Spider venom neurotoxin based bioinsecticides: A novel bioactive for the control of the Asian citrus psyllid Diaphorina citri (Hemiptera).

The Asian citrus psyllid, Diaphorina citri Kuwayama (Hemiptera: Psyllidae), is a key vector of the phloem-limited bacteria Candidatus Liberibacter asiaticus (CLas) associated with huanglongbing (HLB), the most serious and currently incurable disease of citrus worldwide. Here we report the first investigation into the potential use of a spider venom-derived recombinant neurotoxin, ω/κ-HxTx-Hv1h (hereafter HxTx-Hv1h) when delivered alone or when fused to snowdrop lectin (Galanthus nivalis agglutinin; GNA) to control D. citri. Proteins, including GNA alone, were purified from fermented transformed yeast Pichia pastoris cultures. Recombinant HxTx-Hv1h, HxTx-Hv1h/GNA and GNA were all orally toxic to D. citri, with Day 5 median lethal concentrations (LC50) derived from dose-response artificial diet assays of 27, 20 and 52 µM, respectively. Western analysis of whole insect protein extracts confirmed that psyllid mortality was attributable to protein ingestion and that the fusion protein was stable to cleavage by D. citri proteases. When applied topically (either via droplet or spray) HxTx-Hv1h/GNA was the most effective of the proteins causing >70 % mortality 5 days post treatment, some 2 to 3-fold higher levels of mortality as compared to the toxin alone. By contrast, no significant mortality or phenotypic effects were observed for bumble bees (Bombus terrestris L.) fed on the recombinant proteins in acute toxicity assays. This suggests that HxTx-Hv1h/GNA has potential as a novel bioinsecticide for the management of D. citri offering both enhanced target specificity as compared to chemical pesticides and compatibility with integrated pest management (IPM) strategies.

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

The potato/tomato psyllid, Bactericera cockerelli (Sulc), 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.

Bacterial communities of the psyllid pest Bactericera cockerelli (Hemiptera: Triozidae) Central haplotype of tomato crops cultivated at different locations of Mexico.

Background: The psyllid, Bactericera cockerelli, is an insect vector of 'Candidatus Liberibacter' causing "Zebra chip" disease that affects potato and other Solanaceae crops worldwide. In the present study, we analyzed the bacterial communities associated with the insect vector Bactericera cockerelli central haplotype of tomato crop fields in four regions from Mexico. Methods: PCR was used to amplify the mitochondrial cytochrome oxidase I gene (mtCOI) and then analyze the single nucleotide polymorphisms (SNP) and phylogenetic analysis for haplotype identification of the isolated B. cockerelli. Moreover, we carried out the microbial diversity analysis of several B. cockerelli collected from four regions of Mexico through the NGS sequencing of 16S rRNA V3 region. Finally, Wolbachia was detected by the wsp gene PCR amplification, which is the B. cockerelli facultative symbiont. Also we were able to confirm the relationship with several Wolbachia strains by phylogenetic analysis. Results: Our results pointed that B. cockerelli collected in the four locations from Mexico (Central Mexico: Queretaro, and Northern Mexico: Sinaloa, Coahuila, and Nuevo Leon) were identified, such as the central haplotype. Analyses of the parameters of the composition, relative abundance, and diversity (Shannon index: 1.328 ± 0.472; Simpson index 0.582 ± 0.167), showing a notably relatively few microbial species in B. cockerelli. Analyses identified various facultative symbionts, particularly the Wolbachia (Rickettsiales: Anaplasmataceae) with a relative abundance higher. In contrast, the genera of Sodalis and 'Candidatus Carsonella' (Gammaproteobacteria: Oceanospirillales: Halomonadaceae) were identified with a relatively low abundance. On the other hand, the relative abundance for the genus 'Candidatus Liberibacter' was higher only for some of the locations analyzed. PCR amplification of a fragment of the gene encoding a surface protein (wsp) of Wolbachia and phylogenetic analysis corroborated the presence of this bacterium in the central haplotype. Beta-diversity analysis revealed that the presence of the genus 'Candidatus Liberibacter' influences the microbiota structure of this psyllid species. Conclusions: Our data support that the members with the highest representation in microbial community of B. cockerelli central haplotype, comprise their obligate symbiont, Carsonella, and facultative symbionts. We also found evidence that among the factors analyzed, the presence of the plant pathogen affects the structure and composition of the bacterial community associated with B. cockerelli.

Insights into the Molecular Basis of Huanglongbing Tolerance in Persian Lime (Citrus latifolia Tan.) through a Transcriptomic Approach.

Huanglongbing (HLB) is a vascular disease of Citrus caused by three species of the α-proteobacteria "Candidatus Liberibacter", with "Candidatus Liberibacter asiaticus" (CLas) being the most widespread and the one causing significant economic losses in citrus-producing regions worldwide. However, Persian lime (Citrus latifolia Tanaka) has shown tolerance to the disease. To understand the molecular mechanisms of this tolerance, transcriptomic analysis of HLB was performed using asymptomatic and symptomatic leaves. RNA-Seq analysis revealed 652 differentially expressed genes (DEGs) in response to CLas infection, of which 457 were upregulated and 195 were downregulated. KEGG analysis revealed that after CLas infection, some DEGs were present in the plant-pathogen interaction and in the starch and sucrose metabolism pathways. DEGs present in the plant-pathogen interaction pathway suggests that tolerance against HLB in Persian lime could be mediated, at least partly, by the ClRSP2 and ClHSP90 genes. Previous reports documented that RSP2 and HSP90 showed low expression in susceptible citrus genotypes. Regarding the starch and sucrose metabolism pathways, some genes were identified as being related to the imbalance of starch accumulation. On the other hand, eight biotic stress-related genes were selected for further RT-qPCR analysis to validate our results. RT-qPCR results confirmed that symptomatic HLB leaves had high relative expression levels of the ClPR1, ClNFP, ClDR27, and ClSRK genes, whereas the ClHSL1, ClRPP13, ClPDR1, and ClNAC genes were expressed at lower levels than those from HLB asymptomatic leaves. Taken together, the present transcriptomic analysis contributes to the understanding of the CLas-Persian lime interaction in its natural environment and may set the basis for developing strategies for the integrated management of this important Citrus disease through the identification of blanks for genetic improvement.

Predicting the potential global distribution of an invasive alien pest Trioza erytreae (Del Guercio) (Hemiptera: Triozidae).

The impact of invasive alien pests on agriculture, food security, and biodiversity conservation has been worsened by climate change caused by the rising earth's atmospheric greenhouse gases. The African citrus triozid, Trioza erytreae (Del Guercio; Hemiptera: Triozidae), is an invasive pest of all citrus species. It vectors the phloem-limited bacterium "Candidatus Liberibacter africanus", a causal agent of citrus greening disease or African Huanglongbing (HLB). Understanding the global distribution of T. erytreae is critical for surveillance, monitoring, and eradication programs. Therefore, we combined geospatial and physiological data of T. erytreae to predict its global distribution using the CLIMEX model. The model's prediction matches T. erytreae present-day distribution and shows that parts of the Mediterranean region have moderate (0 < EI < 30) to high (EI > 30) suitability for the pest. The model predicts habitat suitability in the major citrus-producing countries, such as Mexico, Brazil, China, India, and the USA. In the Special Report on Emissions Scenarios (SRES) A1B and A2 scenarios, the model predicts a reduction in habitat suitability from the current time to 2070. The findings show that global citrus production will continue to be threatened by T. erytreae. However, our study provides relevant information for biosecurity and risk assessment.

Transgenic Sweet Orange Expressing the Sarcotoxin IA Gene Produces High-Quality Fruit and Shows Tolerance to 'Candidatus Liberibacter asiaticus'.

Huanglongbing (otherwise known as HLB or greening) is currently the most devastating citrus disease worldwide. HLB is primarily associated with the phloem-inhabiting bacterium 'Candidatus Liberibacter asiaticus' (CLas). Currently, there are no citrus species resistant to CLas. Genetic transformation is one of the most effective approaches used to induce resistance against plant diseases. Antimicrobial peptides (AMPs) have shown potential breakthroughs to improve resistance to bacterial diseases in plants. In this paper, we confirm the Agrobacterium-mediated transformation of Pera sweet orange expressing the AMP sarcotoxin IA (stx IA) gene isolated from the flesh fly Sarcophaga peregrina and its reaction to CLas, involving plant performance and fruit quality assessments. Four independent transgenic lines, STX-5, STX-11, STX-12, and STX-13, and a non-transgenic control, were graft-inoculated with CLas. Based on our findings, none of the transgenic plants were immune to CLas. However, the STX-5 and STX-11 lines showed reduced susceptibility to HLB with mild disease symptoms and low incidence of plants with the presence of CLas. Fruit and juice quality were not affected by the genetic transformation. Further, no residues of the sarcotoxin IA protein were found in the juice of the STX-11 and STX-12 fruits, though detected in the juice of the STX-5 and STX-13 lines, as revealed by the immunoblotting test. However, juices from all transgenic lines showed low traces of sarcotoxin IA peptide in its composition. The accumulation of this peptide did not cause any deleterious effects on plants or in fruit/juice. Our findings reinforce the challenges of identifying novel approaches to managing HLB.

A reverse-ecology framework to uncover the potential metabolic interplay among 'Candidatus Liberibacter' species, citrus hosts and psyllid vector.

'Candidatus Liberibacter' species have developed a dependency on essential nutrients and metabolites from the host cell, as a result of substantial genome reduction. Still, it is difficult to state which nutrients they acquire and whether or not they are metabolically reliant. We used a reverse-ecology model to investigate the potential metabolic interactions of 'Ca Liberibacter' species, Citrus, and the psyllid Diaphorina citri in the huanglongbing disease pyramid. Our findings show that hosts (citrus and psyllid) tend to support the nutritional needs of 'Ca. Liberibacter' species, implying that the pathogen's metabolism has become tightly linked to hosts, which may reflect in the parasite lifestyle of this important genus.

Entomopathogenic fungus disrupts the phloem-probing behavior of Diaphorina citri and may be an important biological control tool in citrus.

Citrus is among the most important fruit crops worldwide; however, numerous pests and diseases affect the orchards, increasing production costs. The psyllid Diaphorina citri, is a vector of the phloem-limited bacteria 'Candidatus Liberibacter spp.', the causal agent of Huanglongbing (HLB) disease. The lack of a cure for HLB requires management of the vector, mainly by intensive use of chemical insecticides, leading to the selection of resistant populations. Our study determined the effects of the entomopathogenic fungus Cordyceps fumosorosea on the probing behavior of D. citri at different time points after the fungus was applied by spraying. The electrical penetration graph technique was used to monitor the stylet activities of D. citri after application of the microbiological product. The effects were more pronounced between 30 and 96 h after the insects were sprayed, with significant disruption of the stylet activities related to the phloem and directly associated with the transmission of HLB. Our study indicated that the microbiological product Challenger®, with the active ingredient C. fumosorosea fungus, can significantly change the probing behavior of D. citri, may be helpful in more-sustainable management of the vector, and can be used to reduce the spread of HLB.

Probing the Application of OmpA-Derived Peptides to Disrupt the Acquisition of 'Candidatus Liberibacter asiaticus' by Diaphorina citri.

Huanglongbing (HLB) is currently the most devastating disease of citrus worldwide. Both bacteria 'Candidatus Liberibacter asiaticus' (CLas) and 'Candidatus Liberibacter americanus' (CLam) are associated with HLB in Brazil but with a strong prevalence of CLas over CLam. Conventionally, HLB management focuses on controlling the insect vector population (Diaphorina citri; also known as Asian citrus psyllid [ACP]) by spraying insecticides, an approach demonstrated to be mostly ineffective. Thus, development of novel, more efficient HLB control strategies is required. The multifunctional bacterial outer membrane protein OmpA is involved in several molecular processes between bacteria and their hosts and has been suggested as a target for bacterial control. Curiously, OmpA is absent in CLam in comparison with CLas, suggesting a possible role in host interaction. Therefore, in the current study, we have treated ACPs with different OmpA-derived peptides, aiming to evaluate acquisition of CLas by the insect vector. Treatment of psyllids with 5 µM of Pep1, Pep3, Pep5, and Pep6 in artificial diet significantly reduced the acquisition of CLas, whereas increasing the concentration of Pep5 and Pep6 to 50 µM abolished this process. In addition, in planta treatment with 50 µM of Pep6 also significantly decreased the acquisition of CLas, and sweet orange plants stably absorbed and maintained this peptide for as long as 3 months post the final application. Together, our results demonstrate the promising use of OmpA-derived peptides as a novel biotechnological tool to control CLas.

Identification of a Chromosomal Deletion Mutation and the Dynamics of Two Major Populations of 'Candidatus Liberibacter asiaticus' in Its Hosts.

'Candidatus Liberibacter asiaticus' (Las) is the prominent species of Liberibacter associated with huanglongbing, a devastating disease of citrus worldwide. In this study, we report the identification of an ∼8.3-kb DNA region of the Las genome containing eight putative open reading frames flanked by two inverted repeats, which was not present in the Las str. psy62 genome. Comparisons with other genome sequences established this region as a unique genetic element associated with genome plasticity/instability. Primers specific for both the presence (Las wild type) and absence (Las mutant) of this region were designed to study the population dynamics and host adaptation of the two strains. Las populations with and/or without the wild-type strain were detected and differentiated in >2,300 samples that included psyllids, periwinkle, and several species of citrus. In psyllids, although a mixed population of the wild type and mutant was observed in most samples (88%), the wild-type Las was detected alone at a rate of 11%. In contrast, none of the infected citrus plants were positive for the wild type alone, which harbored either the mutant strain alone (8%) or a mixed population of the mutant and wild type (92%). Furthermore, the dynamics of these two major Las populations varied with different citrus hosts, whereas an in-depth study on grapefruit that did not rapidly succumb to disease revealed that the population of mutant alone increased with time, indicating that the absence of this genetic element is associated with the fitness of Las in planta under the selection pressure of its host.

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.

Protocol for Successful Transmission of 'Candidatus Liberibacter asiaticus' from Citrus to Citrus Using Diaphorina citri.

A protocol to successfully transmit the huanglongbing (HLB) pathogen, 'Candidatus Liberibacter asiaticus', between citrus plants by using the Asian citrus psyllid (ACP) and an alternative way to help growers control ACP are proposed. Best results were obtained when pathogen acquisition by adults reared on fully symptomatic 'Ca. Liberibacter asiaticus'-positive plants, latency, and inoculation occurred at ambient air temperatures ranging from 24 to 28°C and when a single infective adult ACP was confined for 7 days on soft, newly developing vegetative shoots (stages v2 to v4). No infection resulted from confinement of infective ACP adults on mature leaves (stage v6). Under the described conditions, single ACP adults could successfully transmit 'Ca. Liberibacter asiaticus' to an average of 56.5% (35 to 83%) of plantlets with v2 to v4 shoots growing in 0.3-liter tubes and to 80.5% (76 to 86%) of plants with v2 to v4 shoots growing in 4.7-liter pots. The use of single insects and plantlets reduces labor, space, and other resources necessary to undertake transmission tests. It also reduces time needed for transmission studies and should help accelerate research on HLB. The results were used to develop an index for favorability to infection (IFI) to determine orchard vulnerabilities to 'Ca. Liberibacter asiaticus'. The IFI is based on the heterogeneous population of new shoots that occurs on tree canopies and may offer alternative or complementary alternatives to the laborious and costly insect surveys currently used in most instances to determine threshold levels for insecticide applications.

Modeling 'Candidatus Liberibacter asiaticus' Movement Within Citrus Plants.

The phloem-limited 'Candidatus Liberibacter asiaticus' (Las) causes huanglongbing, a destructive citrus disease. Graft-inoculated potted plants were used to assess Las speed of movement in phloem in the greenhouse, and the impacts of temperature on plant colonization in growth-chamber experiments. For assessment of Las speed, plants were inoculated at the main stem and assessed over time by quantitative PCR (qPCR) or symptoms at various distances from the inoculum. For colonization, the plants were inoculated in one of two opposite top branches, maintained at from 8 to 20°C, from 18 to 30°C, or from 24 to 38°C daily range, and assessed by qPCR of samples taken from noninoculated shoots. For all experiments, frequencies of Las-positive sites were submitted to analysis of variance and binomial generalized linear model and logistic regression analyses. Probabilities of detecting Las in greenhouse plants were functions of time and distance from the inoculation site, which resulted in 2.9 and 3.8 cm day-1 average speed of movement. In growth chambers, the temperature impacted plant colonization by Las, new shoot emission, and symptom expression. After a 7-month exposure time, Las was absent in all new shoots in the cooler environment (average three per plant), and present in 70% at the milder environment (six shoots, severe symptoms) and 25% in the warmer environment (eight shoots, no visible symptoms). Temperature of 25.7°C was the optimum condition for plant colonization. This explains the higher impact and incidence of huanglongbing disease during the winter months or regions of milder climates in Brazil.

'Candidatus Liberibacter solanacearum' Infection of Physalis ixocarpa Brot. (Solanales: Solanaceae) in Saltillo, Mexico.

The potato psyllid Bactericera cockerelli (Sulc) (Hemiptera: Triozidae) is a pest of solanaceous crops (order Solanales), including potato (Solanum tuberosum L.) and tomato (S. lycopersicum L.). Feeding by high populations of nymphs causes psyllid yellows while adults and nymphs are vectors of the plant pathogen 'Candidatus Liberibacter solanacearum'. Foliar symptoms that were consistent with either 'Ca. L. solanacearum' infection or psyllid yellows were observed in 2019 on tomatillo (Physalis ixocarpa Brot.; family Solanaceae) grown within an experimental plot located near Saltillo, Mexico. This study had three primary objectives: 9i) determine whether the foliar symptoms observed on tomatillo were associated with 'Ca. L. solanacearum' infection, (ii) identify the haplotypes of 'Ca. L. solanacearum' and potato psyllids present in the symptomatic plot, and (iii) use gut content analysis to infer the plant sources of 'Ca. L. solanacearum'-infected psyllids. Results confirmed that 71% of symptomatic plants and 71% of psyllids collected from the plants were infected with 'Ca. L. solanacearum'. The detection of 'Ca. L. solanacearum' in plants and psyllids and the lack of nymphal populations associated with psyllid yellows strongly suggests that the observed foliar symptoms were caused by 'Ca. L. solanacearum' infection. All infected plants and insects harbored the more virulent 'Ca. L. solanacearum' haplotype B but one psyllid was also coinfected with haplotype A. The potato psyllids were predominantly of the central haplotype but one psyllid was identified as the western haplotype. Molecular gut content analysis of psyllids confirmed the movement of psyllids between noncrop habitats and tomatillo and indicated that 'Ca. L. solanacearum' infection of psyllids was associated with increased plant diversity in their diet.

Genome Sequence Resource of 'Candidatus Liberibacter asiaticus' strain 9PA From Brazil.

'Candidatus Liberibacter asiaticus', an unculturable α-proteobacterium, is associated with citrus huanglongbing (HLB), a devastating disease threatening citrus production in Brazil and worldwide. In this study, a draft whole-genome sequence of 'Ca. L. asiaticus' strain 9PA from a sweet orange (cultivar Pera) tree collected in São Paulo State, Brazil, is reported. The 9PA genome is 1,231,881 bp, including two prophages, with G+C content of 36.7%. This is the first report of a whole-genome sequence of 'Ca. L. asiaticus' from Brazil or South America. The 9PA genome sequence will enrich 'Ca. L. asiaticus' genome resources and facilitate HLB research and control in Brazil and the world.

Genome Sequence Resource of 'Candidatus Liberibacter asiaticus' from Diaphorina citri Kuwayama (Hemiptera: Liviidae) in Colombia.

'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.

Evidence That 'Candidatus Liberibacter asiaticus' Moves Predominantly Toward New Tissue Growth in Citrus Plants.

'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.

Wide-ranging transcriptomic analysis of Poncirus trifoliata, Citrus sunki, Citrus sinensis and contrasting hybrids reveals HLB tolerance mechanisms.

Huanglongbing (HLB), caused mainly by 'Candidatus Liberibacter asiaticus' (CLas), is the most devastating citrus disease because all commercial species are susceptible. HLB tolerance has been observed in Poncirus trifoliata and their hybrids. A wide-ranging transcriptomic analysis using contrasting genotypes regarding HLB severity was performed to identify the genetic mechanism associated with tolerance to HLB. The genotypes included Citrus sinensis, Citrus sunki, Poncirus trifoliata and three distinct groups of hybrids obtained from crosses between C. sunki and P. trifoliata. According to bacterial titer and symptomatology studies, the hybrids were clustered as susceptible, tolerant and resistant to HLB. In P. trifoliata and resistant hybrids, genes related to specific pathways were differentially expressed, in contrast to C. sinensis, C. sunki and susceptible hybrids, where several pathways were reprogrammed in response to CLas. Notably, a genetic tolerance mechanism was associated with the downregulation of gibberellin (GA) synthesis and the induction of cell wall strengthening. These defense mechanisms were triggered by a class of receptor-related genes and the induction of WRKY transcription factors. These results led us to build a hypothetical model to understand the genetic mechanisms involved in HLB tolerance that can be used as target guidance to develop citrus varieties or rootstocks with potential resistance to HLB.

Detección de patógenos asociados a la enfermedad punta morada en los cultivos de papa y tomate en Guatemala / Detection of pathogens associated to potato purple top disease in potato and tomato crops in Guatemala

La punta morada es una enfermedad que afecta la producción de algunas especies de solanáceas como la papa y el tomate, causando enrollamiento en las puntas de las hojas con una marcada coloración morada, decaimiento temprano de la planta y en la papa se observa tuberización aérea. Como patógenos asociados a la enfermedad se consideran al fitoplasma BLTVA y la bacteria Candidatus Liberibacter solanacearum. Dada la similitud en la sin-tomatología foliar que generan ambos patógenos, es difícil precisar cuál de ellos está implicado en la enfermedad. En Guatemala, existen reportes de la sintomatología típica de punta morada en las principales zonas productoras de papa y tomate, desconociéndose el agente asociado. La investigación determinó cuál de los dos patógenos reportados está asociados a la enfermedad en 12 municipios productores de papa y/o tomate en el país. Se realizaron ampli-ficaciones de ADN con cebadores específicos para cada patógeno asociado a la enfermedad. Por la alta incidencia del fitoplasma BLTVA en las muestras de papa (73.9%), en comparación a C. Liberibacter solanacearum (26%), este es considerado como el patógeno asociado más importante en papa. En las muestras de tomate, la incidencia del fitoplasma BLTVA (29.8%) y C. Liberibacter solanacearum del (27.6%) fue similar. Además, sobresale el primer reporte de la detección del fitoplasma BLTVA afectando el cultivo de tomate en Guatemala. Se sugiere un monitoreo constante, mediante métodos moleculares, para un diagnóstico certero y establecer medidas de manejo de la enfermedad para evitar su diseminación hacia zonas aún no afectadas.

The potato purple top is a disease that affects the production of some solanaceous species such as potatoes and tomatoes, causing curl at the tips of the leaves with a marked purple coloration, early decay of the plant, and aerial tuberization is observed in the potato. BLTVA phytoplasma and Candidatus Liberibacter solanacearum are considered as pathogens associated with the disease. Given the similarity in foliar symptoms generated by both pathogens, it is difficult to determine which one is involved in the disease. There are reports of the typical potato purple top symptoms in the main potato and tomato producing areas in Guatemala, being unknown the associated agent. The research determined which of the two reported pathogens is associated with the disease in 12 potatoes and/or tomato producing areas in the country. We performed DNA amplification with specific primers for each disease-associated pathogen. Due to the high incidence of BLTVA phytoplasma in potato samples (73.9%), com-pared to C. liberibacter solanacearum (26%), this is considered the most important associated pathogen in potatoes. In tomato samples, the incidence of BLTVA phytoplasma (29.8%) and C. liberibacter solanacearum (27.6%) was similar. Besides, the first report of the detection of the BLTVA phytoplasma affecting tomato cultivation in Gua-temala stands out. Using molecular methods, constant monitoring is suggested for an accurate diagnosis and to establish management measures for the disease to prevent its spread to areas not yet affected.