The resolution of the present study is relatively low compared to the 70% resolution reported by Fazekas et al. or other plant groups . Factors specific to the evolution of the Pulsatilla and/or the sampling strategy of this study may affect the ability to discriminate between species. Such factors include incomplete lineage sorting and hybridization, the rapid radiation of Pulsatilla species, the variation present at the barcode loci, and the sampling density used in this study. Unlike animal species, many plant species have paraphyletic or polyphyletic origins due to the higher frequency of reticulate evolution, which is facilitated by hybridization and polyploidization . Given that this is the case, barcoding based solely on plastid markers may not reliably distinguish species. For example, in our study, some species are resolved to paraphyletic groups, such as P. patens. In these cases, the use of nuclear DNA sequences may improve the resolution among plant species because nuclear loci have higher overall synonymous substitution rates, thus making nuclear markers such as ITS more sensitive. In our study, P. patens samples, and formed a monophyletic group with sample P. latifolia. However, these samples were not clustered together by chloroplast marker data .Manipulating the innate immune system of insects as a means of inhibiting vector acquisition of pathogens is currently being explored in many insect vectors of human pathogens. To apply these techniques to hemipteran vectors of plant pathogens, the immune systems of these insects must first be understood. The two hemipterans that have been investigated, the pea aphid and brown plant hopper,square pot have differing immune capabilities; with the brown plant hopper having a robust immune system similar to other insects; while the pea aphid has a drastically reduced immune system.
To identify if immune manipulation could be a technique used to inhibit the Asian citrus psyllid transmission of Candidatus Liberibacter asiaticus , we annotated the immune genes of ACP and exposed ACP to different classes of bacteria. Genome annotation revealed a substantially reduced immune system in ACP with an absence of recognition proteins in the classes PGRP and GNBP, the majority of the IMD pathway, and few genes for antimicrobial effectors. These results suggest that ACP have a particularly reduced immune response against gram-negative bacteria. When ACP were exposed to either gram-negative bacteria or grampositive bacteria through oral infections or cuticular punctures, significantly increased mortality was observed in response to gramnegative bacteria. Taken together, the genetic data and the controlled infection results indicate that ACP are not able to detect or eliminate gram-negative bacteria such as CLas. Although innate immune manipulation may have limited utility for inhibiting CLas acquisition by ACP based on these findings, microbial insecticides may prove to be an effective control technique for ACP. Currently, HLB is associated with Candidatus Liberibacter spp., although several phytoplasmas have been found in citrus showing HLBlike symptoms. The aim of this study was to determine if, in addition to Ca. L. asiaticus , phytoplasma species are also associated with HLB in citrus of Mexico. Citrus plants showing HLB-like symptoms were collected in the Mexican States of Nayarit, Colima, and Sinaloa between August 2011 and September 2012. Samples were evaluated for CP and CLas by nested-PCR and conventional PCR, respectively. For actual RFLP, phytoplasma fragments were digested with restriction endonucleases and fractionated using a QIAxcel system . Virtual RFLP analysis was performed on the 16SrDNA sequences using the virtual gel plotting program pDRAW32 . Phylogenetic trees were constructed with the NeighborJoining method, using the MEGA program . A total of 86 HLB-symptomatic samples of Mexican lime , Persian lime , and Valencia sweet orange Osbeck were analyzed. Diffuse chlorosis, blotchy mottle and vein yellowing were symptoms observed, even though we were unable to clearly identify symptoms specifically associated with either bacterium alone or together. Fifty-four out of 86 citrus plants were positive for CLas, 20 were positive for CP, seven were found in mixed infections with both pathogens, and 19 samples were negative for CLas and phytoplasmas.
Actual and virtual RFLP analyses of the 16S rDNA sequences enabled us to classify two HLB phytoplasma strains as members of the aster yellows group Candidatus Phytoplasma asteris , the subgroup B from Nayarit and subgroup S from Colima and Sinaloa, which was confirmed by phylogenetic analysis. In addition, the partial CLas sequences were identical to the strains isolated from several countries affected by HLB. These results confirm the association of CPa with HLB in citrus in Mexico. Huanglongbing , presumably caused by Candidatus Liberibacter asiaticus , is a devastating citrus disease associated with of flavor in orange juice. Relative CLas titer was determined by analyzing the 16S rDNA gene and the LJ primer targeting the CLas prophage in fresh or processed/pasteurized orange juice using qPCR. A method was developed that yielded large quantities of highly purified DNA, using only a small quantity of juice, which was then compared to the sensory characteristics of the juice using linear regression. To this end, orange juice was centrifuged and the pellet was used for qPCR analysis. After lysing the cells in an alkaline and non-ionic detergent Tris-based buffer, the initial DNA precipitation step was accomplished using hexdecetyl trimethyl ammonium bromide and sodium chloride at low concentration to remove polysaccharides like pectin. A trained sensory panel analyzed the same juice for various descriptors for flavor, aroma, mouthfeel, and aftertaste and the chemical components of the juice were also evaluated. By using multiple dilutions of a known amount of standard DNA, a standard curve was generated for log concentration of sensory descriptors against Ct. The amount of nucleic acids in an unknown sample can then be calculated from its Ct value. For the Li primers, Ct values between 35 and 30 indicated a minor decrease in juice flavor quality, but below 30 indicated a more significant flavor decline. Values below 30 indicated flavor decline for the LJ primer in relation to sweet taste or overall orange flavor and conversely to typical HLB flavor scores . This technology also worked for off-flavor causing microorganisms and human pathogens, for example Alicyclobacillus acidoterrestris or Escherichia coli, respectively, in orange juice or apple cider.An opportunity to analyze spatial patterns to determine the underlying biological process has developed from the widespread sampling and testing of Asian citrus psyllids in Texas and California to locate early infections of Huanglongbing disease. The real-time polymerase chain reaction diagnostic methods used to detect the casual agents of HLB, Candidatus Liberibacter asiaticus, are set to run for 40 cycles. The reaction must surpass a set threshold prior to the completion of the run to be considered positive for the presence of CLas.
When diagnostic testing of ACP initially started in Texas and California, the threshold had to be reached at <32 cycles and then later was raised to <37 cycles. Currently,black plastic pots for plants reactions that surpass the threshold at 37 or more cycles have proven impossible to acquire confirmatory conventional PCR bands and DNA sequence data. Thus, these samples are classified as inconclusive. The question we are trying to answer is whether information in the Ct-values between 37 and 40 is also useful for predicting locations with HLBinfected citrus plants. Analysis of 2013 data from California indicates that psyllid samples with high Ct-values tend to be clustered at close ranges and then dispersed at larger scales. Clustering of samples with Ct-values in the 38-39 range is within 1 km of samples with Ct-values below 37. In both Texas and California, spatial clustering of psyllid samples with inconclusive Ct-values have been shown to cluster around known positive HLB-infected trees. One of the main obstacles in advancing the search for antimicrobials against ‘Candidatus Liberibacter asiaticus’ is the inability to culture this pathogen to perform classical growth inhibition tests. Consequently, we centered our research in the identification of chemicals that inactivate critical physiological pathways operating in this bacterium. To achieve our goal, the experimental design used in our laboratories consisted of a deep in silico multifactorial genomic analysis, followed by a conventional biophysical screening of selected targets. Using this approach, we identified new chemicals that targeted a transcriptional regulator belonging to the MarR-family in CLas. The DNA binding sequence of LdtR was identified via DNase I footprinting. In silico analyses using this binding site indicated that LdtR modulates the expression of several genes involved in cell division and cell wall biosynthesis. A gene of particular interest within the regulon is ldtP, which is predicted to encode for an L,D-transpeptidase. This enzyme is involved in the modification of the bacterial cell wall using an alternative pathway and it is resistant to the activity of b-lactam antibiotics. A recombinant LdtP enzyme has been purified and used as a target to identify potential enzymatic inhibitors. Herein, we present the impact of the identified new antimicrobials on the regulatory activity of LdtR and on the enzymatic activity of LdtP. The compounds identified represent an important advance in the identification of chemicals to be used as bactericidal in planta. The results herein described provide important insights to understand CLas regulatory networks and molecular foundations for the design of therapeutics for the treatment of this devastating disease. Since HLB was first reported in Brazil , the EEAOC implemented and strengthened different lines of work in four strategic areas. Provision of sanitized and certified vegetal material: EEAOC is the only sanitation center in northwestern Argentina and is in charge of providing sanitized and certified material to citrus nurseries. Observation and monitoring: a molecular diagnosis laboratory was set up for Candidatus Liberibacter spp. detection for both vegetal and insect samples.
We visually inspected citrus trees from 44 places located in Northwest Argentina and collected leaf samples, displaying HLB-similar symptoms, and insect samples. From 2005 to present, 12,500 samples were analyzed by qPCR TaqMan, 50% corresponded to citrus leaf, 46% to insect, and the remaining 4% to ornamental plants. All samples were negative. To date, there is no evidence that Candidatus Liberibacter spp. are in the NOA; although, 3,000 sticky traps for Diaphorina citri were set across the citrus area in Tucumán province and checked periodically. Furthermore, citrus plantation and alternative hosts in the urban area were monitored too. This monitoring was conducted on a weekly basis, but was intensified in spring and summer. In April 2011, the abovementioned activities resulted in the detection of 11 D. citri specimens on Murraya paniculata plants from the urban area. This case has remained under effective control by the national and provincial phytosanitary authorities and has not led to any subsequent reports. As for the citrus area, over 600,000 shoots and approximately 25,000 colored sticky traps have been checked without detecting the insect vector. These results prove that Tucumán province is free from D. citri. In the north of NOA , where the insect is present, the efficiency of 17 active ingredients was evaluated along with their correspondent fresh fruit residue analysis.One of the principal challenges in managing rapidly spreading epidemics is to identify optimal control strategies. How, when, where, and which control methods should be used to manage disease effectively. Epidemiological modelling can help by providing a means to integrate the current status of knowledge and to provide a set of tools to compare the effectiveness of different control scenarios. The epidemiological approach is also well-suited, taking account of uncertainties in order to inform a risk‐based management of disease. Drawing upon experience gained from modelling a range of emerging pests and pathogens including Huanglongbing and Asian Citrus Psyllid , I propose to show how an ‘epidemiological toolbox’ can be used to predict disease and vector spread, to analyse the effectiveness of control and to compare ‘what‐if’ scenarios for disease management. Successful control of disease requires us to match the scale of control with the inherent spatial and temporal scales of the epidemic. Identification and characterisation of the epidemic scales involve the formulation of mathematical models that capture the essential biological features of disease spread. The spatial distribution of hosts in the landscape is also important along with the effects of environmental variables and anthropomorphic activity, and the degree of stakeholder compliance. Using data for the spread of disease in Southern Gardens and data from psyllid traps in California, I propose to show how it is possible to extract signatures in the form of epidemiological parameters such as transmission rates and dispersal kernels for epidemic and vector spread.