Candidatus Liberibacter Solanacearum Research Articles

Early plant disease detection by Raman spectroscopy: An open-source software designed for the automation of preprocessing and analysis of spectral dataset

This study introduces a reliable, non-coding software named qREAD-Raman, written in the JavaScript® language, for analyzing and interpreting Raman spectral information. It is designed with a focus on the early detection of diseases in tomato plants (S. lycopersicum) during the asymptomatic stage. The platform integrates a set of machine learning algorithms necessary for the preprocessing consisting of outlier removal, baseline correction, fluorescence removal, smoothing, and normalization. For classification, we applied a Consensus of five different classifiers: Multilayer Perceptron (MLP), Partial Least Squares-Discriminant Analysis (PLS-DA), Linear Discriminant Analysis (LDA), Long Short-Term Memory (LSTM), and K-nearest neighbors (kNN). The experiments were conducted on two bacterial diseases: bacterial canker of tomato induced by Clavibacter michiganesis subsp. michiganensis (Cmm), and the tomato vein-greening associated with Candidatus Liberibacter solanacearum (CLso), a non-culturable bacteria transmitted by Bactericera cockerelli insect. Binary models (Cmm-Healthy and CLso-Healthy) demonstrated excellent classification ability. Asymptomatic Cmm-infected plants were distinguished with an accuracy of 88–95 %, while CLso-infected plants showed an accuracy of 68–77 %. The three-class model (CLso-Cmm-Healthy) exhibited acceptable performance in differentiating between Cmm and CLso, with accuracy rates of 71–83% and 58–67%, respectively. The model's performance highlights differences in the relevant spectral regions associated with the biochemical changes induced by each studied disease. The qREAD-Raman software, implemented for the purpose of this research, was found to be a valuable and comprehensive tool that effectively differentiate diseased tomato plants during their asymptomatic stage.

Unbinding the bindweed psyllid (Bactericera maculipennis [Hemiptera: Triozidae]) from its Convolvulus host exposes it to a novel bacterial symbiont.

Psyllids (Hemiptera: Psylloidea) are herbivores that feed and reproduce on narrow subsets of hosts within a few related genera. During surveys of Solanum umbelliferum (Eschsch) (Solanaceae), we collected multiple life stages of Bactericera maculipennis (Crawford), a species exclusively associated with bindweeds (Convolvulaceae). We hypothesized that B. maculipennis has expanded its host range to include this solanaceous host. To test this, we quantified egg to adult development time on S. umbelliferum, 2 other solanaceous hosts, and Convolvulus arvensis L., the most suitable host for B. maculipennis in North America. B. maculipennis failed to develop on additional solanaceous hosts but developed significantly faster on S. umbelliferum than on C. arvensis. We also sampled for B. maculipennis at 27 S. umbelliferum populations and collected 24 individuals directly from S. umbelliferum plants. We confirmed all individuals are B. maculipennis and found that 10/24 were infected with the plant pathogen 'Candidatus Liberibacter solanacearum'(CLso), which is transmitted by the potato psyllid, B. cockerelli (Šulc). Half of infected individuals harbored CLso haplotype B, which is dominant in crops, but rare in S. umbelliferum. The other 50% harbored CLso haplotype Sumb2, previously documented in S. umbelliferum, but never in crops. Our results suggest that the host range of B. maculipennis has expanded to include a key wild host plant of B. cockerelli. This may create opportunities for exchange of multiple haplotypes of CLso between these 2 species, possibly facilitating theemergence of CLso variants as pathogens of plants in the Convolvulaceae.

The perception and evolution of flagellin, cold shock protein and elongation factor Tu from vector-borne bacterial plant pathogens.

Vector-borne bacterial pathogens cause devastating plant diseases that cost billions of dollars in crop losses worldwide. These pathogens have evolved to be host- and vector-dependent, resulting in a reduced genome size compared to their free-living relatives. All known vector-borne bacterial plant pathogens belong to four different genera: 'Candidatus Liberibacter', 'Candidatus Phytoplasma', Spiroplasma and Xylella. To protect themselves against pathogens, plants have evolved pattern recognition receptors that can detect conserved pathogen features as non-self and mount an immune response. To gain an understanding of how vector-borne pathogen features are perceived in plants, we investigated three proteinaceous features derived from cold shock protein (csp22), flagellin (flg22) and elongation factor Tu (elf18) from vector-borne bacterial pathogens as well as their closest free-living relatives. In general, vector-borne pathogens have fewer copies of genes encoding flagellin and cold shock protein compared to their closest free-living relatives. Furthermore, epitopes from vector-borne pathogens were less likely to be immunogenic compared to their free-living counterparts. Most Liberibacter csp22 and elf18 epitopes do not trigger plant immune responses in tomato or Arabidopsis. Interestingly, csp22 from the citrus pathogen 'Candidatus Liberibacter asiaticus' triggers immune responses in solanaceous plants, while csp22 from the solanaceous pathogen 'Candidatus Liberibacter solanacearum' does not. Our findings suggest that vector-borne plant pathogenic bacteria evolved to evade host recognition.

Long intergenic non-coding RNAs modulate proximal protein-coding gene expression and tolerance to Candidatus Liberibacter spp. in potatoes

Long intergenic non-coding RNAs (lincRNAs) are emerging as regulators of protein-coding genes (PCGs) in many plant and animal developmental processes and stress responses. In this study, we characterize the genome-wide lincRNAs in potatoes responsive to a vascular bacterial disease presumably caused by Candidatus Liberibacter solanacearum (CLso). Approximately 4397 lincRNAs were detected in healthy and infected potato plants at various stages of zebra chip (ZC) disease progression. Of them, ~65% (2844) were novel lincRNAs, and less than 1% (9) were orthologs of Arabidopsis and rice based on reciprocal BLAST analysis, suggesting species-specific expansion. Among the proximal lincRNAs within 50 kbp from a PCG, ~49% were transcribed from the same strand, while ~39% and ~15% followed convergent (head-to-head) and divergent (tail-to-tail) orientations, respectively. Approximately 30% (1308) were differentially expressed following CLso infection, with substantial changes occurring 21 days after infection (DAI). Weighted Gene Co-expression Network Analysis (WGCNA) of lincRNAs and PCGs identified 46 highly correlated lincRNA–PCG pairs exhibiting co-up or co-downregulation. Furthermore, overexpression of selected lincRNAs in transgenic potato hairy roots resulted in perturbation of neighboring PCG expression and conferred tolerance to CLso infection. Our results provide novel insights into potato lincRNAs’ identity, expression dynamics, and functional relevance to CLso infection.

Hiding in Plain Sight: A Widespread Native Perennial Harbors Diverse Haplotypes of 'Candidatus Liberibacter solanacearum' and Its Potato Psyllid Vector.

The unculturable bacterium 'Candidatus Liberibacter solanacearum' (CLso) is responsible for a growing number of emerging crop diseases. However, we know little about the diversity and ecology of CLso and its psyllid vectors outside of agricultural systems, which limits our ability to manage crop disease and understand the impacts this pathogen may have on wild plants in natural ecosystems. In North America, CLso is transmitted to crops by the native potato psyllid (Bactericera cockerelli). However, the geographic and host plant range of the potato psyllid and CLso beyond the borders of agriculture are not well understood. A recent study of historic herbarium specimens revealed that a unique haplotype of CLso was present infecting populations of the native perennial Solanum umbelliferum in California decades before CLso was first detected in crops. We hypothesized that this haplotype and other potentially novel CLso variants are still present in S. umbelliferum populations. To test this, we surveyed populations of S. umbelliferum in Southern California for CLso and potato psyllid vectors. We found multiple haplotypes of CLso and the potato psyllid associated with these populations, with none of these genetic variants having been previously reported in California crops. These results suggest that CLso and its psyllid vectors are much more widespread and diverse in North American natural plant communities than suggested by data collected solely from crops and weeds in agricultural fields. Further characterization of these apparently asymptomatic haplotypes will facilitate comparison with disease-causing variants and provide insights into the continued emergence and spread of CLso.

Mitochondrial Genome Resource of the Cottony Ash Psyllid, a Host of a Newly Identified ‘Candidatus Liberibacter’ Bacterium

Cottony ash psyllid (CAP, Psyllopsis discrepans) is an important, invasive insect pest of ash trees in North America, where it has established populations and is the host of a newly identified strain of ‘ Candidatus Liberibacter solanacearum’. However, not much is known about the diversity of its introduced population. In this study, a CAP mitochondrial genome (mitogenome) sequence was obtained from a collection in Saskatoon, Saskatchewan, Canada. The CAP mitogenome is a circular DNA of 18,824 bp, encoding 13 protein-coding genes, 21 transfer RNA genes, and two ribosomal RNA genes. BLAST search using the CAP mitogenome as a query against the GenBank sequence database showed the mitogenome of Euphyllura phillyreae (15,202 bp) was the most similar (query coverage = 77%; percentage identity = 78.90%). The CAP mitogenome is significantly different from other known psyllid mitogenomes with the presence of a 4,357-bp control region. The mitogenome sequence will further the genomic understanding of CAP. [Formula: see text] The author(s) have dedicated the work to the public domain under the Creative Commons CC0 “No Rights Reserved” license by waiving all of his or her rights to the work worldwide under copyright law, including all related and neighboring rights, to the extent allowed by law, 2024.

First report of ‘Candidatus Liberibacter solanacearum’ in carrot plants in mainland Italy

First report of ‘Candidatus Liberibacter solanacearum’ in carrot plants in mainland Italy

A new lysine biosynthetic enzyme from a bacterial endosymbiont shaped by genetic drift and genome reduction.

The effect of population bottlenecks and genome reduction on enzyme function is poorly understood. Candidatus Liberibacter solanacearum is a bacterium with a reduced genome that is transmitted vertically to the egg of an infected psyllid-a population bottleneck that imposes genetic drift and is predicted to affect protein structure and function. Here, we define the function of Ca. L. solanacearum dihydrodipicolinate synthase (CLsoDHDPS), which catalyzes the committed branchpoint reaction in diaminopimelate and lysine biosynthesis. We demonstrate that CLsoDHDPS is expressed in Ca. L. solanacearum and expression is increased ~2-fold in the insect host compared to in planta. CLsoDHDPS has decreased thermal stability and increased aggregation propensity, implying mutations have destabilized the enzyme but are compensated for through elevated chaperone expression and a stabilized oligomeric state. CLsoDHDPS uses a ternary-complex kinetic mechanism, which is to date unique among DHDPS enzymes, has unusually low catalytic ability, but an unusually high substrate affinity. Structural studies demonstrate that the active site is more open, and the structure of CLsoDHDPS with both pyruvate and the substrate analogue succinic-semialdehyde reveals that the product is both structurally and energetically different and therefore evolution has in this case fashioned a new enzyme. Our study suggests the effects of genome reduction and genetic drift on the function of essential enzymes and provides insights on bacteria-host co-evolutionary associations. We propose that bacteria with endosymbiotic lifestyles present a rich vein of interesting enzymes useful for understanding enzyme function and/or informing protein engineering efforts.

Symbiont infection and psyllid haplotype influence phenotypic plasticity during host switching events

Abstract Many herbivorous insect species exhibit phenotypic plasticity when using multiple hosts, which facilitates survival in heterogeneous host environments. Physiological host acclimation is an important part of it, yet the effects of host acclimation on insect feeding behaviour are not well studied, particularly for insect vectors of plant pathogens. We studied the combined effects of host acclimation and infection with a plant pathogenic symbiont on feeding behaviour of Bactericera cockerelli, an oligophagous psyllid widespread in both crop and natural habitats that feed primarily on Solanaceae and transmit an economically important plant pathogen, Candidatus Liberibacter solanacearum (CLso). We used a factorial design and the electrical penetration graphing technique to disentangle the effects of host acclimation, CLso infection and psyllid haplotype on the within‐plant feeding behaviour of B. cockerelli during conspecific and heterospecific host switches. This approach allows to connect phenotypic plasticity with the role of B. cockerelli as a vector by quantifying the frequency and duration of behaviours involved in CLso transmission. We found significant reductions in multiple metrics of B. cockerelli feeding efficiency, exacerbated by infection with CLso, which could lead to reduced transmission of this pathogen. Psyllid genotype was also important; the Central haplotype exhibited less dramatic changes in feeding efficiency than the Western haplotype during heterospecific host switches. Our study shows that host acclimation and heterospecific host switching directly alter feeding behaviours underlying pathogen transmission, and that the magnitude of feeding efficiency reductions depends on both host genotype and infection status.

A Novel Interaction of Nesidiocoris tenuis (Hemiptera: Miridae) as a Biological Control Agent of Bactericera cockerelli (Hemiptera: Triozidae) in Potato.

Nesidiocoris tenuis (Hemiptera: Miridae) is a generalist predator commonly used to control the whitefly Bemisia tabaci in Europe. This mirid has been found and established in South Texas, where it was initially observed feeding on nymphs of the psyllid Bactericera cockerelli (Hemiptera: Triozidae) in open tomato fields. B. cockerelli is the vector of the fastidious bacterium "Candidatus Liberibacter solanacearum" that causes diseases in several solanaceous crops, including zebra chip (ZC) disease in potatoes. There is a need to better understand how this predator impacts the control of important crop pests, such as potato psyllids. We assessed the interactions between N. tenuis and B. cockerelli in three different environmental settings. First, we estimated the numeric response of N. tenuis preying on B. cockerelli under laboratory and greenhouse conditions. Second, we evaluated the predator-prey interaction under controlled field cage conditions. Then, we exposed N. tenuis under controlled field release conditions to the natural occurrence of B. cockerelli. Finally, we assessed the compatibility between the use of N. tenuis as a biological control agent in a field study and its impact on ZC disease incidence, severity in potato tubers, and potato yield. Laboratory and greenhouse experiments resulted in diverse types of functional model responses, including exponential and linear mathematical models. Our findings revealed a significant predation effect exerted by N. tenuis, resulting in a reduction of more than fourfold in the number of B. cockerelli nymphs per cage. Specifically, the nymphal population decreased from 21 ± 3.2 in the absence of N. tenuis to 5 ± 1.6 when N. tenuis was present. Furthermore, the combination of N. tenuis with a reduced insecticide program increased potato yields, but only reduced ZC tuber incidence in one of two potato cultivars evaluated, and in one season. Findings from these studies indicate that N. tenuis could be effective as a biological control agent for B. cockerelli in potato production in South Texas. This is the first report of N. tenuis preying on immature stages of any psyllid species.

Influence of ‘Candidatus Liberibacter solanacearum’ infection on carrot root weight in Germany

Infection with the bacterium ‘Candidatus Liberibacter solanacearum’ (Lso) is suspected to cause severe damage in carrot leading to high carrot weight loss. This study investigates three main aspects: (i) whether there is a reduction of carrot root weight under field conditions due to Lso infection; (ii) the correlation between Lso infection rate in carrot plants and occurrence of the psyllid Trioza apicalis as the known vector for Lso in carrot, and (iii) the comparison between symptoms described in literature and observed symptoms associated with Lso infection. Therefore, field surveys were conducted from 2018 to 2021 on organically managed carrot fields in Lower Saxony, Germany. Two Rebell orange sticky traps were placed per field replaced on a weekly basis. Captured T. apicalis were morphologically identified and counted. Carrot plant samples were collected from T. apicalis infested fields. Discolouration of foliage and further symptoms on carrots were investigated. For detection of Lso in carrot plants and psyllids, the samples were analysed using PCR. The infection rate of carrot plants varied between 2.5% and 80% per field with low abundance of T. apicalis. Of the asymptomatic plants, 39.4% were infected with Lso, while 80.9% of the symptomatic plants were infected with Lso. The weight of Lso-positive carrot samples was not significantly reduced compared to Lso-negative samples. No economically relevant losses of carrot yield were reported by farmers participating in this study. This indicates that there was no effect of Lso infection on carrot root weight during this study. Of the symptoms associated with a Lso infection in carrots, only leaf discolouration could be confirmed. Overall, the bacterium is present in organically grown carrot plants in Lower Saxony but no major carrot root weight losses were observed.

First Report of the Association of the Psyllid Vector Bactericera trigonica (Hemiptera: Triozidae) with 'Candidatus Liberibacter Solanacearum' in Italy.

Psyllids, members of the family Triozidae, represent a potential threat to the cultivation of solanaceous and apiaceous crops worldwide, mainly as vectors of the phloem-restricted bacterium 'Candidatus Liberibacter solanacearum' (Lso). The Lso haplotypes C, D and E are known to affect apiaceous crops, such as carrot and celery, in several European countries. In Italy, data on the incidence and natural spread of both Lso and psyllids have not been reported so far. In this study, the presence of the vectors was investigated in a main Italian district for carrot production, the "Altopiano del Fucino" area (Central Italy). Both occasional and regular surveys were carried out on a total of five carrot fields and one potato field in 2021 and 2022. Bactericera trigonica (Hodkinson), which is known to efficiently transmit Lso to carrots, was found to be well-established in the area. High levels of population density were recorded in the summer period (more than 100 adult specimens per trap caught every two weeks) and then sharply decreased after the carrot harvest, confirming the strict association of this psyllid species with crop availability. In 2022, 27.5% of the total tested psyllid samples resulted in being positive for Lso haplotypes D and E, the latter being prevalent. This survey revealed, for the first time in Italy, the presence of B. trigonica adults associated with Lso in carrot crops. Although this study was limited to a few fields located in one area, it provided important evidence of the risks for Lso outbreaks and prompted further research to assess the spread and incidence of the disease in apiaceous cultivations in Italy.

Sub-optimal temperatures lead to altered expression of stress-related genes and increased 'Candidatus Liberibacter solanacearum' accumulation in potato psyllid.

The potato psyllid Bactericera cockerelli is the insect vector of the fastidious bacterium 'Candidatus Liberibacter solanacearum'. The bacterium infects both B. cockerelli and plant species, causing zebra chip (ZC) disease of potato and vein-greening disease of tomato. Temperatures are known to influence the initiation and progression of disease symptom in the host plant, and seasonal transitions from moderate to high temperatures trigger psyllid dispersal migration to facilitate survival. 'Ca. L. solanacearum' -infected and uninfected psyllids were reared at previously established 'permissible', optimal, and 'non-permissible' and temperatures of 18°C, 24°C, and 30°C, respectively. Gene expression profiles for 'Ca. L. solanacearum'-infected and -uninfected adult psyllids reared at different temperatures were characterized by Illumina RNA-Seq analysis. Bacterial genome copy number was quantified by real-time quantitative-PCR (qPCR) amplification. Relative gene expression profiles varied in psyllids reared at the three experimental temperatures. Psyllids reared at 18°C and 30°C exhibited greater fold-change increased expression of stress- and 'Ca. L. solanacearum' invasion-related proteins. Quantification by qPCR of bacterial genome copy number revealed that 'Ca. L. solanacearum' accumulation was significantly lower in psyllids reared at 18°C and 30°C, compared to 24°C. Temperature is a key factor in the life history of potato psyllid and multiplication/accumulation of 'Ca. L. solanacearum' in both the plant and psyllid host, influences the expression of genes associated with thermal stress tolerance, among others, and may have been instrumental in driving the co-evolution of the pathosystem.

Physalis virginiana as a Wild Field Host of Bactericera cockerelli (Hemiptera: Triozidae) and 'Candidatus Liberibacter solanacearum'.

The potato/tomato psyllid, Bactericera cockerelli (Šulc), is among the most important pests of solanaceous crops as a vector of the pathogen 'Candidatus Liberibacter solanacearum' (Lso). Lso-infected psyllids often arrive in crop fields from various wild species of Solanaceae and Convolvulaceae, especially those that provide early-season hosts for the vector. Physalis species are perennial plants within the family Solanaceae with often broad geographical distributions that overlap those of B. cockerelli, yet the status of many Physalis species as hosts for B. cockerelli or Lso remains unknown. Our objective was to determine whether wild Physalis species that occur in the potato-growing region of Galeana, Nuevo León, Mexico, host B. cockerelli populations and whether they also are susceptible to Lso. Sampling was carried out in the potato-growing zone of Galeana, Nuevo León, Mexico, where unidentified Physalis spp. are common. In March to October 2021, a wild plant identified as Physalis virginiana was observed; eggs, nymphs, and adults of B. cockerelli were observed on these plants throughout the growing season, and nymphs completed development on these plants under laboratory conditions. Lso also was detected in 22 of the 93 (23.7%) wild P. virginiana plants using conventional PCR, while 13.3% of B. cockerelli adults that emerged from P. virginiana cuttings harbored the pathogen. This is the first report that P. virginiana is a host for B. cockerelli and for Lso. These results suggest that P. virginiana is a likely source of Lso-infected psyllids colonizing solanaceous crops in northeastern Mexico. The importance of P. virginiana and other wild hosts on the population dynamics of the vector and pathogen should be investigated to assist in pest management decision-making.

Insights into Bactericera cockerelli and Candidatus Liberibacter solanacearum interaction: a tissue-specific transcriptomic approach.

The tomato-potato psyllid, Bactericera cockerelli (Šulc), belonging to the Hemiptera order, is an insect pest of solanaceous crops and vectors a fastidious bacterium, Candidatus Liberibacter solanacearum (CLso), the presumptive causal agent of zebra chip and vein greening diseases in potatoes and tomatoes, respectively. The genome of B. cockerelli has been sequenced recently, providing new avenues to elucidate mechanistic insights into pathogenesis in vegetable crops. In this study, we performed RNA-sequencing of the critical psyllid organs (salivary glands and ovaries) involved in CLso pathology and transmission to host plants. Transcriptome analysis revealed differentially expressed genes and organ-specific enrichment of gene ontology (GO) terms related to metabolic processes, response to stress/stimulus, phagocytosis, proteolysis, endocytosis, and provided candidate genes encoding transcription factors (TFs). To examine gene regulatory networks across the psyllid organs under CLso(-) and CLso(+) conditions, we performed weighted gene co-expression network analysis (WGCNA), and unique modules differentiating the psyllid organs were identified. A comparative GO analysis of the unique gene modules revealed functional terms enriched in response to stress, gene regulation, and cell division processes in the ovaries. In contrast, respiration, transport, and neuronal transmission-related GO terms were enriched in the salivary glands. Altogether, this study reveals new insights into tissue-specific expression of the psyllid organs in the absence or presence of CLso bacterium. This knowledge can be leveraged to develop new pest and disease management strategies by delineating the regulatory networks involved in the psyllid-CLso interaction.

Accumulation and Transmission of 'Candidatus Liberibacter solanacearum' Haplotypes by the Nymphs of Two Psyllid Vectors.

'Candidatus Liberibacter solanacearum' (Lso) is a plant pathogenic bacterium transmitted by psyllids that causes significant agricultural damage. Several Lso haplotypes have been reported. Among them, LsoA and LsoB are transmitted by the potato psyllid Bactericera cockerelli and infect solanaceous crops, and LsoD is transmitted by the carrot psyllid B. trigonica and infects apiaceous crops. Several studies evaluated the transmission of these haplotypes by adult psyllids. However, fewer data are available on the transmission of different Lso haplotypes by psyllid nymphs. In this study, we investigated the transmission of these three haplotypes by psyllid nymphs to expand our basic understanding of Lso transmission. Specifically, the objective was to determine if the haplotypes differed in their transmission rates by nymphs and if LsoA and LsoB accumulated at different rates in the guts of nymphs as it occurs in adults. First, we quantified LsoA and LsoB titers in the guts of third- and fifth-instar potato psyllid nymphs. We found similar LsoA titers in the two nymphal stages, while LsoB titer was lower in the gut of the third-instar nymphs compared to fifth-instar nymphs. Second, we assessed the transmission efficiency of LsoA and LsoB by third-instar nymphs to tomato plants, revealing that LsoA was transmitted earlier and with higher efficiency than LsoB. Finally, we examined the transmission of LsoD by carrot psyllid nymphs to celery plants and demonstrated an age-related difference in the transmission rate. These findings provide valuable insights into the transmission dynamics of different Lso haplotypes by nymphal vectors, shedding light on their epidemiology and interactions with their psyllid vectors.

RNA interference-mediated knockdown of genes involved in sugar transport and metabolism disrupts psyllid Bactericera cockerelli (Order: Hemiptera) gut physiology and results in high mortality.

The causal agent of zebra chip of potato and vein-greening diseases of tomato is "Candidatus Liberibacter solanacearum" (CLso), a fastidious bacterium transmitted by the potato psyllid. In the absence of disease-resistant cultivars, disease management has relied on minimizing vector population size to reduce CLso transmission, which requires frequent insecticide applications. There is growing interest in the use of RNA interference (RNAi) technology to supplant traditional insecticides with biopesticides. This requires knowledge of genes essential for insect livelihood whose knockdown leads to significant mortality or other phenotypes. Such candidate genes can be evaluated by reverse genetics approaches to further corroborate predicted gene function. Here, five potato psyllid genes involved in sugar homeostasis in the potato psyllid gut, α-glucosidase1 (AGLU1), aquaporin2 (AQP2), facilitated trehalose transporter1 (TRET1), Trehalase1 (TRE1), and Trehalase2 (TRE2), were investigated as candidates for effective gene silencing. Potato psyllid dsRNAs were designed to optimize knockdown of gene targets. Third instar PoP nymphs were given a 48-hr ingestion-access period (IAP) on individual or groups of dsRNA in 20% sucrose. Mortality was recorded 0, 3, 5, 7, and 9 days post-IAP. Gene knockdown was analyzed 9 days post-IAP by quantitative real-time reverse-transcriptase polymerase chain reaction amplification. The individual or stacked dsRNA combinations resulted in 20-60% and 20-40% knockdown, respectively, while subsequent psyllid mortality ranged from 20-40% to >60% for single and stacked dsRNA combinations, respectively. Reverse genetics analysis showed that simultaneous knockdown of the five selected candidate genes with predicted functions in pathways involved in sugar-homeostasis, metabolism, and -transport yielded the highest mortality, when compared with single or combinations of targets. Results confirmed the functions afforded by psyllid gut genes responsible for osmotic homeostasis and sugar metabolism/transport are essential for livelihood, identifying them as potentially lucrative RNAi biopesticide targets and highlighted the translational relevance of targeting multiple nodes in a physiological pathway simultaneously.

The plant pathogenic bacterium Candidatus Liberibacter solanacearum induces calcium-regulated autophagy in midgut cells of its insect vector Bactericera trigonica.

Autophagy plays an important role against pathogen infection in many organisms; however, little has been done with regard to vector-borne plant and animal pathogens, that sometimes replicate and cause deleterious effects in their vectors. Candidatus Liberibacter solanacearum (CLso) is a fastidious gram-negative phloem-restricted plant pathogen and vectored by the carrot psyllid, Bactericera trigonica. The plant disease caused by this bacterium is called carrot yellows and has recently gained much importance due to worldwide excessive economical losses. Here, we demonstrate that calcium ATPase, cytosolic calcium, and most importantly Beclin-1 have a role in regulating autophagy and its association with Liberibacter inside the psyllid. The presence of CLso generates reactive oxygen species and induces the expression of detoxification enzymes in the psyllid midguts, a main site for bacteria transmission. CLso also induces the expression of both sarco/endoplasmic reticulum Ca2+pump (SERCA) and 1,4,5-trisphosphate receptors (ITPR) in midguts, resulting in high levels of calcium in the cellular cytosol. Silencing these genes individually disrupted the calcium levels in the cytosol and resulted in direct effects on autophagy and subsequently on Liberibacter persistence and transmission. Inhibiting Beclin1-phosphorylation through different calcium-induced kinases altered the expression of autophagy and CLso titers and persistence. Based on our results obtained from the midgut, we suggest the existence of a direct correlation between cytosolic calcium levels, autophagy, and CLso persistence and transmission by the carrot psyllid. IMPORTANCE Plant diseases caused by vector-borne Liberibacter species are responsible for the most important economic losses in many agricultural sectors. Preventing these diseases relies mostly on chemical sprays against the insect vectors. Knowledge-based interference with the bacteria-vector interaction remains a promising approach as a sustainable solution. For unravelling how Liberibacter exploits molecular pathways in its insect vector for transmission, here, we show that the bacterium manipulates calcium levels on both sides of the endoplasmic reticulum membrane, resulting in manipulating autophagy. Silencing genes associated with these pathways disrupted the calcium levels in the cytosol and resulted in direct effects on autophagy and Liberibacter transmission. These results demonstrate major pathways that could be exploited for manipulating and controlling the disease transmission.

A 'Candidatus Liberibacter solanacearum' Haplotype B-Specific Family of Candidate Bacterial Effectors.

'Candidatus Liberibacter solanacearum' (Lso) is a phloem-limited pathogen associated with devastating diseases in members of the Solanaceae and Apiaceae and vectored by several psyllid species. Different Lso haplotypes have been identified, and LsoA and LsoB are responsible for diseases in Solanaceae crops. Our efforts are aimed at identifying pathogenicity factors used by this bacterium to thrive in different hosts. Bacterial secreted proteins can play a role in host colonization or the manipulation of the host immune responses; these proteins are called effectors. In this study, we identified six LsoB-specific proteins with a conserved secretion motif as well as a conserved N-terminal domain in the mature protein. These proteins had different expression and secretion patterns but a similar subcellular localization in Nicotiana benthamiana leaves, suggesting that they play different roles regardless of their conserved secretion motif. One of these proteins, CKC_04425, was expressed at high levels in the insect vector and the host plant, indicating that it could play a role in both the plant and insect hosts, whereas the others were mainly expressed in the plant. One protein, CKC_05701, was able to efficiently suppress programmed cell death and reactive oxygen species production, suggesting that it may have a virulence role in LsoB-specific pathogenesis.

Biology, Ecology, and Management of the Potato Psyllid, Bactericera cockerelli (Hemiptera: Triozidae), and Zebra Chip Disease in Potato.

The potato psyllid, Bactericera cockerelli (Šulc) (Hemiptera: Triozidae), transmits the pathogen "Candidatus liberibacter solanacearum" (Lso), the putative causal agent of zebra chip disease (ZC). ZC is a disease of potato that reduces yield and quality and has disrupted integrated pest management programs in parts of the Americas and New Zealand. Advances in our understanding of the ecological factors that influence ZC epidemiology have been accelerated by the relatively recent identification of Lso and motivated by the steady increase in ZC distribution and the potential for devastating economic losses on a global scale. Management of ZC remains heavily reliant upon insecticides, which is not sustainable from the standpoint of insecticide resistance, nontarget effects on natural enemies, and regulations that may limit such tools. This review synthesizes the literature on potato psyllids and ZC, outlining recent progress, identifying knowledge gaps, and proposing avenues for further research on this important pathosystem of potatoes.