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.

ß-carotene genetically-enriched lyophilized orange juice increases antioxidant capacity and reduces ß-amyloid proteotoxicity and fat accumulation in Caenorhabditis elegans.

Citrus sinensis orange juice is an excellent dietary source of ß-carotene, a well-known antioxidant. However, ß-carotene concentrations are relatively low in most cultivars. We developed a new orange through metabolic engineering strategy (GS) with 33.72-fold increase in ß-carotene content compared to its conventional counterpart (CV). Using Caenorhabditis elegans, we found that animals treated with GS showed a greater reduction in intracellular reactive oxygen species (ROS) which is associated with a greater resistance to oxidative stress and induction of the expression of antioxidant genes. Moreover, animals treated with GS orange showed a more effective protection against ß-amyloid proteotoxicity and greater hypolipidemic effect under high glucose diet compared to animals treated with CV. These data demonstrate that the increased amount of ß-carotene in orange actually provides a greater beneficial effect in C. elegans and a valuable proof of principle to support further studies in mammals and humans.

Insight into resistance to 'Candidatus Liberibacter asiaticus,' associated with Huanglongbing, in Oceanian citrus genotypes.

Huanglongbing (HLB), the most destructive citrus disease, is associated with unculturable, phloem-limited Candidatus Liberibacter species, mainly Ca. L. asiaticus (Las). Las is transmitted naturally by the insect Diaphorina citri. In a previous study, we determined that the Oceanian citrus relatives Eremocitrus glauca, Microcitrus warburgiana, Microcitrus papuana, and Microcitrus australis and three hybrids among them and Citrus were full-resistant to Las. After 2 years of evaluations, leaves of those seven genotypes remained Las-free even with their susceptible rootstock being infected. However, Las was detected in their stem bark above the scion-rootstock graft union. Aiming to gain an understanding of the full-resistance phenotype, new experiments were carried out with the challenge-inoculated Oceanian citrus genotypes through which we evaluated: (1) Las acquisition by D. citri fed onto them; (2) Las infection in sweet orange plants grafted with bark or budwood from them; (3) Las infection in sweet orange plants top-grafted onto them; (4) Las infection in new shoots from rooted plants of them; and (5) Las infection in new shoots of them after drastic back-pruning. Overall, results showed that insects that fed on plants from the Oceanian citrus genotypes, their canopies, new flushes, and leaves from rooted cuttings evaluated remained quantitative real-time polymerase chain reaction (qPCR)-negative. Moreover, their budwood pieces were unable to infect sweet orange through grafting. Furthermore, sweet orange control leaves resulted infected when insects fed onto them and graft-receptor susceptible plants. Genomic and morphological analysis of the Oceanian genotypes corroborated that E. glauca and M. warburgiana are pure species while our M. australis accession is an M. australis × M. inodora hybrid and M. papuana is probably a M. papuana × M. warburgiana hybrid. E. glauca × C. sinensis hybrid was found coming from a cross between E. glauca and mandarin or tangor. Eremocitrus × Microcitrus hybrid is a complex admixture of M. australasica, M. australis, and E. glauca while the last hybrid is an M. australasica × M. australis admixture. Confirmation of consistent full resistance in these genotypes with proper validation of their genomic parentages is essential to map properly genomic regions for breeding programs aimed to generate new Citrus-like cultivars yielding immunity to HLB.

Resistance of True Citrus species to Diaphorina citri.

BACKGROUND: Host genetic resistance is a promising strategy for the management of Diaphorina citri Kuwayama (Hemiptera: Psyllidae), and consequently Huanglongbing (HLB). To date, no study has investigated the resistance to D. citri in the clonal and vegetatively propagated plants of the Microcitrus, Eremocitrus, and Atalantia genera. This study assesses Near and True Citrus genotype antixenosis and antibiosis against D. citri, with trichome density and volatile emission as possible mechanisms of resistance. RESULTS: All genotypes were oviposited by D. citri, however, 8 of 14 genotypes were less oviposited than Citrus × sinensis 'Valencia' (susceptible control). Diaphorina citri nymphs had lower nymphal viability in E. glauca (31%) and M. warburgiana (58%) than that in Citrus × sinensis (77%). The behavioral assay showed that 30% of D. citri nymphs in the last instars evaded E. glauca shoots, whereas no nymphs evaded Citrus × sinensis shoots. A higher trichome density was observed in E. glauca shoots compared to the other genotypes. Chemical analysis revealed differences in the volatile profiles of E. glauca and Citrus × sinensis. CONCLUSION: Eremocitrus glauca and M. warburgiana genotypes were more resistant to D. citri than Citrus × sinensis. Higher trichome density in the shoots may negatively influence the development of D. citri nymphs. Eremocitrus glauca volatiles may also be involved in their resistance to D. citri. © 2022 Society of Chemical Industry.

Generation of Transfer-DNA-Free Base-Edited Citrus Plants.

To recover transgenic citrus plants in the most efficient manner, the use of selection marker genes is essential. In this work, it was shown that the mutated forms of the acetolactate synthase (ALS) gene in combination with the herbicide selection agent imazapyr (IMZ) added to the selection medium may be used to achieve this goal. This approach enables the development of cisgenic regenerants, namely, plants without the incorporation of those bacterial genes currently employed for transgenic selection, and additionally it allows the generation of edited, non-transgenic plants with altered endogenous ALS genes leading to IMZ resistance. In this work, the citrus mutants, in which ALS has been converted into IMZ-resistant forms using a base editor system, were recovered after cocultivation of the explants with Agrobacterium tumefaciens carrying a cytidine deaminase fused to nSpCas9 in the T-DNA and selecting regenerants in the culture medium supplemented with IMZ. Analysis of transgene-free plants indicated that the transient expression of the T-DNA genes was sufficient to induce ALS mutations and thus generate IMZ-resistant shoots at 11.7% frequency. To our knowledge, this is the first report of T-DNA-free edited citrus plants. Although further optimization is required to increase edition efficiency, this methodology will allow generating new citrus varieties with improved organoleptic/agronomic features without the need to use foreign genes.

From the smallest to the largest subcellular plant pathogen: Citrus tristeza virus and its unique p23 protein.

Knowledge on diseases caused by Citrus tristeza virus (CTV) has greatly increased in last decades after their etiology was demonstrated in the past seventies. Professor Ricardo Flores substantially contributed to these advances in topics like: i) improvement of virus purification to obtain biologically active virions, ii) sequencing mild CTV isolates for genetic comparisons with sequences of moderate or severe isolates and genetic engineering, iii) analysis of genetic variation of both CTV genomic RNA ends and features of the highly variable 5' end that allow accommodating this variation within a conserved secondary structure, iv) studies on the structure, subcellular localization and biological functions of the CTV-unique p23 protein, and v) potential use of p23 and other 3'-proximal regions of the CTV genome to develop transgenic citrus resistant to the virus. Here we review his main achievements on these topics and how they contributed to deeper understanding of CTV biology and to new potential measures for disease control.

Improvement of Antioxidant Properties in Fruit from Two Blood and Blond Orange Cultivars by Postharvest Storage at Low Temperature.

Numerous studies have revealed the remarkable health-promoting activities of citrus fruits, all of them related to the accumulation of bioactive compounds, including vitamins and phytonutrients. Anthocyanins are characteristic flavonoids present in blood orange, which require low-temperature for their production. Storage at low-temperature of blood oranges has been proven to be a feasible postharvest strategy to increase anthocyanins in those countries with warm climates. To our knowledge, no studies comparing the effect of postharvest storage effect on phenylpropanoid accumulation in cultivars with and without anthocyanins production have been published. We have investigated the effect of postharvest cold storage in flavonoid accumulation in juice from Citrus sinensis L. Osbeck in two different oranges: Pera, a blond cultivar, and Moro, a blood one. Our findings indicate a different response to low-temperature of fruit from both cultivars at biochemical and molecular levels. Little changes were observed in Pera before and after storage, while a higher production of phenylpropanoids (3.3-fold higher) and flavonoids (1.4-fold higher), including a rise in anthocyanins from 1.3 ± 0.7 mg/L to 60.0 ± 9.4 mg/L was observed in Moro concurrent with an upregulation of the biosynthetic genes across the biosynthetic pathway. We show that postharvest storage enhances not only anthocyanins but also other flavonoids accumulation in blood oranges (but not in blond ones), further stimulating the interest in blood orange types in antioxidant-rich diets.

Cultural Management of Huanglongbing: Current Status and Ongoing Research.

Huanglongbing (HLB), formerly known as greening, is a bacterial disease restricted to some Asian and African regions until two decades ago. Nowadays, associated bacteria and their vectors have spread to almost all citrus-producing regions, and it is currently considered the most devastating citrus disease. HLB management can be approached in terms of prevention, limiting or avoiding pathogen and associated vectors to reach an area, or in terms of control, trying to reduce the impact of the disease by adopting different cultural strategies depending on infestation/infection levels. In both cases, control of psyllid populations is currently the best way to stop HLB spread. Best cultural actions (CHMAs, TPS system) to attain this goal and, thus, able to limit HLB spread, and ongoing research in this regard is summarized in this review.

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.

The Genome of "Candidatus Liberibacter asiaticus" Is Highly Transcribed When Infecting the Gut of Diaphorina citri.

The Asian citrus psyllid, Diaphorina citri, is the vector of the bacterium "Candidatus Liberibacter asiaticus" (Las), associated with the devastating, worldwide citrus disease huanglongbing. In order to explore the molecular interactions of this bacterium with D. citri during the vector acquisition process, cDNA libraries were sequenced on an Illumina platform, obtained from the gut of adult psyllids confined in healthy (H) and in Las-infected young shoots (Las) for different periods of times (I = 1/2 days, II = 3/4 days, and III = 5/6 days). In each sampling time, three biological replicates were collected, containing 100 guts each, totaling 18 libraries depleted in ribosomal RNA. Reads were quality-filtered and mapped against the Chinese JXGC Las strain and the Floridian strain UF506 for the analysis of the activity of Las genome and SC1, SC2, and type 3 (P-JXGC-3) prophages of the studied Las strain. Gene activity was considered only if reads of at least two replicates for each acquisition access period mapped against the selected genomes, which resulted in coverages of 44.4, 79.9, and 94.5% of the JXGC predicted coding sequences in Las I, Las II, and Las III, respectively. These genes indicate an active metabolism and increased expression according to the feeding time in the following functional categories: energy production, amino acid metabolism, signal translation, cell wall, and replication and repair of genetic material. Pilins were among the most highly expressed genes regardless of the acquisition time, while only a few genes from cluster I of flagella were not expressed. Furthermore, the prophage region had a greater coverage of reads for SC1 and P-JXGC-3 prophages and low coverage in SC2 and no indication of activity for the lysis cycle. This research presents the first descriptive analysis of Las transcriptome in the initial steps of the D. citri gut colonization, where 95% of Las genes were active.

Engineering of citrus to obtain huanglongbing resistance.

Huanglongbing (HLB) disease is threatening the sustainability of citriculture in affected regions because of its rapid spread and the severity of the symptoms it induces. Herein, we summarise the main research findings that can be exploited to develop HLB-resistant cultivars. A major bottleneck has been the lack of a system for the ex vivo cultivation of HLB-associated bacteria (CLs) in true plant hosts, which precludes the evaluation of target genes/metabolites in reliable plant/pathogen/vector environments. With regard to HLB vectors, several biotechnologies which have been proven in laboratory settings to be effective for insect control are presented. Finally, new genotypes that are resistant to CLs or their insect vectors are described, and the most relevant strategies for fighting HLB are highlighted.

Early Population Dynamics of "Candidatus Liberibacter asiaticus" in Susceptible and Resistant Genotypes After Inoculation With Infected Diaphorina citri Feeding on Young Shoots.

Huanglongbing is a highly destructive citrus disease associated with "Candidatus Liberibacter asiaticus" (Las), a phloem-limited and non-culturable bacterium, naturally transmitted by the psyllid Diaphorina citri. Although diverse approaches have been used to understand the molecular mechanisms involved in the pathogen-host interaction, such approaches have focused on already infected and/or symptomatic plants, missing early events in the initial days post-inoculation. This study aimed to identify the time course of Las multiplication and whole-plant colonization immediately following inoculation by infected psyllids feeding for 2 days. Thus, the experimental approach was to track Las titers after psyllid inoculation in new shoots (NS) of Citrus × sinensis (susceptible), Murraya paniculata (partially resistant), and Bergera koenigii (fully resistant). Soon after psyllid removal, Las titers dropped until the 10-12th days in all three species. Following this, Las titers increased exponentially only in C. × sinensis and M. paniculata, indicating active bacterial multiplication. In C. × sinensis, Las reached a stationary phase at ∼5 log Las cells/g of tissue from the 40th day onward, while in M. paniculata, Las increased at a lower rate of up to ∼3 log Las cells/g of tissue between the 40th and 60th days, decreasing gradually thereafter and becoming undetectable from the 160th day onward. In B. koenigii, Las titers decreased from the start and remained undetectable. In C. × sinensis, an average of 2.6 log of Las cells/g of tissue was necessary for Las to move out of 50% of the NS in 23.6 days and to colonize the rest of the plant, causing a successful infection. Conversely, the probability of Las moving out of the NS remained below 50% in M. paniculata and zero in B. koenigii. To our knowledge, this is the first study on Las dynamics and whole-plant colonization during the earliest stages of infection. Identification of critical time-points for either successful multiplication or Las resistance may help to elucidate initial events of Las-host interactions that may be missed due to longer sampling intervals and at later stages of infection.

Biological and molecular characterization of linalool-mediated field resistance against Xanthomonas citri subsp. citri in citrus trees.

The biological and molecular traits of the Ponkan mandarin (Citrus reticulata Blanco) were characterized in an investigation of the mechanisms of field resistance against citrus canker disease caused by the bacterial pathogen, Xanthomonas citri subsp. citri (Xcc). Various conventional citrus varieties that show diverse responses to Xcc were investigated, and the temporal changes in Xcc titer in response to linalool concentrations among the varieties revealed differences in Xcc proliferation trends in the inoculated leaves of the immune, field-resistant and susceptible varieties. In addition, increased linalool accumulation was inversely related to Xcc titers in the field-resistant varieties, which is likely caused by host--pathogen interactions. Quantitative trait locus (QTL) analysis using the F1 population of the resistant Ponkan mandarin and susceptible 'Harehime' ('E-647' × 'Miyagawa-wase') cultivar revealed that linalool accumulation and Xcc susceptibility QTLs overlapped. These results provide novel insights into the molecular mechanisms of linalool-mediated field resistance to Xcc, and suggest that high linalool concentrations in leaves has an antibacterial effect and becomes a candidate-biomarker target for citrus breeding to produce seedlings with linalool-mediated field resistance against Xcc.

Engineered Orange Ectopically Expressing the Arabidopsis ß-Caryophyllene Synthase Is Not Attractive to Diaphorina citri, the Vector of the Bacterial Pathogen Associated to Huanglongbing.

Huanglongbing (HLB) is a destructive disease, associated with psyllid-transmitted phloem-restricted pathogenic bacteria, which is seriously endangering citriculture worldwide. It affects all citrus species and cultivars regardless of the rootstock used, and despite intensive research in the last decades, there is no effective cure to control either the bacterial species (Candidatus Liberibacter spp.) or their insect vectors (Diaphorina citri and Trioza erytreae). Currently, the best attempts to manage HLB are based on three approaches: (i) reducing the psyllid population by intensive insecticide treatments; (ii) reducing inoculum sources by removing infected trees, and (iii) using nursery-certified healthy plants for replanting. The economic losses caused by HLB (decreased fruit quality, reduced yield, and tree destruction) and the huge environmental costs of disease management seriously threaten the sustainability of the citrus industry in affected regions. Here, we have generated genetically modified sweet orange lines to constitutively emit (E)-ß-caryophyllene, a sesquiterpene repellent to D. citri, the main HLB psyllid vector. We demonstrate that this alteration in volatile emission affects behavioral responses of the psyllid in olfactometric and no-choice assays, making them repellent/less attractant to the HLB vector, opening a new alternative for possible HLB control in the field.

Curing and low-temperature combined post-harvest storage enhances anthocyanin biosynthesis in blood oranges.

Anthocyanins are pigments present in blood oranges which can be enriched by post-harvest cold storage. Additionally, citrus fruits contain appreciable levels of other flavonoids, whose content increases under post-harvest heat treatments. Here, we investigated the effects of curing (37 °C for 3 days) and storage at low-temperature (9 °C) during 15, 30 and 45 days on accumulation of anthocyanins and other flavonoids in Moro and Sanguinelli Polidori blood oranges (Citrus sinensis L. Osbeck). Cured fruits reached up to 191.4 ± 1.4 mg/L of anthocyanins in their juice after cold storage and a 3-fold enrichment of other flavonoids such as flavones and flavanones, compared to 85.7 ± 3.3 mg/L anthocyanins from fruits with cold storage alone. Concomitantly, qPCR analysis showed that curing enhanced upregulation of the main structural and transcription factor genes regulating the flavonoid pathway. GC-MS analysis showed that no unpleasant compounds were generated in the cured plus cold-stored juice volatilome.

Behavioral responses of Diaphorina citri to host plant volatiles in multiple-choice olfactometers are affected in interpretable ways by effects of background colors and airflows.

In several phytophagous hemipterans, behavior appears to be mediated by both visual and chemical cues. For the Asian citrus psyllid (ACP) Diaphorina citri (Hemiptera: Liviidae), olfactometric assays are generally difficult to interpret owing to the low proportion of individuals responding to odors (~30-40%), which compromises the efficiency and reliability of the results of behavioral tests. In the present study, the ACP behavioral response to emitted odors from sweet orange (Citrus sinensis L. Osbeck) flushes in a 4-arm olfactometer using different colors (four white-, two white- and two yellow- on opposite sides, or four yellow-colored fields), and the role of the airflow in the concentration of volatile organic compounds (VOCs) were assessed at two airflows [0.4 and 0.1 L/min (LPM)]. Exposure to 'Pera' sweet orange or clean air in treatments with four yellow-colored-fields increased the response rate of ACP females to the odor sources compared with exposure to 'Pera' sweet orange or clean air in treatments with four white-colored-fields, independently of the odor source and airflow tested. For the assays using two white- and two yellow-colored fields on opposite sides and 0.4 or 0.1 LPM airflow, the residence time of ACP females to odors ('Pera' sweet orange or clean air) was similar or higher in treatments using yellow- than those using white-colored fields. For both assays (VOCs and olfactometric behavioral parameters), the reduction in airflow from 0.4 to 0.1 LPM greatly changed the airborne concentration and ACP behavior. Quantitative chemical analyses revelead that the concentration of most compounds emitted by 'Pera' sweet orange flushes for the headspace using 0.1 LPM airflow were greater than the concentrations measured using 0.4 LPM airflow. Therefore, this treatment design provides an useful tool to assess the ACP behavioral response to the odors from citrus plants, and it can also help in the discrimination of dose-response screenings for VOCs or conspecific insects.

Resistance to 'Candidatus Liberibacter asiaticus,' the Huanglongbing Associated Bacterium, in Sexually and/or Graft-Compatible Citrus Relatives.

Huanglongbing (HLB) is the most destructive, yet incurable disease of citrus. Finding sources of genetic resistance to HLB-associated 'Candidatus Liberibacter asiaticus' (Las) becomes strategic to warrant crop sustainability, but no resistant Citrus genotypes exist. Some Citrus relatives of the family Rutaceae, subfamily Aurantioideae, were described as full-resistant to Las, but they are phylogenetically far, thus incompatible with Citrus. Partial resistance was indicated for certain cross-compatible types. Moreover, other genotypes from subtribe Citrinae, sexually incompatible but graft-compatible with Citrus, may provide new rootstocks able to restrict bacterial titer in the canopy. Use of seedlings from monoembryonic species and inconsistencies in previous reports likely due to Las recalcitrance encouraged us to evaluate more accurately these Citrus relatives. We tested for Las resistance a diverse collection of graft-compatible Citrinae species using an aggressive and consistent challenge-inoculation and evaluation procedure. Most Citrinae species examined were either susceptible or partially resistant to Las. However, Eremocitrus glauca and Papua/New Guinea Microcitrus species as well as their hybrids and those with Citrus arose here for the first time as full-resistant, opening the way for using these underutilized genotypes as Las resistance sources in breeding programs or attempting using them directly as possible new Las-resistant Citrus rootstocks or interstocks.

Reunion overseas: introduced wild boars and cultivated orange trees interact in the Brazilian Atlantic Forest

Introduction: Little is known concerning novel interactions between species that typically interact in their native range but, as a consequence of human activity, are also interacting out of their original distribution under new ecological conditions. Objective: We investigate the interaction between the orange tree and wild boar, both of which share Asian origins and have been introduced to the Americas (i.e. the overseas). Methods: Specifically, we assessed whether i) wild boars consume orange (Citrus sinensis) fruits and seeds in orchards adjacent to a remnant of the Atlantic Forest of Brazil, ii) the orange seeds are viable after passing through boar's digestive tract and iii) whether the orange tree may naturalise in the forest remnant assisted by wild boars. Results: Our camera surveys indicated that wild boar was by far the most frequent consumer of orange fruits (40.5 % of camera trap-days). A considerable proportion of sown orange seeds extracted from fresh boar feces emerged seedlings (27.8 %, N = 386) under controlled greenhouse conditions. Further, 37.6 % of sown seeds (N = 500) in the forest remnant emerged seedlings in July 2015; however, after ~4 years (March 2019) only 9 seedlings survived (i.e. 4.8 %, N = 188). Finally, 52 sweet orange seedlings were found during surveys within the forest remnant which is intensively used by wild boars. This study indicates a high potential of boars to act as effective seed dispersers of the sweet orange. However, harsh competition with native vegetation and the incidence of lethal diseases, which quickly kill sweet orange trees under non-agricultural conditions, could seriously limit orange tree establishment in the forest. Conclusions: Our results have important implications not only because the wild boar could be a vector of potential invasive species, but also because they disperse seeds of some native species (e.g. the queen palm, Syagrus romanzofiana) in defaunated forests, where large native seed dispersers are missing; thus, wild boars could exert critical ecological functions lost due to human activity.

Introducción: Se conoce relativamente poco sobre las llamadas 'interacciones noveles' entre especies que típicamente interactúan en su área de distribución nativa pero que, como consecuencia de la actividad humana, también interactúan fuera de su distribución original bajo nuevas condiciones ecológicas. Objetivo: Investigamos la interacción entre el naranjo y el jabalí, ambos con origen asiático e introducidos en las Américas (es decir, del extranjero). Métodos: Específicamente, evaluamos si i) los jabalíes consumen frutas y semillas del naranjo (Citrus sinensis) en naranjales adyacentes a un parche remanente del bosque atlántico de Brasil, ii) las semillas de naranja son viables tras pasar por el tracto digestivo del jabalí, y iii) si el naranjo puede llegar a naturalizarse en el parche de bosque gracias a los jabalíes. Resultados: Los resultados de nuestro fototrampeo indicaron que el jabalí fue, con mucho, el consumidor más frecuente de las naranjas (40.5 % cámaras trampa-días). Una proporción considerable de semillas de naranjo extraídas de heces de jabalí frescas y sembradas emergieron plántulas bajo condiciones de invernadero controladas (27.8 %, N = 386). Además, del 37.6 % de las semillas sembradas (N = 500) en el parche remanente de bosque emergieron plántulas en julio 2015; sin embargo, después de ~ 4 años (marzo 2019) solo sobrevivieron 9 plántulas (es decir, 4.8 %, N = 188). Finalmente, se encontraron 52 plántulas de naranja dulce durante varias prospecciones dentro del parche de bosque que es utilizado intensivamente por los jabalíes. Este estudio indica un alto potencial de los jabalíes para actuar como dispersores de semillas eficaces del naranjo dulce. Sin embargo, la severa competencia con la abundante vegetación nativa y la incidencia de enfermedades letales, que matan rápidamente los naranjos dulces en condiciones no agrícolas, podrían limitar seriamente el establecimiento de naranjos en el bosque. Conclusiones: Nuestros resultados tienen implicaciones importantes no solo porque el jabalí podría ser un vector de posibles especies de plantas invasoras, sino también porque dispersan semillas de algunas especies nativas (p.e., la palmera reina, Syagrus romanzofiana) en estos bosques defaunados, donde faltan dispersores nativos de semillas de gran tamaño. Por ello, los jabalíes podrían ejercer funciones ecológicas críticas que se han perdido debido a la actividad humana.

Murraya paniculata and Swinglea glutinosa as Short-Term Transient Hosts of 'Candidatus Liberibacter asiaticus' and Implications for the Spread of Huanglongbing.

Murraya paniculata and Swinglea glutinosa are aurantioid hosts of the Asian citrus psyllid (ACP) Diaphorina citri, the principal vector of 'Candidatus Liberibacter asiaticus' (Las). Las is the pathogen associated with huanglongbing (HLB), the Asian form of which is the most devastating disease of Citrus species and cultivars (Rutaceae: Aurantioideae). M. paniculata is a common ornamental and S. glutinosa is grown as an ornamental, a citrus rootstock, and a hedgerow fence plant. Because of the uncertain status of these plants as reservoirs of Las, a series of cross-inoculation bioassays were carried out in different environments, using infected Valencia sweet orange (Citrus × aurantium) infected shoot tops as a source of inoculum and D. citri nymphs and adults reared on M. paniculata and S. glutinosa to inoculate pathogen-free Valencia orange plantlets. In contrast to sweet orange, Las was more unevenly distributed and reached much lower titers in M. paniculata and S. glutinosa. Infections in M. paniculata and S. glutinosa were also transient. Very few insects that successfully acquired Las from M. paniculata and S. glutinosa were able to transmit the pathogen to healthy citrus. Transmission rates were low from M. paniculata (1.0%) and S. glutinosa (2.0%) and occurred only in a controlled environment highly favorable to Las and ACP using 10-day-old adults that completed their life cycle on Las-positive plants. Our study showed that in HLB-endemic areas, M. paniculata and S. glutinosa can be deemed as epidemiologically dead-end hosts for Las and are not important alternative hosts of the pathogen for transmission to citrus. However, under a combination of conditions highly favorable to Las infection and transmission and in the absence of effective quarantine procedures, these plants could eventually serve as carriers of Las to regions currently free from HLB.