Prevalent Transmission of 'Candidatus Liberibacter asiaticus' over 'Ca. Liberibacter americanus' in a Long-Term Controlled Environment.

In Brazil, citrus huanglongbing (HLB) is associated with 'Candidatus Liberibacter americanus' (CLam) and 'Ca. Liberibacter asiaticus' (CLas). However, there are few studies about HLB epidemiology when both Liberibacter spp. and its insect vector, the Asian citrus psyllid (ACP, Diaphorina citri), are present. The objective of this work was to compare the transmission of HLB by ACP when both CLam and CLas are present as primary inoculum. Two experiments were performed under screenhouse conditions from April 2008 to January 2012 (experiment 1) and from February 2011 to December 2015 (experiment 2). The experiments were carried out with sweet orange plants infected with CLam or CLas as inoculum source surrounded by sweet orange healthy plants. One hundred Liberibacter-free adult psyllids were monthly confined to the source of inoculum plants for 7 days with subsequent free movement inside the screenhouse. Fortnightly, nymphs and adults of psyllids were monitored. Psyllid and leaf samples were collected periodically for Liberibacter detection by PCR or quantitative PCR. CLas was detected more frequently than CLam in both psyllid and leaf samples. No mixed infections were detected in the psyllids. A clear prevalence of CLas over CLam was observed in both experiments. The final HLB incidences were 16.7 and 14.5% of Liberibacter-positive test plants, and CLas was detected in 92.3 and 93.1% of these infected plants. Mixed infection was observed only in 3.8% of infected test plants in experiment 1. These results endorse the shift in the prevalence of CLam to CLas observed in citrus orchards of São Paulo, Brazil.

The molecular interplay of the establishment of an infection - gene expression of Diaphorina citri gut and Candidatus Liberibacter asiaticus.

BACKGROUND: Candidatus Liberibacter asiaticus (CLas) is one the causative agents of greening disease in citrus, an unccurable, devastating disease of citrus worldwide. CLas is vectored by Diaphorina citri, and the understanding of the molecular interplay between vector and pathogen will provide additional basis for the development and implementation of successful management strategies. We focused in the molecular interplay occurring in the gut of the vector, a major barrier for CLas invasion and colonization. RESULTS: We investigated the differential expression of vector and CLas genes by analyzing a de novo reference metatranscriptome of the gut of adult psyllids fed of CLas-infected and healthy citrus plants for 1-2, 3-4 and 5-6 days. CLas regulates the immune response of the vector affecting the production of reactive species of oxygen and nitrogen, and the production of antimicrobial peptides. Moreover, CLas overexpressed peroxiredoxin, probably in a protective manner. The major transcript involved in immune expression was related to melanization, a CLIP-domain serine protease we believe participates in the wounding of epithelial cells damaged during infection, which is supported by the down-regulation of pangolin. We also detected that CLas modulates the gut peristalsis of psyllids through the down-regulation of titin, reducing the elimination of CLas with faeces. The up-regulation of the neuromodulator arylalkylamine N-acetyltransferase implies CLas also interferes with the double brain-gut communication circuitry of the vector. CLas colonizes the gut by expressing two Type IVb pilin flp genes and several chaperones that can also function as adhesins. We hypothesized biofilm formation occurs by the expression of the cold shock protein of CLas. CONCLUSIONS: The thorough detailed analysis of the transcritome of Ca. L. asiaticus and of D. citri at different time points of their interaction in the gut tissues of the host led to the identification of several host genes targeted for regulation by L. asiaticus, but also bacterial genes coding for potential effector proteins. The identified targets and effector proteins are potential targets for the development of new management strategies directed to interfere with the successful utilization of the psyllid vector by this pathogen.

The Genome of "Candidatus Liberibacter asiaticus" Is Highly Transcribed When Infecting the Gut of Diaphorina citri.

The Asian citrus psyllid, Diaphorina citri, is the vector of the bacterium "Candidatus Liberibacter asiaticus" (Las), associated with the devastating, worldwide citrus disease huanglongbing. In order to explore the molecular interactions of this bacterium with D. citri during the vector acquisition process, cDNA libraries were sequenced on an Illumina platform, obtained from the gut of adult psyllids confined in healthy (H) and in Las-infected young shoots (Las) for different periods of times (I = 1/2 days, II = 3/4 days, and III = 5/6 days). In each sampling time, three biological replicates were collected, containing 100 guts each, totaling 18 libraries depleted in ribosomal RNA. Reads were quality-filtered and mapped against the Chinese JXGC Las strain and the Floridian strain UF506 for the analysis of the activity of Las genome and SC1, SC2, and type 3 (P-JXGC-3) prophages of the studied Las strain. Gene activity was considered only if reads of at least two replicates for each acquisition access period mapped against the selected genomes, which resulted in coverages of 44.4, 79.9, and 94.5% of the JXGC predicted coding sequences in Las I, Las II, and Las III, respectively. These genes indicate an active metabolism and increased expression according to the feeding time in the following functional categories: energy production, amino acid metabolism, signal translation, cell wall, and replication and repair of genetic material. Pilins were among the most highly expressed genes regardless of the acquisition time, while only a few genes from cluster I of flagella were not expressed. Furthermore, the prophage region had a greater coverage of reads for SC1 and P-JXGC-3 prophages and low coverage in SC2 and no indication of activity for the lysis cycle. This research presents the first descriptive analysis of Las transcriptome in the initial steps of the D. citri gut colonization, where 95% of Las genes were active.

Engineered Orange Ectopically Expressing the Arabidopsis ß-Caryophyllene Synthase Is Not Attractive to Diaphorina citri, the Vector of the Bacterial Pathogen Associated to Huanglongbing.

Huanglongbing (HLB) is a destructive disease, associated with psyllid-transmitted phloem-restricted pathogenic bacteria, which is seriously endangering citriculture worldwide. It affects all citrus species and cultivars regardless of the rootstock used, and despite intensive research in the last decades, there is no effective cure to control either the bacterial species (Candidatus Liberibacter spp.) or their insect vectors (Diaphorina citri and Trioza erytreae). Currently, the best attempts to manage HLB are based on three approaches: (i) reducing the psyllid population by intensive insecticide treatments; (ii) reducing inoculum sources by removing infected trees, and (iii) using nursery-certified healthy plants for replanting. The economic losses caused by HLB (decreased fruit quality, reduced yield, and tree destruction) and the huge environmental costs of disease management seriously threaten the sustainability of the citrus industry in affected regions. Here, we have generated genetically modified sweet orange lines to constitutively emit (E)-ß-caryophyllene, a sesquiterpene repellent to D. citri, the main HLB psyllid vector. We demonstrate that this alteration in volatile emission affects behavioral responses of the psyllid in olfactometric and no-choice assays, making them repellent/less attractant to the HLB vector, opening a new alternative for possible HLB control in the field.

Mass spectrometry imaging as a potential technique for diagnostic of Huanglongbing disease using fast and simple sample preparation.

Huanglongbing (HLB) is a disease of worldwide incidence that affects orange trees, among other commercial varieties, implicating in great losses to the citrus industry. The disease is transmitted through Diaphorina citri vector, which inoculates Candidatus Liberibacter spp. in the plant sap. HLB disease lead to blotchy mottle and fruit deformation, among other characteristic symptoms, which induce fruit drop and affect negatively the juice quality. Nowadays, the disease is controlled by eradication of sick, symptomatic plants, coupled with psyllid control. Polymerase chain reaction (PCR) is the technique most used to diagnose the disease; however, this methodology involves high cost and extensive sample preparation. Mass spectrometry imaging (MSI) technique is a fast and easily handled sample analysis that, in the case of Huanglongbing allows the detection of increased concentration of metabolites associated to the disease, including quinic acid, phenylalanine, nobiletin and sucrose. The metabolites abieta-8,11,13-trien-18-oic acid, suggested by global natural product social molecular networking (GNPS) analysis, and 4-acetyl-1-methylcyclohexene showed a higher distribution in symptomatic leaves and have been directly associated to HLB disease. Desorption electrospray ionization coupled to mass spectrometry imaging (DESI-MSI) allows the rapid and efficient detection of biomarkers in sweet oranges infected with Candidatus Liberibacter asiaticus and can be developed into a real-time, fast-diagnostic technique.

Behavioral responses of Diaphorina citri to host plant volatiles in multiple-choice olfactometers are affected in interpretable ways by effects of background colors and airflows.

In several phytophagous hemipterans, behavior appears to be mediated by both visual and chemical cues. For the Asian citrus psyllid (ACP) Diaphorina citri (Hemiptera: Liviidae), olfactometric assays are generally difficult to interpret owing to the low proportion of individuals responding to odors (~30-40%), which compromises the efficiency and reliability of the results of behavioral tests. In the present study, the ACP behavioral response to emitted odors from sweet orange (Citrus sinensis L. Osbeck) flushes in a 4-arm olfactometer using different colors (four white-, two white- and two yellow- on opposite sides, or four yellow-colored fields), and the role of the airflow in the concentration of volatile organic compounds (VOCs) were assessed at two airflows [0.4 and 0.1 L/min (LPM)]. Exposure to 'Pera' sweet orange or clean air in treatments with four yellow-colored-fields increased the response rate of ACP females to the odor sources compared with exposure to 'Pera' sweet orange or clean air in treatments with four white-colored-fields, independently of the odor source and airflow tested. For the assays using two white- and two yellow-colored fields on opposite sides and 0.4 or 0.1 LPM airflow, the residence time of ACP females to odors ('Pera' sweet orange or clean air) was similar or higher in treatments using yellow- than those using white-colored fields. For both assays (VOCs and olfactometric behavioral parameters), the reduction in airflow from 0.4 to 0.1 LPM greatly changed the airborne concentration and ACP behavior. Quantitative chemical analyses revelead that the concentration of most compounds emitted by 'Pera' sweet orange flushes for the headspace using 0.1 LPM airflow were greater than the concentrations measured using 0.4 LPM airflow. Therefore, this treatment design provides an useful tool to assess the ACP behavioral response to the odors from citrus plants, and it can also help in the discrimination of dose-response screenings for VOCs or conspecific insects.

Drench Application of Systemic Insecticides Disrupts Probing Behavior of Diaphorina citri (Hemiptera: Liviidae) and Inoculation of Candidatus Liberibacter asiaticus.

Candidatus Liberibacter asiaticus (CLas) is a phloem-limited bacterium that is associated with the Huanglongbing (HLB) disease of citrus and transmitted by the psyllid, Diaphorina citri. There are no curative methods to control HLB and the prevention of new infections is essential for HLB management. Therefore, the objective of our study was to determine the effects of systemic insecticides, such as the neonicotinoids imidacloprid, thiamethoxam, and a mixture of thiamethoxam and chlorantraniliprole (diamide) on the probing behavior of CLas-infected D. citri and their effect on CLas transmission. The electrical penetration graph (EPG-DC) technique was used to monitor the stylet penetration activities of CLas-infected D. citri on sweet orange [Citrus sinensis (L.) Osbeck] 'Valencia' treated with systemic insecticides. Systemic insecticides disrupted the probing behavior of CLas-infected D. citri, in a way that affected CLas transmission efficiency, particularly by negatively affecting the stylet activities related to the phloem phase. All insecticides reduced (by 57-73%) the proportion of psyllids that exhibited sustainable phloem ingestion (waveform E2 > 10 min), with significant differences observed on plants treated with thiamethoxam and thiamethoxam + chlorantraniliprole. The transmission rate of CLas with high inoculum pressure (five CLas-infected D. citri per plant and a seven-day inoculation access period) to untreated control plants was 93%. In contrast, CLas transmission was reduced to 38.8% when test plants were protected by systemic insecticides. Our results indicated that all insecticides tested presented a potential to reduce CLas inoculation by an average of 59%; therefore, these insecticides can be used to reduce the spread of HLB.

Genetic Diversity of 'Candidatus Liberibacter asiaticus' Revealed by Short Tandem Repeats and Prophage Typing Indicates Population Homogeneity in Brazil.

'Candidatus Liberibacter asiaticus' is the most common huanglongbing-associated bacteria, being present in Asia, South, Central, and North America. Genomic approaches enabled sequencing of 'Ca. L. asiaticus' genomes, allowing for a broader assessment of its genetic variability with the application of polymerase chain reaction (PCR)-based tools such as microsatellite or short tandem repeat (STR) analysis. Although these tools contributed to a detailed analysis of strains from Japan, China, and the United States, Brazilian strains were analyzed in either too few samples with several STRs or in several strains with only a single microsatellite and a single PCR marker. We used 573 'Ca. L. asiaticus' strains, mainly collected from São Paulo State (SPS), in our genetic analyses, employing three STRs and several prophage PCR markers. STR revealed a homogeneous population regardless of sampling year or geographic regions of SPS. Thirty-eight haplotypes were recognized with a predominance of VNTR_005 higher than 10 repeats, with VNTR_002 and VNTR_077 containing 11 and 8 repeats, respectively. This haplotype is indicated as class HE, which comprised 80.28% of strains. Classes HA and HB, predominant in Florida, were not found. A new genomic organization in the junction of prophages SC2 and SC1 is prevalent in Brazilian strains, indicating gene rearrangement and a widespread occurrence of a type 1 prophage as well as the presence of a type 2-like prophage. Our results indicate that 'Ca. L. asiaticus' populations are homogeneous and harbor a new genomic organization in prophages type 1 and 2.

Molecular Characterization and Detection of 16SrIII Group Phytoplasma Associated with Huanglongbing Symptoms.

When huanglongbing (HLB) was found in Brazil in 2004, 'Candidatus Liberibacter americanus' was infecting most of the trees while 'Ca. L. asiaticus' was present in a minor proportion. Currently, 'Ca. L. asiaticus' is the predominant bacterium associated with HLB in citrus trees in São Paulo (SP) and Minas Gerais (MG) States, the major citrus-growing regions in Brazil. A phytoplasma from the 16SrIX group was associated with HLB symptoms in Brazil in 2007, in plants free of Liberibacter spp. In this report, HLB samples testing negative for 'Ca. L. asiaticus', 'Ca. L. americanus', and 16SrIX phytoplasma were infected with 16SrIII phytoplasmas. Coinfection with 'Ca. L. asiaticus' and 16SrIII was also found. The 16S ribosomal RNA (rRNA) gene sequences from 22 samples were obtained and sequenced, confirming that the 16SrIII group phytoplasma is associated with HLB symptoms in SP and MG States. Ten single-nucleotide polymorphisms (SNPs) were found in the 1,427-bp 16S rRNA gene sequences from 16SrIII phytoplasmas from citrus, whereas none was detected in 16S rRNA gene sequences among 16SrIX phytoplasma from citrus. Ribosomal protein (rp) rpsSrplVrpsC gene sequences were amplified with 16SrIII group-specific primers, sequenced from a subset of nine samples, and assembled into three groups based on eight SNPs. SNPs in 16S rRNA gene and rp gene sequences are common in 16SrIII phytoplasmas from other hosts and this phytoplasma group is widespread in South America. 16SrIII phytoplasmas highly related are commonly found in Melia azedarach, a widespread tree in Brazil and Argentina. The finding of a new phytoplasma associated with HLB symptoms belonging to the 16SrIII group reinforces the need to develop diagnostic tools to assess HLB-associated microbiomes.

ß-caryophyllene emitted from a transgenic Arabidopsis or chemical dispenser repels Diaphorina citri, vector of Candidatus Liberibacters.

Production of citrus, the main fruit tree crop worldwide, is severely threatened by Huanglongbing (HLB), for which as yet a cure is not available. Spread of this bacterial disease in America and Asia is intimately connected with dispersal and feeding of the insect vector Diaphorina citri, oligophagous on rutaceous host plants. Effective control of this psyllid is an important component in successful HLB management programs. Volatiles released from the non-host guava have been shown to be repellent to the psyllid and to inhibit its response to citrus odour. By analysing VOC emission from guava we identified one volatile compound, (E)-ß-caryophyllene, which at certain doses exerts a repellent effect on D. citri. Non-host plant rejection mediated by (E)-ß-caryophyllene is demonstrated here by using Arabidopsis over-expression and knock-out lines. For the first time, results indicate that genetically engineered Arabidopsis plants with modified emission of VOCs can alter the behaviour of D. citri. This study shows that transgenic plants with an inherent ability to release (E)-ß-caryophyllene can potentially be used in new protection strategies of citrus trees against HLB.

Influence of culture medium pH on growth, aggregation, and biofilm formation of Xylella fastidiosa.

The major feature of Xylella fastidiosa growing in its hosts, as well as in culture media, is its cellular aggregation and biofilm formation, leading to partial obstruction of the xylem causing water stress in the plant. We report that growth, aggregation, and biofilm formation of X. fastidiosa are influenced by the medium pH. We have verified that X. fastidiosa cell aggregation is reversibly inhibited by decreasing the medium pH from 6.6 to 6.4. Biofilm formation on glass walls was affected as well, and a concomitant decrease in cell multiplication was observed below pH 6.4. The manipulation of culture medium pH can be used as a tool for the cloning of X. fastidiosa strains isolated from plant hosts, because different strains can inhabit the same plant. Also, X. fastidiosa mutants produced by gene manipulation can be isolated from cell aggregates containing transformed and untransformed cells.

Expression and purification of cellulase Xf818 from Xylella fastidiosa in Escherichia coli.

Xylella fastidiosa was the first plant pathogen whose complete genome sequence was available. X. fastidiosa causes citrus variegated chlorosis, but the physiological basis of the disease in unknown. Through comparative sequence analysis, several putative plant cell wall-degrading enzymes were identified on the X. fastidiosa genome. We have cloned Xf818, a putative endoglucanase ORF, into expression vectors pET20b and pET28b, and purified a recombinant form of Xf818 containing a His(6) tag. Through biochemical assays, we have characterized the endoglucanase activity of this protein. The best conditions for hydrolysis over carboxymethyl cellulose (CMC) were on pH 5.2 at 65 degrees C. Xf818 hydrolyzed CMC, acid swollen cellulose, Avicel, birch wood, oat spels xylans, and the oligosaccharides cellotetraose and cellopentaose. Xf818 carried out transglycosylation and had a functional cellulose-binding domain.