Electrochemical Impedance Spectroscopy (EIS) is a non-destructive technique that is sensitive to the working interface, used to monitor changes on the electrode surfaces during each construction step of electrochemical biosensors. In this study, EIS was employed to analyze the changes at the electrode/solution interface during the development of an immunosensor for the diagnosis of Huanglongbing (HLB). HLB is a devastating citrus disease of worldwide importance, caused by the uncultured bacterium Candidatus Liberibacter asiaticus (CLas) and vectored by the psyllid Diaphorina citri. Management of HLB relies on recognition of symptomatic plants and the visual diagnosis of HLB in groves is performed by observing symptoms on the leaves. The gold standard diagnosis is the quantitative polymerase chain reaction (qPCR). However, there is a need for a test less dependent on laboratory equipment and suitable for tree-side development. This work developed an impedimetric immunosensor for the detection of CLas in citrus leaf samples. An antibody specific to CLas outer membrane protein was immobilized via Schiff base reaction on the surface of iron oxide magnetic nanoparticles (mNP) modified with the aminosilane (APTES). The resultant properties of nanoparticles surface analyzed with EIS in the behavior of the electrode/solution interface enabled the understanding of kinetic processes and mass transport in the system. This, in turn, allowed the adjustment of the equivalent electrical circuit (EECs) and the correlation of interfacial events with electrical components. The developed device successfully distinguished HLB-positive from negative samples, validated by qPCR.Comparison was made of the results obtained using the proposed methodology and the gold-standard qPCR technique, revealing excellent agreement that was statistically validated.

Among citrus diseases, Huanglongbing (HLB) is recognized as the most destructive and economically damaging worldwide. It is mainly associated with 'Candidatus Liberibacter asiaticus' (CLas) being transmitted by Diaphorina citri. There are no curative treatments or commercial citrus varieties resistant to CLas. Wild Aurantioideae species have been widely screened and, recently, Oceanian genotypes graft- and sexually compatible with Citrus were identified as HLB-resistant; however, there is no information regarding their use as interstocks of commercial varieties yet. Under greenhouse conditions, six HLB-resistant genotypes were evaluated as interstocks between 'Valencia' sweet orange scion and 'Rangpur' lime rootstock, both susceptible to HLB, with 'Valencia' interstock as the control. Rootstocks were nucellar seedlings, and the scion was a commercial accession preimmunized with a mild CTV strain. Plants were assessed for CLas infection and titer in leaves, stem bark, and roots up to 12-24 months after graft-inoculation in the scion and compared to non-inoculated controls. Furthermore, molecular, anatomical, and biometric variables were investigated. As expected, the scion variety was colonized by CLas regardless of the genotype evaluated as interstock. Although bacteria were detected in the roots of most CLas-inoculated plants, CLas movement from the scion to the roots was blocked in 42% and 86% of composite plants when using a F1 hybrid of C. australis × C. inodora or an admixture hybrid of C. glauca, C. australis, and C. australasica as interstocks. Overall, CLas titers were similar in infected plant tissues among the evaluated genotypes, but titers were lower in interstock bark tissues compared to scion and rootstock ones. After one-two years of CLas + CTV infection (experiments I and II, respectively), the dry weight of the root system decreased by 50% in infected trees compared to control trees for most genotypes, and CLas + CTV infection was associated with changes in the sieve phloem and gene expression. These findings suggest that, despite CTV infection, interstocks derived from some hybrids of Australian citrus types have the potential to restrict the movement of CLas from the scion into the roots of infected citrus trees. Long-term evaluation of composite plants in field conditions is necessary to assess tree performance and, ultimately, the impact of CLas blockage by interstocking on HLB disease damage.

Huanglongbing (HLB) is considered the most serious Citrus disease for which there are currently no effective control methods. The putative agents of HLB, Candidatus Liberibacter asiaticus (CLas), and the vector Diaphorina citri, are widespread in citrus regions, causing losses in citrus cultivation worldwide. Studying the interactions between D. citri and CLas can elucidate disease epidemiology and determine specific targets for HLB control. This work aimed to improve our understanding of the host-bacteria (D. citri-CLas) relationship, focusing on cysteine peptidase (DcCathL) and its inhibition by citrus cystatin (CsinCPI-2). In this study, a combination of qPCR, FISH, and immunolocalization techniques was employed to detect DcCathL genes or proteins in CLas-free or -infected D. citri samples. To verify the effect of DcCathL inhibition on insect survival, an artificial diet assay containing recombinant CsinCPI-2 was performed with infected and healthy D. citri nymphs and adults. Accordingly, a relative expression of DcCathL 1.23 times higher and an approximately 3.3 times greater DcCathL transcripts in gut tissue were demonstrated in CLas-infected compared to CLas-free insects. Furthermore, the presence of DcCathL protein was detected in the gut, ovary, and salivary glands of D. citri, concentrated at the peripheral regions of the cells. The fluorescence signal associated with DcCathL indicates that the gut of infected D. citri contains 4.81 times more protein than the gut of a healthy insect. Similarly, the protein levels in the salivary glands and ovaries of infected insects were 1.38 and 1.1 times higher, respectively, compared to those of CLas-free insects. The efficacy of the artificial diet in delivering the recombinant protein to the insect was demonstrated by the detection of green fluorescent protein (GFP) in the intestinal tract of nymphs and adults. Finally, the CsinCPI-2 demonstrated a substantial increase in mortality among CLas-free nymphs (df = 1, p < 0.0001) and CLas-free or CLas-infected adults (df = 1, p = 0.0001). Thus, the development of inhibitors that can disrupt the interactions between bacteria and vectors by blocking DcCathL activity may represent a promising strategy to prevent the spread of HLB disease.

Citrus black spot (CBS) is a fungal disease caused by Phyllosticta citricarpa, which affects a broad range of citrus species. The species Citrus latifolia (‘Tahiti’ lime) has been reported as resistant to CBS, whereas C. sinensis (sweet orange) is considered susceptible. However, there are some studies reporting C. aurantium (sour orange) as resistant and others as susceptible. To elucidate this contradiction, a field trial was performed over two seasons to characterize CBS symptoms expressed on these three citrus species. Fruits of ‘Tahiti’ lime, ‘Valencia’ sweet orange, and ‘São Paulo’ sour orange were inoculated with P. citricarpa conidia at three developmental stages. CBS incidence and severity were assessed, and incubation period and symptom types in each species were described. Sweet orange showed typical CBS symptoms, such as false melanosis and hard spot (with pycnidia), whereas no symptom was observed in ‘Tahiti’ lime. For the first time, atypical microlesions were observed in sour orange inoculated fruit. These microlesions did not produce pycnidia and were smaller when compared with typical false melanosis symptoms in sweet orange. There were differences among the three species regarding CBS incidence and severity. In the first season, CBS incidence and severity on sweet orange fruit reached 95

One of the prominent bacterial diseases impacting orange production and trade is citrus canker, caused by the bacterium Xanthomonas citri subsp. citri (X. citri). The management of citrus canker involves deploying copper products as a protective measure to control the development of symptoms, which carries the risk of selecting strains that are resistant to copper. The objective of this report was to evaluate four peptides with potential antibiotic activity against X. citri in vitro and in planta. In growth inhibition assays, the peptides Gr01, Guavanin 2, K-13, and Lin1 had an inhibitory effect on X. citri at concentrations below 12.5 µmol liter-1. Minimal bactericidal effects were observed at peptide concentrations of 100 and 50 µmol liter-1 for Lin1 and Guavanin 2, respectively, and 12.5 µmol liter-1 for Gr01 and K-13. Membrane mimetics coupled with fluorescence spectroscopy assays showed that Guavanin 2, Gr01, and K-13, but not Lin1, act by promoting bacterial membrane lysis. Moreover, the Gr01, K-13, and Lin1 peptides triggered a prolonged induction of genes associated with the activation of the jasmonic acid and salicylic acid pathways, suggesting a dual function of these peptides in promoting a priming effect. The severity of citrus canker in plants treated with Gr01, K-13, and Guavanin 2 was 0.2, 0.3, and 0.4 lesions/cm2, respectively. These values were similar to those recorded in plants treated with copper (0.3 lesions/cm2) and significantly lower than the 1.5 lesions/cm2 observed in untreated plants. Gr01 and K-13 peptides are promising dual-activity alternatives in the management of citrus canker.

Huanglongbing (HLB) is a devastating disease of citrus plants caused by the non-culturable phloem-inhabiting bacterium Candidatus Liberibacter ssp., being Ca. Liberibacter asiaticus (CLas) the most aggressive species. CLas is vectored by the psyllid Diaphorina citri and introduced into sieve cells, establishing a successful infection in all Citrus species. Partial or complete resistance has been documented in the distant relatives Murraya paniculata and Bergera koenigii, respectively, providing excellent systems to investigate the molecular basis of HLB-resistance. It has been shown previously that the first weeks after bacterial release into the phloem are critical for the establishment of the bacterium. In this study, a thorough transcriptomic analysis of young flushes exposed to CLas-positive and negative psyllids has been performed in Citrus × sinensis, as well as in the aforementioned resistant species, along the first eight weeks after exposure. Our results indicate that the resistant species do not deploy a classical immunity response upon CLas recognition. Instead, transcriptome changes are scarce and only a few genes are differentially expressed when flushes exposed to CLas-positive and negative psyllid are compared. Functional analysis suggests that primary metabolism and other basic cellular functions could be rewired in the resistant species to limit infection. Transcriptomes of young flushes of the three species are very different, supporting the existence of distinct biochemical niches for the bacterium. These findings suggest that both intrinsic metabolic inadequacies to CLas survival, as well as inducible reprogramming of physiological functions upon CLas recognition, could orchestrate together restriction of bacterial multiplication in these resistant hosts.

Citriculture is facing challenges due to the spread of various diseases, among which the most threatening, with worldwide occurrence, is Huanglongbing (HLB), caused by the bacteria Candidatus Liberibacter spp., vectored by psyllids. In orchards, HLB-infected plants are identified by visual observation of symptoms. For laboratory diagnosis, the gold standard is the polymerase chain reaction (PCR), requiring expertise and specific equipment, and with high financial cost. In this work, a selective impedimetric immunosensor was developed for the detection and determination of Ca. L. asiaticus (CLas) in citrus samples. An anti-HLB antibody against the outer membrane protein (OMP) sequence of CLas was obtained and immobilized on previously synthesized and characterized magnetic nanoparticles. The immobilized antibody was presented to various citrus leaf sample extracts. After the affinity reaction with the antigen, a washing step was performed to minimize matrix effects. The affinity reaction was monitored by electrochemical impedance spectroscopy, using a glassy carbon working electrode containing a neodymium magnet. The developed device was able to distinguish HLB-positive samples from HLB-negative samples and those with other infections. The results obtained with the proposed methodology were in good agreement with quantitative PCR (qPCR).

The occurrence of 'Candidatus Liberibacter' spp. and 'Ca. Phytoplasma' spp. associated with blotchy mottle symptoms poses challenges to huanglongbing (HLB) diagnosis using molecular techniques. The ability to detect multiple targets simultaneously and specifically is a key aspect met by quantitative PCR (qPCR). A set of primers and hydrolysis probes useful in either single or multiplex reactions for the detection and quantification of HLB-associated bacteria were developed. Sequences from conserved genes of the ribosomal proteins for Liberibacter and phytoplasma circumvent the lack of specificity and cross-reactivity problems related to 16Sr DNA gene amplification, allowing precise and specific detection of HLB-associated bacteria in citrus and in the Liberibacter vector, Diaphorina citri. The triplex reaction exhibited high quality and precision as a robust tool for quantifying 'Ca. L. asiaticus' (CLas), 'Ca. L. americanus' (CLam), and 16Sr IX phytoplasma. Triplex qPCR showed consistent results and comparable sensitivity to the ribonuclease reductase test, although quantification cycle (Cq) values were higher when compared with 16SrDNA qPCR. Detection tests using field samples indicate that the qPCR triplex can identify HLB-associated bacteria in samples with varying levels of symptoms, ranging from typical to asymptomatic. Assessment of field samples from growers indicated more than 78.6% had Cq lower than 35.0, below the cutoff established for qPCR reactions used in this work. qPCR triplex is a safe, specific, and sufficiently sensitive technique for detecting CLas, CLam, and 16Sr IX phytoplasma simultaneously, in both citrus and D. citri samples. Its application is of importance in assisting growers in making decisions for HLB management.

Abstract The Asian Citrus Psyllid (ACP), Diaphorina citri, severely threatens citrus production worldwide by transmitting the greening (= Huanglongbing)—causing bacterium Candidatus Liberibacter asiaticus. There is growing evidence that the push-pull strategy is suitable to partially mitigate HLB by repelling ACP with transgenic plants engineered to produce repellents and attracting the vector to plants with a minimal disease transmission rate. Species that pull ACP away from commercial citrus plants have been identified, and transgenic plants that repel ACP have been developed. The concept of a repellent-producing plant was first demonstrated with an Arabidopsis line engineered to overexpress a gene controlling the synthesis of β-caryophyllene and other sesquiterpenes. We have analyzed the volatile organic compounds released by this Arabidopsis line and identified α-humulene, α-copaene, and trace amounts of β-elemene, in addition to β-caryophyllene. Behavioral measurements demonstrated that α-copaene repels ACP at doses ca. 100× lower than those needed for β-caryophyllene repellence. In contrast, α-humulene is innocuous at the level emitted by the transgenic plant. We confirmed that a mixture of the three sesquiterpenes in the ratio 1:100:10 repels ACP. Likewise, a commercial sample of copaiba oil containing the three sesquiterpenes, in a proportion similar to that in the transgenic plant, repelled ACP.

"Candidatus Liberibacter asiaticus" (CLas) is associated with citrus huanglongbing, a severe disease with global importance that affects citrus production in Brazil. This study reports the first complete genome of a Brazilian strain of CLas. The genomic structure comparison of strain 9PA with those of 13 complete CLas genomes revealed 9,091 mismatches and 992 gaps/insertions, highlighting eight locally colinear blocks, among which six are in the prophage region. Phylogenetic analysis categorized 13 CLas genomes into two clusters with 9PA clustered with strains from China and the United States. Whole-genomic comparison identified diverse hypervariable genomic regions (HGRs). Three HGRs in the chromosomal region and three in the prophage region were selected and investigated by polymerase chain reaction. HGRs assessed from 68 samples, from medium- to high-huanglongbing incidence areas in Sao Paulo state, were grouped into haplotypes A to P. Haplotype A, which includes strain 9PA, is the second most prevalent, representing 19.1% of the samples. Haplotype B, the most common, accounts for 42.6%. Together with haplotype C, these make up 72% of the evaluated samples. The 9PA strain has prophage P-9PA-1, both integrated and circularized, and P-9PA-3, only found in a circularized form. Prophages show high identity with SC1 (83%) and P-JXGC-3 (98%). Co-occurrence of both type 1 and 3 prophages was observed in field samples. The approach employed provides insights into the Brazilian CLas population, providing markers for population studies and highlighting the prevalence of type 1 and 3 prophages in the population. IMPORTANCE CLas is a destructive pathogen responsible for causing the severe citrus disease known as huanglongbing. Our study presents the first fully sequenced Brazilian strain of CLas, designated as 9PA, and includes an analysis of two prophages occurring in this strain. The main objective of our research was to compare the genome features of this Brazilian strain with other fully sequenced genomes and to identify its hypervariable genetic regions. These regions were subsequently used to assess genomic variability within both the chromosomal and prophage regions in Brazilian isolates of CLas. Our findings offer valuable insights into the diversified adaptation of CLas.

The Asian Citrus Psyllid (ACP), Diaphorina citri, is a vector of the pathological bacterium Candidatus Liberibacter asiaticus (CLas), which causes the most devastating disease to the citrus industry worldwide, known as greening or huanglongbing (HLB). Earlier field tests with an acetic acid-based lure in greening-free, ‘Valencia’ citrus orange groves in California showed promising results. The same type of lures tested in São Paulo, Brazil, showed unsettling results. During the unsuccessful trials, we noticed a relatively large proportion of females in the field, ultimately leading us to test field-collected males and females for Wolbachia and CLas. The results showed high rates of Wolbachia and CLas infection in field populations. We then compared the olfactory responses of laboratory-raised, CLas-free, and CLas-infected males to acetic acid. As previously reported, CLas-uninfected males responded to acetic acid at 1 µg. Surprisingly, CLas-infected males required 50 × higher doses of the putative sex pheromone, thus explaining the failure to capture CLas-infected males in the field. CLas infection was also manifested in electrophysiological responses. Electroantennogram responses from CLas-infected ACP males were significantly higher than those obtained with uninfected males. To the best of our knowledge, this is the first report of a pathogen infection affecting a vector’s response to a sex attractant.

IntroductionUseful germplasm for citrus breeding includes all sexually compatible species of the former genera Citrus, Clymenia, Eremocitrus, Fortunella, Microcitrus, Oxanthera, and Poncirus, now merged in the single Citrus genus. An improved knowledge on the synteny/collinearity between the genome of these different species, and on their recombination landscapes, is essential to optimize interspecific breeding schemes.MethodWe have performed a large comparative genetic mapping study including several main clades of the Citrus genus. It concerns five species (C. maxima, C. medica, C. reticulata, C. trifoliata and C. glauca), two horticultural groups resulting from interspecific admixture (clementine and lemon) and two recent interspecific hybrids (C. australis x C. australasica and C. maxima x C. reticulata). The nine individual genetic maps were established from GBS data of 1,216 hybrids.Results and discussionThe number of SNPs mapped for each parent varies from 760 for C. medica to 4,436 for the C. maxima x C. reticulata hybrid, with an average of 2,162.3 markers by map. Their comparison with C. clementina v1.0 assembly and inter-map comparisons revealed a high synteny and collinearity between the nine genetic maps. Non-Mendelian segregation was frequent and specific for each parental combination. The recombination landscape was similar for the nine mapped parents, and large genomic regions with very low recombination were identified. A consensus genetic map was successfully established. It encompasses 10,756 loci, including 7,915 gene-based markers and 2,841 non-genic SNPs. The anchoring of the consensus map on 15 published citrus chromosome-scale genome assemblies revealed a high synteny and collinearity for the most recent assemblies, whereas discrepancies were observed for some older ones. Large structural variations do not seem to have played a major role in the differentiation of the main species of the Citrus genus. The consensus genetic map is a useful tool to check the accuracy of genome assemblies, identify large structural variation and focus on analyzing potential relationships with phenotypic variations. It should also be a reference framework to integrate the positions of QTLs and useful genes identified in different analyses.

Huanglongbing (or HLB) is the most devastating citrus disease that is widespread throughout the world, causing enormous economic damage to the citrus industry and making oranges and the juice produced with them of lower quality for consumption.The presumptive causal agent of the disease is the bacteria Candidatus Liberibacter spp. that has not yet been cultivated in vitro, with three species: Ca.L. asiaticus, Ca.L. americanus and Ca.L. africanus.Currently, there is no cure for HLB disease.The disease has been managed by the use of healthy nursery trees; monitoring and controlling the psyllid vector; and scouting and eradication of symptomatic trees.One of the alternatives to reduce the problem, currently in use in Florida, USA, has been the prolonged use of antibiotics; however, the long-term practice may be harmful to human health and to the environment.In this sense, the main objective of the present review is to discuss strategies applied to gain deeper understanding concerning chemical and biochemical aspects of HLB disease and review different methodologies to be potentially applied to control the spreading of the disease in the fields and to avoid the increase of the incidence in the orchards.

An updated real-time multiplex quantitative polymerase chain reaction (qPCR) assay was designed and validated for the simultaneous detection of three ‘ Candidatus Liberibacter species’ (CLsp), ‘ Ca. Liberibacter asiaticus’ (CLas), ‘africanus’ (CLaf), and ‘americanus’ (CLam), associated with the huanglongbing disease of citrus. The multiplex assay was designed based on the qPCR assay published in 2006 by Li et al., considering all available CLsp 16S rRNA gene sequences in GenBank and the MIQE guidelines and workflow for qPCR optimization, which became available after 2006. When using the updated multiplex CLsp qPCR assay compared with singleplex qPCR, no significant increase in quantitative cycle (Cq) values was detected. The specificity and sensitivity of the updated qPCR assay was optimal, and measuring the intra- and interassay variations confirmed the reproducibility and repeatability of the assay. The assay was also successfully used with a large number of diverse samples at independent laboratories in four countries, thus demonstrating its transferability, applicability, practicability, and robustness as different qPCR reaction conditions or instruments had a minor effect on Cq values. This updated multiplex CLsp qPCR assay can be used in a variety of citrus surveys, germplasm, or nursery stock programs that require different pathogen detection tools for their successful operation. [Formula: see text] Copyright © 2023 The Author(s). This is an open access article distributed under the CC BY-NC-ND 4.0 International license .

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.

Diseases caused by bacteria are an important and widespread constraint, occurring in almost all crops, including vegetables, pulses, cereals, ornamentals, fruit and forages. Although several strategies have been developed to obtain disease resistance in plants using genetic engineering, few studies have effectively demonstrated the control of bacterial diseases. It has previously been reported that a gene encoding a sphingomyelinase (ThSMase), identified in Trichoderma harzianum, is up-regulated during biocontrol against phytopathogens, and this may have biotechnological applications. SMases are involved in multiple cellular functions, including the immune response against pathogens. We hypothesized that the expression of this gene from fungi in transgenic tobacco could generate resistance to plant pathogens. ThSMase was cloned under control of the double CaMV 35S promoter plus a leader sequence from alfalfa mosaic virus (AMV), and stably introduced and expressed in tobacco. Reverse transcription-quantitative PCR analysis revealed that the ThSMase gene was expressed at similar levels in all transgenic lines tested. Our results showed no statistically significant difference in susceptibility after challenging transgenic and nontransgenic lines with the fungus Sclerotinia sclerotiorum. However, transgenic tobacco plants revealed a significant resistance to the bacterium Pseudomonas syringae pv. tabaci, and a tolerance to Xylella fastidiosa. Our results demonstrated the strong potential of ThSMase in biotechnological processes such as molecular breeding of the plants.

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.

‘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.

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.