Publications

4374

‘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.

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.

Abstract Candidatus Accumulibacter phosphatis is an important microorganism for enhanced biological phosphorus removal (EBPR). In a previous study, we found a remarkable flexibility regarding salinity, since this same microorganism could thrive in both freshwater- and seawater-based environments, but the mechanism for the tolerance to saline conditions remained unknown. Here, we identified and described the role of trehalose as an osmolyte in Ca. Accumulibacter phosphatis. A freshwater-adapted culture was exposed to a single batch cycle of hyperosmotic and hypo-osmotic shock, which led to the release of trehalose up to 5.34 mg trehalose/g volatile suspended solids (VSS). Long-term adaptation to 30% seawater-based medium in a sequencing batch reactor (SBR) gave a stable operation with complete anaerobic uptake of acetate and propionate along with phosphate release of 0.73 Pmol/Cmol, and complete aerobic uptake of phosphate. Microbial analysis showed Ca. Accumulibacter phosphatis clade I as the dominant organism in both the freshwater- and seawater-adapted cultures (> 90% presence). Exposure of the seawater-adapted culture to a single batch cycle of hyperosmotic incubation and hypo-osmotic shock led to an increase in trehalose release upon hypo-osmotic shock when higher salinity is used for the hyperosmotic incubation. Maximum trehalose release upon hypo-osmotic shock was achieved after hyperosmotic incubation with 3× salinity increase relative to the salinity in the SBR adaptation reactor, resulting in the release of 11.9 mg trehalose/g VSS. Genome analysis shows the possibility of Ca. Accumulibacter phosphatis to convert glycogen into trehalose by the presence of treX, treY, and treZ genes. Addition of trehalose to the reactor led to its consumption, both during anaerobic and aerobic phases. These results indicate the flexibility of the metabolism of Ca. Accumulibacter phosphatis towards variations in salinity. Key points • Trehalose is identified as an osmolyte in Candidatus Accumulibacter phosphatis. • Ca. Accumulibacter phosphatis can convert glycogen into trehalose. • Ca. Accumulibacter phosphatis clade I is present and active in both seawater and freshwater.

‘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.

AbstractCarrot cultivation in Europe is suffering from infections with “Candidatus Liberibacter solanacearum” (CLso), a psyllid‐transmitted bacterial pathogen. In this study, field experiments were carried out in Finland to separately measure the effects of psyllid feeding damage and CLso infection on the carrot root growth and to reveal the dynamics of the spreading of CLso within the field. Most of the experiments were carried out during the summers 2016 and 2017, and a follow‐up sampling was performed in 2018. Carrot psyllid (Trioza apicalis) flight activity was monitored and carrots were sampled at 25 points within the field. Early in the season a clear spatial correlation was found between the sampling sites showing the psyllid feeding damage, that is, leaf‐curling, up to the range of 40–60 m, indicating aggregation behaviour of the psyllids. No CLso infections were detected in the first sampling, which was performed before the psyllid flight peak in both years. Later, a positive correlation between the psyllid feeding damage and the CLso titre was observed. An increase in the CLso titre occurred approximately a month after the psyllid flight peak, and this increase correlated with the accumulating effective temperature sum. In 2016, both the psyllid feeding damage and CLso infection had a significant effect on the carrot root weight. The effect of CLso titre on root weight was nonlinear, that is, it intensified rapidly at the highest bacterial titres. During the colder summer of 2017 the CLso titres did not reach high enough levels in the plants to cause substantial visible symptoms and root growth reduction. Thus, it seems that in the Nordic conditions the effect of CLso infection on carrot yield is strongly dependent on the weather conditions during the growing season.

Sugarcane Grassy Shoot (SCGS) disease is known to be related to Rice Yellow Dwarf (RYD) phytoplasmas (16SrXI-B group) which are found predominantly in sugarcane growing areas of the Indian subcontinent and South-East Asia. The 16S rRNA gene sequences of SCGS phytoplasma strains belonging to the 16SrXI-B group share 98.07 % similarity with ‘Ca. Phytoplasma cynodontis’ strain BGWL-C1 followed by 97.65 % similarity with ‘Ca. P. oryzae’ strain RYD-J. Being placed distinctly away from both the phylogenetically related species, the taxonomic identity of SCGS phytoplasma is unclear and confusing. We attempted to resolve the phylogenetic positions of SCGS phytoplasma based on the phylogenetic analysis of 16S rRNA gene (>1500 bp), nine housekeeping genes (>3500 aa), core genome phylogeny (>10 000 aa) and OGRI values. The draft genome sequences of SCGS phytoplasma (strain SCGS) and Bermuda Grass White leaf (BGWL) phytoplasma (strain LW01), closely related to ‘Ca. P. cynodontis’, were obtained. The SCGS genome was comprised of 29 scaffolds corresponding to 505 173 bp while LW01 assembly contained 21 scaffolds corresponding to 483 935 bp with the fold coverages over 330× and completeness over 90 % for both the genomes. The G+C content of SCGS was 19.86 % while that of LW01 was 20.46 %. The orthoANI values for the strain SCGS against strains LW01 was 79.42 %, and dDDH values were 22. Overall analysis reveals that SCGS phytoplasma forms a distant clade in RYD group of phytoplasmas. Based on phylogenetic analyses and OGRI values obtained from the genome sequences, a novel taxon ‘Candidatus Phytoplasma sacchari’ is proposed.