Study on the citrus greening disease: Current challenges and novel therapies.

The unprecedented worldwide spread of the Citrus greening disorder, called Huanglongbing (HLB), has urged researchers for rapid interventions. HLB poses a considerable threat to global citriculture owing to its devastating impact on citrus species. This disease is caused by Candidatus Liberibacter species (CLs), primarily transferred through psyllid insects, such as Trioza erytreae and Diaphorina citri. It results in phloem malfunction, root decline, and altered plant source-sink relationships, leading to a deficient plant with minimal yield before it dies. Thus, many various techniques have been employed to eliminate HLB and control vector populations through the application of insecticides and antimicrobials. The latter have evidenced short-term efficiency. While nucleic acid-based analyses and symptom-based identification of the disease have been used for detection, they suffer from limitations such as false negatives, complex sample preparation, and high costs. To address these challenges, secreted protein-based biomarkers offer a promising solution for accurate, rapid, and cost-effective disease detection. This paper presents an overview of HLB symptoms in citrus plants, including leaf and fruit symptoms, as well as whole tree symptoms. The differentiation between HLB symptoms and those of nutrient deficiencies is discussed, emphasizing the importance of precise identification for effective disease management. The elusive nature of CLs and the challenges in culturing them in axenic cultures have hindered the understanding of their pathogenic mechanisms. However, genome sequencing has provided insights into CLs strains' metabolic traits and potential virulence factors. Efforts to identify potential host target genes for resistance are discussed, and a high-throughput antimicrobial testing method using Citrus hairy roots is introduced as a promising tool for rapid assessment of potential treatments. This review summarizes current challenges and novel therapies for HLB disease. It highlights the urgency of developing accurate and efficient detection methods and identifying the complex relations between CLs and their host plants. Transgenic citrus in conjunction with secreted protein-based biomarkers and innovative testing methodologies could revolutionize HLB management strategies toward achieving a sustainable citrus cultivation. It offers more reliable and practical solutions to combat this devastating disease and safeguard the global citriculture industry.

Huanglongbing and Citrus Variegated Chlorosis Integrated Management Based on Favorable Periods for Vector Population Increase and Symptom Expression.

Huanglongbing (HLB, associated with Candidatus Liberibacter asiaticus and transmitted by the Asian citrus psyllid Diaphorina citri) and citrus variegated chlorosis (CVC, caused by Xylella fastidiosa subsp. pauca and transmitted by sharpshooter species) have been managed by vector control and removal of symptomatic trees. Although vectors and new symptomatic trees can be detected year round, peaks of vector populations are higher in spring and summer, and the most symptomatic trees are found in autumn and winter. This work aimed to compare the management of both diseases during these favorable periods. The experiment was conducted during 5 years in a commercial orchard and had a three-by-two factorial design. The factor "vector control" had three levels: monthly vector control year round (VCYR), monthly vector control in spring and summer (VCSS), and vector control when a threshold level of 10% occupancy was detected (VCOT). The factor "inoculum removal" had two levels: monthly eradication year round (TEYR) and monthly eradication in autumn and winter (TEAW). Host flush, both HLB and CVC vector populations, and the number of symptomatic citrus plants were visually assessed. The level of vectors over the seasons, as measured using the average area under the curve (AUC), was similar for all treatments with the exception of psyllid abundance, which was approximately 4.5 times higher for VCSS than for other treatments. For both diseases, no difference in the average AUC of disease progress and disease final incidence was observed. VCOT or adjusted VCSS associated with TEAW could be integrated for sustainable citrus production.

Antibacterial FANA oligonucleotides as a novel approach for managing the Huanglongbing pathosystem.

Candidatus Liberibacter asiaticus (CLas), a bacterium transmitted by the Asian citrus psyllid, Diaphorina citri, is the causal agent of citrus greening disease, or Huanglongbng (HLB). Currently, vector population suppression with insecticides and tree removal are the most effective strategies for managing the HLB pathosystem. In this study, we assessed the bactericidal capabilities of 2'-deoxy-2'-fluoro-D-arabinonucleic acid antisense oligonucleotides (FANA ASO) both in vitro and in vivo by (1) confirming their capacity to penetrate insect cells, (2) silencing bacterial essential genes, and (3) quantifying reductions in bacterial titer and D. citri transmission. We confirmed that FANA ASO are able to penetrate insect cells without the use of a delivery agent. Expression of an essential gene in the D. citri endosymbiont, Wolbachia (wDi), significantly decreased by 30% following incubation with a wDi-specific FANA ASO. Viability of isolated wDi cells also decreased in response to the FANA ASO treatment. Delivery of a CLas-specific FANA ASO to infected adult D. citri in feeding assays resulted in significant silencing of a CLas essential gene. CLas relative density and transmission were significantly lower among D. citri fed FANA ASO in diet compared to untreated insects. Root infusions of a CLas-specific FANA ASO into infected Citrus trees significantly reduced CLas titer during a 30-day trial. Our results suggest that FANA ASO targeting insect-transmitted plant bacteria or insect endosymbionts may be useful tool for integrated management of agricultural pathogens.

Development on Citrus medica infected with 'Candidatus Liberibacter asiaticus' has sex-specific and -nonspecific impacts on adult Diaphorina citri and its endosymbionts.

Huanglongbing (HLB) is a deadly, incurable citrus disease putatively caused by the unculturable bacterium, 'Candidatus Liberibacter asiaticus' (CLas), and transmitted by Diaphorina citri. Prior studies suggest D. citri transmits CLas in a circulative and propagative manner; however, the precise interactions necessary for CLas transmission remain unknown, and the impact of insect sex on D. citri-CLas interactions is poorly understood despite reports of sex-dependent susceptibilities to CLas. We analyzed the transcriptome, proteome, metabolome, and microbiome of male and female adult D. citri reared on healthy or CLas-infected Citrus medica to determine shared and sex-specific responses of D. citri and its endosymbionts to CLas exposure. More sex-specific than shared D. citri responses to CLas were observed, despite there being no difference between males and females in CLas density or relative abundance. CLas exposure altered the abundance of proteins involved in immunity and cellular and oxidative stress in a sex-dependent manner. CLas exposure impacted cuticular proteins and enzymes involved in chitin degradation, as well as energy metabolism and abundance of the endosymbiont 'Candidatus Profftella armatura' in both sexes similarly. Notably, diaphorin, a toxic Profftella-derived metabolite, was more abundant in both sexes with CLas exposure. The responses reported here resulted from a combination of CLas colonization of D. citri as well as the effect of CLas infection on C. medica. Elucidating these impacts on D. citri and their endosymbionts contributes to our understanding of the HLB pathosystem and identifies the responses potentially critical to limiting or promoting CLas acquisition and propagation in both sexes.

Asian citrus psyllid adults inoculate huanglongbing bacterium more efficiently than nymphs when this bacterium is acquired by early instar nymphs.

The Asian citrus psyllid (Diaphorina citri) transmits the bacterium 'Candidatus Liberibacter asiaticus' (CLas), which causes huanglongbing (citrus greening) disease, in a circulative-propagative manner. We compared CLas inoculation efficiency of D. citri nymphs and adults into healthy (uninfected) citron leaves when both vector stages were reared from eggs on infected plants. The proportion of CLas-positive leaves was 2.5% for nymphs and 36.3% for adults. CLas acquisition by early instar nymphs followed by dissections of adults and 4th instar nymphs revealed that CLas bacterium had moved into the head-thorax section (containing the salivary glands) in 26.7-30.0% of nymphs and 37-45% of adults. Mean Ct values in these sections were 31.6-32.9 and 26.8-27.0 for nymphs and adults, respectively. Therefore, CLas incidence and titer were higher in the head-thorax of adults than in nymphs. Our results suggest that following acquisition of CLas by early instar D. citri nymphs, emerging adults inoculate the bacteria into citrus more efficiently than nymphs because adults are afforded a longer latent period necessary for multiplication and/or translocation of CLas into the salivary glands of the vector. We propose that CLas uses D. citri nymphs mainly for pathogen acquisition and multiplication, and their adults mainly for pathogen inoculation and spread.

Unexpected conservation and global transmission of agrobacterial virulence plasmids.

The accelerated evolution and spread of pathogens are threats to host species. Agrobacteria require an oncogenic Ti or Ri plasmid to transfer genes into plants and cause disease. We developed a strategy to characterize virulence plasmids and applied it to analyze hundreds of strains collected between 1927 and 2017, on six continents and from more than 50 host species. In consideration of prior evidence for prolific recombination, it was surprising that oncogenic plasmids are descended from a few conserved lineages. Characterization of a hierarchy of features that promote or constrain plasticity allowed inference of the evolutionary history across the plasmid lineages. We uncovered epidemiological patterns that highlight the importance of plasmid transmission in pathogen diversification as well as in long-term persistence and the global spread of disease.

First Report of HLB Causal Agent in Psyllid in State of Bahia, Brazil.

The state of Bahia ranks fourth in the national rank for citrus production, and the region of Chapada Diamantina is emerging an important producer of orange for fresh fruit market. Huanglongbing (HLB) is the major phytosanitary threat to Bahia citriculture. In Brazil, the disease was first reported in 2004 in São Paulo state. The bacterium Candidatus Liberibacter asiaticus (CLas) is one of the causal agents of HLB, which is transmitted by the insect vector Diaphorina citri Kuwayama (Hemiptera: Liviidae). Bahia is a HLB-free area; therefore, it is essential to monitor its citrus-producing areas to early detect any possible introduction of the CLas. This study aimed to monitor the presence of the bacteria in the insect vector. Diaphorina citri samples were collected from 2011 to 2014 in different cities located at Chapada Diamantina region and tested by qPCR for the presence of CLas. Three samples were considered positive to bacterium, and all from psyllids collected on Murraya paniculata in the city of Seabra.

Heterologous Expression of CLIBASIA_03915/CLIBASIA_04250 by Tobacco Mosaic Virus Resulted in Phloem Necrosis in the Senescent Leaves of Nicotiana benthamiana.

Huanglongbing (HLB), also known as citrus greening, is the most notorious citrus disease worldwide. Candidatus Liberibacter asiaticus (CaLas) is a phloem-restricted bacterium associated with HLB. Because there is no mutant library available, the pathogenesis of CaLas is obscure. In this study, we employed tobacco mosaic virus (TMV) to express two mature secretion proteins CLIBASIA_03915 (m03915) and CLIBASIA_04250 (m04250) in Nicotiana benthamiana (N. benthamiana). Phloem necrosis was observed in the senescent leaves of N. benthamiana that expressed the two low molecular weight proteins, while no phloem necrosis was observed in the plants that expressed the control, green fluorescent protein (GFP). Additionally, no phloem necrosis was observed in the senescent leaves of N. benthamiana that expressed the null mutation of m03915 and frameshifting m04250. The subcellular localizations of m03915 and m04250 were determined by fusion with GFP using confocal microscopy. The subcellular localization of m03915 was found to be as free GFP without a nuclear localization sequence (NLS). However, m04250 did have an NLS. Yeast two-hybrid (Y2H) was carried out to probe the citrus proteins interacting with m03915 and m04250. Six citrus proteins were found to interact with m03915. The identified proteins were involved in the metabolism of compounds, transcription, response to abiotic stress, ubiquitin-mediated protein degradation, etc. The prey of m04250 was involved in the processing of specific pre-mRNAs. Identification of new virulence factors of CaLas will give insight into the pathogenesis of CaLas, and therefore, it will eventually help develop the HLB-resistant citrus.

Comparative iTRAQ proteomic profiling of sweet orange fruit on sensitive and tolerant rootstocks infected by 'Candidatus Liberibacter asiaticus'.

Citrus Huanglongbing (HLB), which is also known as citrus greening, is a destructive disease continuing to devastate citrus production worldwide. Although all citrus varieties can be infected with 'Candidatus Liberibacter asiaticus' (CaLas), a certain level of HLB tolerance of scion varieties can be conferred by some rootstocks. To understand the effects of rootstock varieties on orange fruit under CaLas stress, comparative iTRAQ proteomic profilings were conducted, using fruit from 'Valencia' sweet orange grafted on the sensitive ('Swingle') and tolerant rootstocks (a new selection called '46x20-04-48') infected by CaLas as experimental groups, and the same plant materials without CaLas infection as controls. The symptomatic fruit on 'Swingle' had 573 differentially-expressed (DE) proteins in comparison with their healthy fruit on the same rootstock, whereas the symptomatic fruit on '46x20-04-48' had 263 DE proteins. Many defense-associated proteins were down-regulated in the symptomatic fruit on 'Swingle' rootstock that were seldom detected in the symptomatic fruit on the '46x20-04-48' rootstock, especially the proteins involved in the jasmonate biosynthesis (AOC4), jasmonate signaling (ASK2, RUB1, SKP1, HSP70T-2, and HSP90.1), protein hydrolysis (RPN8A and RPT2a), and vesicle trafficking (SNAREs and Clathrin) pathways. Therefore, we predict that the down-regulated proteins involved in the jasmonate signaling pathway and vesicle trafficking are likely to be related to citrus sensitivity to the CaLas pathogen.

What makes or breaks a campaign to stop an invading plant pathogen?

Diseases in humans, animals and plants remain an important challenge in our society. Effective control of invasive pathogens often requires coordinated concerted action of a large group of stakeholders. Both epidemiological and human behavioural factors influence the outcome of a disease control campaign. In mathematical models that are frequently used to guide such campaigns, human behaviour is often ill-represented, if at all. Existing models of human, animal and plant disease that do incorporate participation or compliance are often driven by pay-offs or direct observations of the disease state. It is however very well known that opinion is an important driving factor of human decision making. Here we consider the case study of Citrus Huanglongbing disease (HLB), which is an acute bacterial disease that threatens the sustainability of citrus production across the world. We show how by coupling an epidemiological model of this invasive disease with an opinion dynamics model we are able to answer the question: What makes or breaks the effectiveness of a disease control campaign? Frequent contact between stakeholders and advisors is shown to increase the probability of successful control. More surprisingly, we show that informing stakeholders about the effectiveness of control methods is of much greater importance than prematurely increasing their perceptions of the risk of infection. We discuss the overarching consequences of this finding and the effect on human as well as plant disease epidemics.

UV reflective properties of magnesium oxide increase attraction and probing behavior of Asian citrus psyllids (Hemiptera: Liviidae).

Asian citrus psyllid (Diaphorina citri) vectors the bacterium Candidatus Liberibacter asiaticus, the causal pathogen of citrus greening disease that is devastating citrus industries worldwide. Suppressing psyllid populations is crucial to prevent disease spread. An attract-and-kill trap based on psyllid behavior would fill a niche for monitoring and control. To optimize visual attraction of psyllids, the ultraviolet (UV) reflective properties of magnesium oxide (MgO) and/or barium sulfate (BaSO4) were assessed for potential application to a trap surface. Under low UV, high UV and natural sunlight conditions, the reflectance, attraction, and probing behaviors of psyllids were evaluated on surfaces containing magnesium oxide or barium sulfate. Magnesium oxide added to yellow sticky traps enhanced visual response of D. citri. Probing assays demonstrated that magnesium oxide alone or as a mixture with a phagostimulant blend, increased the UV reflectance of substrates, as well as, attraction and probing by psyllids. Results demonstrated that psyllids respond to both short (UV) and long (yellow) wavelengths during orientation, and that these inert compounds can increase UV reflectance and improve attractiveness of an attract-and-kill device.

Metabolomics Analysis of the Peels of Different Colored Citrus Fruits (Citrus reticulata cv. 'Shatangju') During the Maturation Period Based on UHPLC-QQQ-MS.

Citrus is a globally consumed fruit with great popularity. Mandarin (Citrus reticulata cv. 'Shatangju') is a local variety, and its planting area and yield are the greatest regarding fruit tree planting in Guangdong Province, China. However, its resistance to Huanglongbing (HLB) is weak. After infection by HLB, the fruits cannot develop normally. In this study, four kinds of fruits were classified as HBG, XQG, ZQG, and DHG, according to the color of their peels. The metabolomes of the three abnormally colored groups (HBG, XQG, and ZQG) and the normally colored group (DHG) were compared using a UPLC-QQQ-MS-based metabolomics approach. In total, 913 metabolites were identified and classified into 23 different categories, including phenylpropanoids and flavonoids; among them, 215 (HBG, 177; XQG, 124; and ZQG, 62) metabolites showed differential accumulation in the three comparison groups (HBG/XQG/ZQG versus DHG). A total of 2 unique metabolites, O-caffeoyl maltotriose and myricetin were detected only in DHG samples. When comparing HBG with DHG, there were 109 decreased and 68 increased metabolites; comparing XQG with DHG, there were 88 decreased and 36 increased metabolites; comparing ZQG with DHG, 41 metabolites were decreased, and 21 metabolites were increased. Metabolic pathway enrichment analysis of these differential metabolites showed significant enrichment of the "phenylpropanoid biosynthesis" pathway in all comparison groups. The hierarchical cluster analysis of the differential metabolites of the four groups showed a clear grouping patterns. The relative contents of three phenylpropanoids, four flavonoids, two alkaloids, one anthocyanin, and two other metabolites were significantly different between each comparison group. This study might provide fundamental insight for the isolation and identification of functional compounds from the peels of citrus fruit infected with HLB and for in-depth research on the effect of HLB on the formation of fruits pigment and the development of HLB-resistant citrus varieties.

Comparative Genomics Screen Identifies Microbe-Associated Molecular Patterns from 'Candidatus Liberibacter' spp. That Elicit Immune Responses in Plants.

Citrus huanglongbing (HLB), caused by phloem-limited 'Candidatus Liberibacter' bacteria, is a destructive disease threatening the worldwide citrus industry. The mechanisms of pathogenesis are poorly understood and no efficient strategy is available to control HLB. Here, we used a comparative genomics screen to identify candidate microbe-associated molecular patterns (MAMPs) from 'Ca. Liberibacter' spp. We identified the core genome from multiple 'Ca. Liberibacter' pathogens, and searched for core genes with signatures of positive selection. We hypothesized that genes encoding putative MAMPs would evolve to reduce recognition by the plant immune system, while retaining their essential functions. To efficiently screen candidate MAMP peptides, we established a high-throughput microtiter plate-based screening assay, particularly for citrus, that measured reactive oxygen species (ROS) production, which is a common immune response in plants. We found that two peptides could elicit ROS production in Arabidopsis and Nicotiana benthamiana. One of these peptides elicited ROS production and defense gene expression in HLB-tolerant citrus genotypes, and induced MAMP-triggered immunity against the bacterial pathogen Pseudomonas syringae. Our findings identify MAMPs that boost immunity in citrus and could help prevent or reduce HLB infection.

Identification of the Virulence Factors of Candidatus Liberibacter asiaticus via Heterologous Expression in Nicotiana benthamiana using Tobacco Mosaic Virus.

Huanglongbing (HLB), also known as citrus greening, is the most destructive disease of citrus worldwide. HLB is associated with the non-culturable bacterium, Candidatus Liberibacter asiaticus (CaLas) in the United States. The virulence mechanism of CaLas is largely unknown, partly because of the lack of a mutant library. In this study, Tobacco mosaic virus (TMV) and Nicotiana benthamiana (N. benthamiana) were used for large-scale screening of the virulence factors of CaLas. Agroinfiltration of 60 putative virulence factors in N. benthamiana led to the identification of four candidates that caused severe symptoms in N. benthamiana, such as growth inhibition and cell death. CLIBASIA_05150 and CLIBASIA_04065C (C-terminal of CLIBASIA_04065) could cause cell death in the infiltrated leaves at five days post infiltration. Two low-molecular-weight candidates, CLIBASIA_00470 and CLIBASIA_04025, could inhibit plant growth. By converting start codon to stop codon or frameshifting, the four genes lost their harmful effects to N. benthamiana. It indicated that the four virulence factors functioned at the protein level rather than at the RNA level. The subcellular localization of the four candidates was determined by confocal laser scanning microscope. CLIBASIA_05150 located in the Golgi apparatus; CLIBASIA_04065 located in the mitochondrion; CLIBASIA_00470 and CLIBASIA_04025 distributed in cells as free GFP. The host proteins interacting with the four virulence factors were identified by yeast two-hybrid. The host proteins interacting with CLIBASIA_00470 and CLIBASIA_04025 were overlapping. Based on the phenotypes, the subcellular localization and the host proteins identified by yeast two-hybrid, CLIBASIA_00470 and CLIBASIA_04025, functioned redundantly. The hypothesis of CaLas virulence was proposed. CaLas affects citrus development and suppresses citrus disease resistance, comprehensively, in a complicated manner. Ubiquitin-mediated protein degradation might play a vital role in CaLas virulence. Deep characterization of the interactions between the identified virulence factors and their prey will shed light on HLB. Eventually, it will help in developing HLB-resistant citrus and save the endangered citrus industry worldwide.

Physiochemical changes mediated by "Candidatus Liberibacter asiaticus" in Asian citrus psyllids.

Plant pathogenic bacteria interact with their insect host(s)/vector(s) at the cellular and molecular levels. This interaction may alter the physiology of their insect vector, which may also promote the growth and transmission of the bacterium. Here we studied the effect of "Candidatus Liberibacter asiaticus" ("Ca. L. asiaticus") on physiochemical conditions within its insect vector, the Asian citrus psyllid (ACP), and whether these changes were beneficial for the pathogen. The local microenvironments inside ACPs were quantified using microelectrodes. The average hemolymph pH was significantly higher in infected ACPs (8.13 ± 0.21) than in "Ca. L. asiaticus"-free ACPs (7.29 ± 0.15). The average hemolymph oxygen tension was higher in "Ca. L. asiaticus"-free ACPs than in infected ACPs (67.13% ± 2.11% vs. 35.61% ± 1.26%). Oxygen tension reduction and pH increase were accompanied by "Ca. L. asiaticus" infection. Thus, oxygen tension of the hemolymph is an indicator of infection status, with pH affected by the severity of the infection.

Liberibacter crescens biofilm formation in vitro: establishment of a model system for pathogenic 'Candidatus Liberibacter spp.'

The Liberibacter genus comprises insect endosymbiont bacterial species that cause destructive plant diseases, including Huanglongbing in citrus and zebra chip in potato. To date, pathogenic 'Candidatus Liberibacter spp.' (CLs) remain uncultured, therefore the plant-associated Liberibacter crescens (Lcr), only cultured species of the genus, has been used as a biological model for in vitro studies. Biofilm formation by CLs has been observed on the outer midgut surface of insect vectors, but not in planta. However, the role of biofilm formation in the life cycle of these pathogens remains unclear. Here, a model system for studying CLs biofilms was developed using Lcr. By culture media modifications, bovine serum albumin (BSA) was identified as blocking initial cell-surface adhesion. Removal of BSA allowed for the first time observation of Lcr biofilms. After media optimization for biofilm formation, we demonstrated that Lcr attaches to surfaces, and form cell aggregates embedded in a polysaccharide matrix both in batch cultures and under flow conditions in microfluidic chambers. Biofilm structures may represent excellent adaptive advantages for CLs during insect vector colonization helping with host retention, immune system evasion, and transmission. Future studies using the Lcr model established here will help in the understanding of the biology of CLs.

Laser-induced breakdown spectroscopy (LIBS) as a novel technique for detecting bacterial infection in insects.

To prevent the spread of diseases in humans, animals or plants, determining whether potential vectors are infected is crucial. For example, early detection of the citrus disease Huanglongbing, which has been a scourge on the citrus industries around the world, is a critical need. This vector-borne disease is transmitted by Diaphorina citri, the Asian citrus psyllid, which carries the putative bacterial phytopathogen, Candidatus Liberibacter asiaticus (CLas). In this investigation, we introduced Laser-Induced Breakdown Spectroscopy (LIBS) to reveal key biochemical differences between CLas-infected and non-infected psyllids. The emission spectra captured from laser ablation of CLas-infected and healthy psyllids were processed through the principal component analysis (PCA) method and compared. Thirteen peaks from seven different elements were detected in D. citri. The t-test showed that CLas-infected D. citri were deficients in zinc, iron, copper, magnesium, calcium, and nitrogen. The PCA showed that LIBS can successfully differentiate between CLas-infected and healthy D. citri by comparing their elemental profile. In this work, we demonstrated a method that allows for a fast and precise compositional microanalysis of an insect vector which can contribute to the early detection of citrus huanglongbing.

Genetic Diversity of 'Candidatus Liberibacter asiaticus' Revealed by Short Tandem Repeats and Prophage Typing Indicates Population Homogeneity in Brazil.

'Candidatus Liberibacter asiaticus' is the most common huanglongbing-associated bacteria, being present in Asia, South, Central, and North America. Genomic approaches enabled sequencing of 'Ca. L. asiaticus' genomes, allowing for a broader assessment of its genetic variability with the application of polymerase chain reaction (PCR)-based tools such as microsatellite or short tandem repeat (STR) analysis. Although these tools contributed to a detailed analysis of strains from Japan, China, and the United States, Brazilian strains were analyzed in either too few samples with several STRs or in several strains with only a single microsatellite and a single PCR marker. We used 573 'Ca. L. asiaticus' strains, mainly collected from São Paulo State (SPS), in our genetic analyses, employing three STRs and several prophage PCR markers. STR revealed a homogeneous population regardless of sampling year or geographic regions of SPS. Thirty-eight haplotypes were recognized with a predominance of VNTR_005 higher than 10 repeats, with VNTR_002 and VNTR_077 containing 11 and 8 repeats, respectively. This haplotype is indicated as class HE, which comprised 80.28% of strains. Classes HA and HB, predominant in Florida, were not found. A new genomic organization in the junction of prophages SC2 and SC1 is prevalent in Brazilian strains, indicating gene rearrangement and a widespread occurrence of a type 1 prophage as well as the presence of a type 2-like prophage. Our results indicate that 'Ca. L. asiaticus' populations are homogeneous and harbor a new genomic organization in prophages type 1 and 2.

Targeted Early Detection of Citrus Huanglongbing Causal Agent 'Candidatus Liberibacter asiaticus' Before Symptom Expression.

Citrus Huanglongbing (HLB) is the most severe disease of citrus plants caused by 'Candidatus Liberibacter asiaticus' and transmitted by the insect vector Asian citrus psyllid (ACP). No effective curative measure is available against HLB. For citrus production areas without HLB or with low HLB disease incidence, removal of 'Ca. L. asiaticus' inoculum is critical to prevent HLB spread. Such a strategy requires robust early diagnosis of HLB for inoculum removal to prevent ACP acquisition and transmission of 'Ca. L. asiaticus'. However, early diagnosis of HLB is challenging, because the citrus trees remain asymptomatic for several months to years after 'Ca. L. asiaticus' transmission by ACP. In this study, we report a new method for targeted early detection of 'Ca. L. asiaticus' in cultivar Valencia sweet orange (Citrus sinensis) before HLB symptom expression. We take advantage of the fact that 'Ca. L. asiaticus' remains around the ACP feeding site immediately after transmission into the young flush and before flush maturation. ACPs secrete salivary sheaths at their feeding sites, which can be visualized using Coomassie brilliant blue staining owing to the presence of salivary sheaths secreted by ACP. Epifluorescence and confocal microscopy indicate the presence of salivary sheaths beneath the blue spots on ACP-fed leaves. Quantitative real-time polymerase chain reaction (PCR) and conventional PCR assays are able to detect 'Ca. L. asiaticus' in the ACP feeding surrounding areas as early as 2 to 20 days after ACP feeding. This finding lays a foundation to develop much-needed tools for early diagnosis of HLB before symptom expression, thus assisting 'Ca. L. asiaticus' inoculum removal and preventing HLB from spreading.

Modeling Citrus Huanglongbing transmission within an orchard and its optimal control.

Citrus Huanglongbing (HLB) is the most devastating citrus disease worldwide. In this paper, a deterministic dynamical model is proposed to explore the transmission dynamics of HLB between citrus tree and Asian citrus psyllid (ACP). Using the theory of dynamical system, the dynamics of the model are rigorously analyzed. The results show that the disease-free equilibrium is globally asymptotically stable when the basic reproduction number $\mathscr{R}_0 < 1$, and when $\mathscr{R}_0 > 1$ the system is uniformly persistent. Applying the global sensitivity analysis of $\mathscr{R}_0$, some parameters that have the greatest impact on HLB transmission dynamics are obtained. Furthermore, the optimal control theory is applied to the model to study the corresponding optimal control problem. Both analytical and numerical results show that: (1) the infected ACP plays a decisive role in the transmission of HLB in citrus trees, and eliminating the ACP will be helpful to curtail the spread of HLB; (2) optimal control strategy is superior to the constant control strategy in decreasing the prevalence of the diseased citrus trees, and the cost of implementing optimal control is much lower than that of the constant control strategy; and (3) spraying insecticides is more effective than other control strategies in reducing the number of ACP in the early phase of the transmission of HLB. These theoretical and numerical results may be helpful in making public policies to control HLB in orchards more effectively.