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The development of sustainable methods to control Diaphorina citri Kuwayama (Hemiptera: Liviidae) is crucial to improve management of huanglongbing (HLB), the most important citrus disease worldwide. In a flushing Citrus × sinensis L. Osbeck (Rutaceae) orchard, this study assessed the combined effects of insecticide-treated Bergera koenigii L. (Rutaceae) trees in the orchard border, as a lethal trap crop, and the application of processed kaolin to the citrus crop, as a repellent (push-pull and kill strategy), in reducing D. citri infestation and dispersal. Additionally, the volatile organic compound (VOC) emission profile and attractiveness of B. koenigii shoots were investigated in indoor experiments. The push-pull and kill strategy reduced the number of D. citri adults on citrus trees by 95, 84, and 63% compared to the untreated control, trap crop only, and kaolin only treatments, respectively. These high reductions can be attributed to the combination of attractive stimuli (VOCs) and repellent stimuli by the trap crop and kaolin, respectively. The number of psyllids observed on citrus trees subjected to treatments with trap crop and kaolin separately was 69 and 86% lower than on untreated control trees, respectively. In addition, all treatments reduced and delayed D. citri dispersal inside the orchard, especially the push-pull and kill strategy. In olfactometric assays, D. citri females showed a significant preference for B. koenigii odors over C. × sinensis cv. ‘Valencia’ flush odors, likely related with the different VOC emission profiles of the two plants. This study shows the effectiveness of push-pull and kill as a new management strategy to control D. citri and consequently HLB.

Citrus relatives are a relevant source of valuable traits for use in citrus breeding, including resistance to diseases such as Huanglongbing (HLB). Resistant rootstocks may impact tree responses to HLB. This requires graft compatibility, which has been poorly investigated within the Aurantioideae. In this study, the biometric characteristics and the anatomy of the graft union of 86 scion/rootstock combinations were assessed. This comprised 18 genotypes/species and 8 genera from Citrinae, Balsamocitrinae, and Clauseninae subtribes sensu Swingle and Reece. Most graft combinations were found to be noncompatible. Phylogenetic proximity did not ensure successful grafting as, for example, Orange jasmine autografts failed, whereas some intergeneric grafts were successful (>60% of graft-take). Plant scion height was directly related to graft-take, but the correlation between the scion and rootstock stem diameters was not a reliable indicator of graft compatibility. Rangpur/Tabog, Tabog/Rangpur, Wampee/Rangpur, Wampee/Pomeroy, Wampee/Swingle, Pomeroy/Wampee, and Swingle/Wampee were the most compatible intergeneric graft combinations. Graft-take success for this was at similar levels to those of sweet orange grafted on common citrus rootstocks. The position as a scion or rootstock in the combination affected the performance and was specific to the genotypes tested. The lack of differentiation between xylem-derived calli and the accumulation of phenolic compounds at the graft union were clear anatomical and biochemical markers, respec-tively, of incompatibility for most Aurantioideae combinations. In the field, within a set of the ten most promising combinations, Hamlin/Rangpur (control) was the only one that became infected by ‘Candidatus Liberibacter asiaticus’. This was first observed 12 months after planting. Overall, the assessment of biometric traits and anatomy of the graft union allowed Aurantioideae genotypes to be divided into four clusters, with respect to their gra

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.

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. Er

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.

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.

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.

[EN] 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)-beta-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.

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.

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 co