Publications

4492

HighlightsA portable system based on real-time fluorescence analysis was developed for field detection of Candidatus Liberibacter asiaticus within 40 min from sample to answer.A smartphone-assisted device was designed for easy operation, reliable nucleic acid amplification, and highly sensitive fluorescence detection, with sensitivity comparable to that of a commercial instrument.A novel homemade 3D printed box was used for in-field reagent storage, and it could maintain low temperature (<4°C) for about 8 hours without power supply.This fully integrated system is stable, easy to use, inexpensive, and has great application prospects in resource-limited areas.Abstract. Candidatus Liberibacter asiaticus (Las) is a main causal agent of huanglongbing (HLB), a destructive disease that has greatly reduced citrus yields and quality. Instruments with high sensitivity and portability are urgently required for on-site testing. In this study, a novel sample-to-answer optical system for on-site detection of Las was developed. Three major functions, including DNA extraction, amplification, and detection, are integrated into a portable case. This system mainly consists of (1) a specially designed 3D printed box for on-site reagent storage that can maintain low temperature (below 4°C) for 7.5 h at ambient temperature (35°C); (2) a custom device, called the IF-Device, for DNA amplification and detection of HLB, with an optimized optical structure, a sensitive signal processing circuit, and a precise temperature control algorithm with an accuracy of ±0.1°C; and (3) a battery-based power supply for the whole system. In a typical test using sodium fluorescein as a standard model, the results showed that the sensitivity of this system (1.0 nM) could easily meet the requirements of fluorescence biosensors. The feasibility of this homemade system was evaluated with samples extracted from infected citrus leaves based on the loop-mediated isothermal amplification (LAMP) method, and the limit of detection (LOD) was approximately 1.0 × 10-4 ng µL-1. The whole detection process for eight samples could be simultaneously accomplished within 40 min, and the results could be displayed on a smartphone in real-time. Moreover, the portable case is anti-interference, low cost, and only 2 kg in weight. Considering its sensitivity, stability, and portability, this highly integrated system possesses promising prospects for in-field detection. Keywords: Field detection, Fluorescence biosensor, Huanglongbing, Isothermal amplification, Sample-to-answer.

‘Candidatus Liberibacter asiaticus’ (CLas) is an unculturable phloem-restricted α-proteobacterium associated with huanglongbing (HLB). Here, we provide the genome sequence of CLas strain CoFLP1 from its insect vector Diaphorina citri (Hemiptera: Liviidae) collected in the department of La Guajira, Colombia. The CoFLP1 strain is composed of 1,231,639 bp with G+C 36.5% content. This study reports the first CLas genome sequence from Colombia, which will add to CLas genome resources and help to elucidate our understanding of the introduction pathway of HLB in South America.

iaphorina citri is considered the most dangerous citrus pest because it transmits Candidatus Liberibacter asiaticus, the causal agent of Huanglongbing (HLB). Like other hemiptera insects, D. citri has developed mutualistic interactions with prokaryotic organisms known as endosymbionts. This symbiosis can be obligatory, when the interaction occurs with a primary endosymbiont, or facultative, when it is a secondary endosymbiont. Symbiosis is essential for various physiological functions, but some endosymbionts can adversely affect the psyllid's abilities. D. citri is associated with a great diversity of endosymbionts, with Candidatus Carsonella ruddii, Candidatus Profftella armatura, Candidatus Wolbachia spp. and Candidatus Liberibacter spp standing out. The aim o symbiotic relationship, prokaryotes, Candidatus Wolbachia, transmission of Candidatus Liberibacter asiaticus.

"Candidatus Liberibacter solanacearum" is a gram-negative, fastidious bacterium that inhabits and multiplies in the phloem of host plants. The bacterium causes economically important diseases of solanaceous crops such as "zebra chips" of potatoes, and chlorotic weakening of cultivated plants of the Apiaceae family. Beside potatotes, this pathogen causes significant economic losses in tobacco, peper and tomato production. Insect vectors classified in the family Triozidae play an important role in the epidemiology of this bacterium. Insects transfer bacterial cells from infected to healthy plants by feeding on the plant juice from the phloem tissue. This pathogenic bacterium could be transmitted by infected seeds which result in long-distance spread. Based on the analysis of single nucleotide polymorphism (SNP), nine haplotypes (A, B, C, D, E, F, G, H, U) from different geographical regions, host plants and insect vectors have been described. Considering to impossibility of isolation of this bacterium on a nutrient medium, detection and identification are based on the application of molecular methods (conventional PCR, real-time PCR, multiplex PCR, nested PCR and LAMP PCR). Taking into consideration the increasing spread of "Candidatus Liberibacter solanacearum" in Europe, as well as its recent detection in Serbia, the aim of this paper was to raise an awareness about importance of this bacterium and summarize the protocols and molecular methods for its detection and identification.

Wheat blue dwarf (WBD) is one of the most economically damaging cereal crop diseases in northwestern PR China. The agent associated with the WBD disease is a phytoplasma affiliated with the aster yellows (AY) group, subgroup C (16SrI-C). Since phytoplasma strains within the AY group are ecologically and genetically diverse, it has been conceived that the AY phytoplasma group may consist of more than one species. This communication presents evidence to demonstrate that, while each of the two 16 rRNA genes of the WBD phytoplasma shares >97.5 % sequence similarity with that of the ‘CandidatusPhytoplasma asteris’ reference strain, the WBD phytoplasma clearly represents an ecologically separated lineage: the WBD phytoplasma not only has its unique transmitting vector (Psammotettix striatus) but also elicits a distinctive symptom in its predominant plant host (wheat). In addition, the WBD phytoplasma possesses molecular characteristics that further manifest its significant divergence from ‘Ca. P. asteris’. Such molecular characteristics include lineage-specific antigenic membrane proteins and a lower than 95 % genome-wide average nucleotide identity score with ‘Ca. P. asteris’. These ecological, molecular and genomic evidences justify the recognition of the WBD phytoplasma as a novel taxon, ‘CandidatusPhytoplasma tritici’.

‘Candidatus Liberibacter asiaticus’ (Las) is an unculturable, phloem-limited, insect-transmitted bacterium associated with the Asiatic form of huanglongbing (HLB), the most destructive citrus disease. In Asia and the Americas, it is transmitted by the Asian citrus psyllid (Diaphorina citri Kuwavama). Despite considerable research, little is known about the processes involved in plant infection and colonization by Las. This study was conducted to determine whether the basal portion (below girdling) of the plant is an important route for Las to move laterally from a point of inoculation on a branch to pathogen-free branches elsewhere in the canopy, and to quantify the influence of actively growing tissues on vertical upward (acropetally) or downward (basipetally) movement of Las. Nongirdled and fully or partially girdled stems of potted plants of ‘Pera’ sweet orange, graft-inoculated above or below girdling, were sampled in distinct regions and assessed by qPCR, 6 months postinoculation. Las invaded all regions of partially and nongirdled plants but remained restricted to the inoculated regions of fully girdled plants, evidence that in planta bacterium movement is limited to the phloem. In fully girdled plants, starch accumulated above the girdling site, probably because of changes in flow of phloem sap. To study the influence of actively growing tissues, inoculated ‘Valencia’ sweet orange plants were kept intact or were top- or root-pruned to force production of new tissues, and sampled at 15-day intervals. Las migrated rapidly and most predominantly toward newly developing root and leaf tissues. The rapid and predominant movement of Las to newly developed shoots and roots would explain failures of canopy heat treatments and pruning to cure HLB-affected trees, and reinforces the need to protect rapidly growing new shoots from feeding by D. citri in order to minimize transmission and spread of the pathogen by the vector within and between orchards.