator of transport of iron and heavy metals in the phloem?

Recent work has demonstrated that minerals in plants are circulated between root and shoot. This occurs during the whole life time and renders possible response to changing environmental conditions. This mineral circulation occurs through intensive solute exchange between xylem and phloem in roots, stems, and leaves. The transport form of heavy metals such as iron, manganes, zinc and copper in the phloem, whether ionic or chelated, is unclear in most cases.
The unusual amino acid nicotianamine (NA) is ubiquitous throughout the plant kingdom. It is a chelator of several divalent transition metals. Its physiological role was investigated with the tomato mutant chloronerva, the only known NA-free multicellular plant. The mutant also exhibits disturbances of its iron metabolism and that of other heavy metals. This leads, among others, to a typical intercostal chlorosis and progressive iron accumulation in the leaves. From the heavy metal chelating properties of NA and from the phenotype of the mutant chloronerva it is concluded that NA is needed for normal distribution of heavy metals in young growing tissues fed via the phloem. This function could be fulfilled by mediating phloem loading or unloading of heavy metals as well as by preventing their precipitation in the alkaline phloem sap. An attempt is made to explain the chloronerva phenotype in the light of the phloem transport hypothesis of chelated iron.     

Infection Density Dynamics of the Citrus Greening Bacterium “Candidatus Liberibacter asiaticus” in Field Populations of the Psyllid Diaphorina citri and Its Relevance to the Efficiency of Pathogen Transmission to Citrus Plants

Huanglongbing, or citrus greening, is a devastating disease of citrus plants recently spreading worldwide, which is caused by an uncultivable bacterial pathogen, “Candidatus Liberibacter asiaticus,” and vectored by a phloem-sucking insect, Diaphorina citri. We investigated the infection density dynamics of “Ca. Liberibacter asiaticus” in field populations of D. citri with experiments using field-collected insects to address how “Ca. Liberibacter asiaticus” infection density in the vector insect is relevant to pathogen transmission to citrus plants. Of 500 insects continuously collected from “Ca. Liberibacter asiaticus”-infected citrus trees with pathological symptoms in the spring and autumn of 2009, 497 (99.4%) were “Ca. Liberibacter asiaticus” positive. The infections were systemic across head-thorax and abdomen, ranging from 10³ to 10⁷ bacteria per insect. In spring, the infection densities were low in March, at ∼10³ bacteria per insect, increasing up to 10⁶ to 10⁷ bacteria per insect in April and May, and decreasing to 10⁵ to 10⁶ bacteria per insect in late May, whereas the infection densities were constantly ∼10⁶ to 10⁷ bacteria per insect in autumn. Statistical analysis suggested that several factors, such as insect sex, host trees, and collection dates, may be correlated with “Ca. Liberibacter asiaticus” infection densities in field D. citri populations. Inoculation experiments with citrus seedlings using field-collected “Ca. Liberibacter asiaticus”-infected insects suggested that (i) “Ca. Liberibacter asiaticus”-transmitting insects tend to exhibit higher infection densities than do nontransmitting insects, (ii) a threshold level (∼10⁶ bacteria per insect) of “Ca. Liberibacter asiaticus” density in D. citri is required for successful transmission to citrus plants, and (iii) D. citri attaining the threshold infection level transmits “Ca. Liberibacter asiaticus” to citrus plants in a stochastic manner. These findings provide valuable insights into understanding, predicting, and controlling this notorious citrus pathogen.     

Characterization of “Candidatus Liberibacter Asiaticus” Populations by Double-Locus Analyses

“Candidatus Liberibacter asiaticus” (CaLas) is associated with citrus Huanglongbing (HLB, yellow shoot disease), which is highly destructive to world citrus production. Understanding the relationships of CaLas isolates from different geographical regions is important for HLB research and development of disease management strategies. In this study, 301 CaLas isolates [85 Brazil, 132 China, and 84 U.S. (83 Florida and 1 California)] were collected, and genomic variations among them were evaluated based on the analyses of two genomic loci: trn1, characteristic of variable tandem repeat numbers (TRNs), and snp1, characteristic of single nucleotide polymorphisms (SNPs). Locus trn1 revealed the homogeneity of all Brazilian isolates, and locus snp1 revealed the homogeneity of most Florida isolates. When the two loci were analyzed simultaneously, i.e., double-locus (DL) analyses, CaLas isolates were clustered mostly according to geographical origins. DL genotype 1 included 97 % of the Chinese isolates, DL genotype 2 included all Brazilian isolates, and DL genotype 3 included 93 % of the U.S. isolates. DL analyses successfully revealed inter-continental overlapping or movement pattern of CaLas isolates. The isolate recently found in California belonged to Asiatic DL genotype 1.     

Convenient Detection of the Citrus Greening (Huanglongbing) Bacterium ‘Candidatus Liberibacter asiaticus’ by Direct PCR from the Midrib Extract

A phloem-limited bacterium, ‘Candidatus Liberibacter asiaticus’ (Las) is a major pathogen of citrus greening (huanglongbing), one of the most destructive citrus diseases worldwide. The rapid identification and culling of infected trees and budwoods in quarantine are the most important control measures. DNA amplification including conventional polymerase chain reaction (PCR) has commonly been used for rapid detection and identification. However, long and laborious procedures for DNA extraction have greatly reduced the applicability of this method. In this study, we found that the Las bacterial cells in the midribs of infected leaves were extracted rapidly and easily by pulverization and centrifugation with mini homogenization tubes. We also found that the Las bacterial cells in the midrib extract were suitable for highly sensitive direct PCR. The performance of direct PCR using this extraction method was not inferior to that of conventional PCR. Thus, the direct PCR method described herein is characterized by its simplicity, sensitivity, and robustness, and is applicable to quarantine testing.     

Is uronic acid oxidase involved in the hormonal regulation of abscission in explants of citrus leaves and fruits?

The role of uronic acid oxidase in abscission was studied in explants of citrus ( Citrus sinensis L. Osbeck; var. Shamouti) leaves and fruits. In leaf explants, activity of uronic acid oxidase prior to onset of abscission and the rate of abscission were markedly accelerated by ethylene and delayed by 2,4-dichlorophenoxyacetie acid. Similar results were obtained for uronic acid oxidase activity in the exocellular fraction of young fruit explants. In mature fruit explants, treated with ethylene, an immediate increase in activity was evidenty in the non-active shoot/peduncle abscission zone, whereas in the calyx abscission zone the rise in activity occurred after a prolonged exposure to ethylene, when most of the fruits had already abscised. Whenever ethylene enhanced uronic acid oxidase activity, 2,4-dichlorophenoxyacetic acid delayed it. A gradient of decreasing activity or uronic acid oxidase was recorded from both sides of the abscission zone in leaves and fruits toward the separation line, where activity was the lowest as compared with the activity found in adjacent tissues. It is suggested that uronic acid oxidase is involved in senescence and cell wall degradation. However, it is yet questionable whether this enzyme is directly related to the control mechanism of abscission.