RESUMEN
Huanglongbing (HLB) is a fatal citrus disease that is currently threatening citrus varieties worldwide. One putative causative agent, Candidatus Liberibacter asiaticus (CLas), is vectored by Diaphorina citri, known as the Asian citrus psyllid (ACP). Understanding the details of CLas infection in HLB disease has been hindered by its Candidatus nature and the inability to confidently detect it in diseased trees during the asymptomatic stage. To identify early changes in citrus metabolism in response to inoculation of CLas using its natural psyllid vector, leaves from Madam Vinous sweet orange (Citrus sinensis (L.) Osbeck) trees were exposed to CLas-positive ACP or CLas-negative ACP and longitudinally analyzed using transcriptomics (RNA sequencing), proteomics (liquid chromatography-tandem mass spectrometry; data available in Dryad: 10.25338/B83H1Z), and metabolomics (proton nuclear magnetic resonance). At 4 weeks postexposure (wpe) to psyllids, the initial HLB plant response was primarily to the ACP and, to a lesser extent, the presence or absence of CLas. Additionally, analysis of 4, 8, 12, and 16 wpe identified 17 genes and one protein as consistently differentially expressed between leaves exposed to CLas-positive ACP versus CLas-negative ACP. This study informs identification of early detection molecular targets and contributes to a broader understanding of vector-transmitted plant pathogen interactions.
Asunto(s)
Citrus sinensis , Hemípteros , Enfermedades de las Plantas , Proteómica , Rhizobiaceae , Transcriptoma , Animales , Citrus sinensis/genética , Citrus sinensis/metabolismo , Citrus sinensis/microbiología , Citrus sinensis/parasitología , Hemípteros/microbiología , Hemípteros/genética , Hemípteros/metabolismo , Insectos Vectores/microbiología , Insectos Vectores/metabolismo , Liberibacter/patogenicidad , Liberibacter/genética , Liberibacter/metabolismo , Metabolómica/métodos , Enfermedades de las Plantas/microbiología , Hojas de la Planta/microbiología , Hojas de la Planta/metabolismo , Proteoma/metabolismo , Proteoma/análisis , Proteómica/métodos , Rhizobiaceae/patogenicidad , Rhizobiaceae/genética , Rhizobiaceae/fisiologíaRESUMEN
Citrus Huanglongbing (HLB), which is caused by 'Candidatus Liberibacter asiaticus' (CLas), is one of the most destructive citrus diseases worldwide, and defense-related Citrus sinensis gene resources remain largely unexplored. Calcium signaling plays an important role in diverse biological processes. In plants, a few calcium-dependent protein kinases (CDPKs/CPKs) have been shown to contribute to defense against pathogenic microbes. The genome of C. sinensis encodes dozens of CPKs. In this study, the role of C. sinensis calcium-dependent protein kinases (CsCPKs) in C. sinensis defense was investigated. Silencing of CsCPK6 compromised the induction of defense-related genes in C. sinensis. Expression of a constitutively active form of CsCPK6 (CsCPK6CA) triggered the activation of defense-related genes in C. sinensis. Complementation of CsCPK6 rescued the defense-related gene induction in an Arabidopsis thaliana cpk4/11 mutant, indicating that CsCPK6 carries CPK activity and is capable of functioning as a CPK in Arabidopsis. Moreover, an effector derived from CLas inhibits defense induced by the expression of CsCPK6CA and autophosphorylation of CsCPK6, which suggests the involvement of CsCPK6 and calcium signaling in defense. These results support a positive role for CsCPK6 in C. sinensis defense against CLas, and the autoinhibitory regulation of CsCPK6 provides a potential genome-editing target for improving C. sinensis defense. [Formula: see text] Copyright © 2024 The Author(s). This is an open access article distributed under the CC BY-NC-ND 4.0 International license.
Asunto(s)
Citrus sinensis , Regulación de la Expresión Génica de las Plantas , Enfermedades de las Plantas , Proteínas de Plantas , Proteínas Quinasas , Citrus sinensis/genética , Citrus sinensis/microbiología , Enfermedades de las Plantas/microbiología , Enfermedades de las Plantas/inmunología , Proteínas Quinasas/metabolismo , Proteínas Quinasas/genética , Proteínas de Plantas/genética , Proteínas de Plantas/metabolismo , Arabidopsis/genética , Arabidopsis/microbiología , Arabidopsis/inmunología , Resistencia a la Enfermedad/genética , Liberibacter/genética , Liberibacter/fisiologíaRESUMEN
The evolution of insect vector-pathogen relationships has long been of interest in the field of molecular ecology. One system of special relevance, due to its economic impacts, is that between Diaphorina citri and 'Candidatus Liberibacter asiaticus' (CLas), the cause of the severe Asian form of huanglongbing. CLas-positive D. citri are more fecund than their CLas-negative counterparts, boosting opportunities for pathogens to acquire new vector hosts. The molecular mechanism behind this life-history shift remains unclear. Here, we found that CLas promoted ovarian development and increased the expression of the vitellogenin receptor (DcVgR) in ovaries. DcVgR RNAi significantly decreased fecundity and CLas titer in ovaries, extended the preoviposition period, shortened the oviposition period and blocked ovarian development. Given their importance in gene regulation, we explored the role of miRNAs in shaping these phenotypes and their molecular triggers. Our results showed that one miRNA, miR-275, suppressed DcVgR expression by binding to its 3' UTR. Overexpression of miR-275 knocked down DcVgR expression and CLas titer in ovaries, causing reproductive defects that mimicked DcVgR knockdown phenotypes. We focused, further, on roles of the Juvenile Hormone (JH) pathway in shaping the observed fecundity phenotype, given its known impacts on ovarian development. After CLas infection, this pathway was upregulated, thereby increasing DcVgR expression. From these combined results, we conclude that CLas hijacks the JH signalling pathway and miR-275, thereby targeting DcVgR to increase D. citri fecundity. These changes simultaneously increase CLas replication, suggesting a pathogen-vector host mutualism, or a seemingly helpful, but cryptically costly life-history manipulation.
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Citrus , Hemípteros , Liberibacter , MicroARNs , Rhizobiaceae , Animales , Femenino , Rhizobiaceae/genética , Citrus/genética , Enfermedades de las Plantas/genética , Hemípteros/genética , Fertilidad/genética , MicroARNs/genética , Proliferación CelularRESUMEN
AIMS: Huanglongbing (citrus greening) is a plant disease putatively caused by the unculturable Gram-negative bacterium Candidatus Liberibacter asiaticus (CLas), and it has caused severe damage to citrus plantations worldwide. There are no definitive treatments for this disease, and conventional disease control techniques have shown limited efficacy. This work presents an in silico evaluation of using specifically targeting anti-microbial peptides (STAMPs) consisting of a targeting segment and an antimicrobial segment to inhibit citrus greening by inhibiting the BamA protein of CLas, which is an outer membrane protein crucial for bacterial viability. METHODS AND RESULTS: Initially, a set of peptides with a high affinity toward BamA protein were screened and evaluated via molecular docking and molecular dynamics simulations and were verified in vitro via bio-layer interferometry (BLI). In silico studies and BLI experiments indicated that two peptides, HASP2 and HASP3, showed stable binding to BamA. Protein structures for STAMPs were created by fusing known anti-microbial peptides (AMPs) with the selected short peptides. The binding of STAMPs to BamA was assessed using molecular docking and binding energy calculations. The attachment of high-affinity short peptides significantly reduced the free energy of binding for AMPs, suggesting that it would make it easier for the STAMPs to bind to BamA. Efficacy testing in vitro using a closely related CLas surrogate bacterium showed that STAMPs had greater inhibitory activity than AMP alone. CONCLUSIONS: In silico and in vitro results indicate that the STAMPs can inhibit CLas surrogate Rhizobium grahamii more effectively compared to AMPs, suggesting that STAMPs can achieve better inhibition of CLas, potentially via enhancing the site specificity of AMPs.
Asunto(s)
Citrus , Hemípteros , Rhizobiaceae , Animales , Péptidos Antimicrobianos , Simulación del Acoplamiento Molecular , Liberibacter , Citrus/microbiología , Enfermedades de las Plantas/prevención & control , Enfermedades de las Plantas/microbiología , Hemípteros/microbiologíaRESUMEN
Fast and effective monitoring and surveillance techniques are crucial for the swift implementation of control methods to prevent the spread of Huanglongbing, a devastating citrus disease, and its invasive psyllid vector, Asian citrus psyllid, Diaphorina citri, into South Africa, as well as to control the native vector, African citrus triozid, Trioza erytreae. Monitoring for citrus psyllid pests can be improved by using semiochemical odorants to augment already visually attractive yellow sticky traps. However, environmental variables such as temperature and humidity could influence odorant release rates. Five field cages were used to test the ability of a selection of odorants to improve yellow sticky trap efficacy in capturing citrus psyllids. Environmental effects on odorant loss from the dispensers were also investigated. The odorants that most improved yellow sticky trap captures in field cages were then tested under open field conditions alongside lower concentrations of those same lures. Gas chromatography-mass spectrometry was used to calculate odorant release rates as well as to determine if any contamination occurred under field conditions. None of the odorants under field cage or field conditions significantly improved psyllid capture on yellow sticky traps. Temperature influenced odorant loss, and release rate from polyethylene bulbs decreased over time. Based on these results, the use of unbaited yellow sticky traps seems to be the most effective method for monitoring of Huanglongbing vectors.
RESUMEN
Citrus greening disease, or Huanglongbing (HLB), has devastated citrus crops globally in recent years. The causal bacterium, 'Candidatus Liberibacter asiaticus', presents a sampling issue for qPCR diagnostics and results in a high false negative rate. In this work, we compared six metabolomics assays to identify HLB-infected citrus trees from leaf tissue extracted from 30 control and 30 HLB-infected trees. A liquid chromatography-mass spectrometry-based assay was most accurate. A final partial least squares-discriminant analysis (PLS-DA) model was trained and validated on 690 leaf samples with corresponding qPCR measures from three citrus varieties (Rio Red grapefruit, Hamlin sweet orange, and Valencia sweet orange) from orchards in Florida and Texas. Trees were naturally infected with HLB transmitted by the insect vector Diaphorina citri. In a randomized validation set, the assay was 99.9% accurate to classify diseased from nondiseased samples. This model was applied to samples from trees receiving plant defense-inducer compounds or biological treatments to prevent or cure HLB infection. From two trials, HLB-related metabolite abundances and PLS-DA scores were tracked longitudinally and compared with those of control trees. We demonstrate how our assay can assess tree health and the efficacy of HLB treatments and conclude that no trialed treatment was efficacious.
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Citrus sinensis , Citrus , Hemípteros , Liberibacter , Rhizobiaceae , Citrus/microbiología , Rhizobiaceae/genética , Enfermedades de las Plantas/prevención & control , Enfermedades de las Plantas/microbiología , ÁrbolesRESUMEN
Although huanglongbing (HLB) is a devastating citrus disease, improved tolerant cultivars, such as Sugar Belle (SB) mandarin, have been identified. To understand the responses that HLB-affected SB undergoes, we compared 14CO2 fixation, carbohydrate export, phloem callose accumulation, relative expression of plant defense activators, and anatomical changes between healthy and infected SB trees versus susceptible Pineapple (PA) sweet orange. Eight- to ten-week-old leaves of infected SB showed a 2.5-fold increase in 14CO2 fixation and a 13% decrease in 14C-carbohydrate export, whereas HLB-affected PA presented a decrease of 33 and 50%, respectively. The mean distance of a callose deposit to its closest neighbor was 36% smaller in infected SB versus healthy, whereas in HLB-affected PA, it was 33% higher. Expression of papain-like cysteine proteases (PLCPs) was upregulated in SB but downregulated in PA. Infected SB showed minor alterations in the number of xylem vessels, a 16% larger xylem vessel lumen area, and a 14% increase in the proportional area of the xylem. In contrast, PA showed a 2.4-fold increase in the xylem vessel number and a 2% increase in the proportional xylem area. Three complementary mechanisms of tolerance in SB are hypothesized: (i) increased carbohydrate availability induced by greater CO2 fixation, mild effect in carbohydrate export, and local accumulation of callose in the phloem; (ii) activation of defense response via upregulation of PLCPs, and (iii) increased investment in the xylem structure. Thus, phloem and xylem modifications seem to be involved in SB tolerance.
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Floema , Azúcares , Floema/fisiología , Dióxido de Carbono , Enfermedades de las Plantas , XilemaRESUMEN
Citrus Huanglongbing (HLB) caused by 'Candidatus Liberibacter asiaticus' (CLas) is the most devastating citrus disease worldwide. CLas induces systemic and chronic reactive oxygen species (ROS) production, which has been suggested to be a primary cause of cell death in phloem tissues and subsequent HLB symptoms. Mitigating oxidative stress caused by CLas using horticultural approaches has been suggested as a useful strategy to reduce HLB damages. To provide information regarding the application timing to mitigate ROS, we investigated monthly dynamics of CLas concentration, CLas-triggered ROS, and phloem cell death in the bark tissues of asymptomatic and symptomatic branches of HLB-positive Hamlin and Valencia sweet orange trees in the field. Healthy branches in the screenhouse were used as controls. CLas concentration exhibited significant variations over the course of the year, with two distinct peaks observed in Florida citrus groves-late spring/early summer and late fall. Within both Hamlin and Valencia asymptomatic tissues, CLas concentration demonstrated a negative correlation with the deviation between the monthly average mean temperature and the optimal temperature for CLas colonization in plants (25.7°C). However, such a correlation was not evident in symptomatic tissues of Hamlin or Valencia sweet oranges. ROS levels were consistently higher in symptomatic or asymptomatic branches than in healthy branches in most months. ROS concentrations were higher in symptomatic branches than in asymptomatic branches in most months. CLas triggered significant increases in ion leakage in most months for asymptomatic and symptomatic branches compared with healthy controls. In asymptomatic branches of Hamlin, a positive correlation was observed between CLas concentration and ROS concentrations, CLas concentration and ion leakage levels, as well as ROS and ion leakage. Intriguingly, such a relationship was not observed in Valencia asymptomatic branches or in the symptomatic branches of Hamlin and Valencia. This study sheds light on the pathogenicity of CLas by providing useful information on the temporal dynamics of ROS production, phloem cell death, and CLas growth, as well as provides useful information in determining the timing for application of antioxidants and antimicrobial agents to control HLB.
Asunto(s)
Citrus sinensis , Enfermedades de las Plantas , Especies Reactivas de Oxígeno , Rhizobiaceae , Especies Reactivas de Oxígeno/metabolismo , Citrus sinensis/microbiología , Enfermedades de las Plantas/microbiología , Rhizobiaceae/fisiología , Floema/microbiología , Corteza de la Planta/microbiología , Liberibacter , Iones/metabolismoRESUMEN
Citrus Huanglongbing, one of the most devastating citrus diseases, is caused by 'Candidatus Liberibacter asiaticus' (CLas). Polyamines are aliphatic nitrogen-containing compounds that play important roles in disease resistance and are synthesized primarily by two pathways: an arginine decarboxylation pathway and an ornithine decarboxylation pathway. However, it is unclear whether polyamines play a role in the tolerance of citrus to infection by CLas and, if so, whether one or both of the core polyamine metabolic pathways are important. We used high-performance liquid chromatography and ultra-performance liquid chromatography-quadrupole time-of-flight mass spectrometry to detect the contents of nine polyamine metabolism-related compounds in six citrus cultivars with varying levels of tolerance to CLas. We also systematically detected the changes in polyamine metabolism-related compounds and H2O2 contents and compared the gene expression levels and the activities of enzymes involved in the polyamine metabolic pathway among healthy, asymptomatic, and symptomatic leaves of Newhall navel oranges infected with CLas. The tolerant and moderately tolerant varieties showed higher polyamine metabolism-related compound levels than those of susceptible varieties. Compared with the healthy group, the symptomatic group showed significantly increased contents of arginine, ornithine, γ-aminobutyric acid, and putrescine by approximately 180, 19, 1.5, and 0.2 times, respectively, and upregulated expression of biosynthetic genes. Arginase and ornithine decarboxylase enzyme activities were the highest in the symptomatic group, whereas arginine decarboxylase and agmatine deiminase enzyme activities were the highest in the asymptomatic group. The two polyamine biosynthetic pathways showed different trends with the increase of the CLas titer, indicating that polyamines were mainly synthesized through the arginine decarboxylase pathway in the asymptomatic leaves and were synthesized via the ornithine decarboxylase pathway in symptomatic leaves. These findings provide new insight into the changes in polyamine metabolism in citrus infected with CLas.
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Citrus , Enfermedades de las Plantas , Poliaminas , Rhizobiaceae , Poliaminas/metabolismo , Enfermedades de las Plantas/microbiología , Citrus/microbiología , Rhizobiaceae/fisiología , Hojas de la Planta/microbiología , Hojas de la Planta/metabolismo , Peróxido de Hidrógeno/metabolismo , Ornitina Descarboxilasa/metabolismo , Ornitina Descarboxilasa/genética , Liberibacter/fisiología , Regulación de la Expresión Génica de las Plantas , Redes y Vías MetabólicasRESUMEN
Nodule-specific cysteine-rich (NCR) peptides, encoded in the genome of the Mediterranean legume Medicago truncatula (barrelclover), are known to regulate plant-microbe interactions. A subset of computationally derived 20-mer peptide fragments from 182 NCR peptides was synthesized to identify those with activity against the unculturable vascular pathogen associated with citrus greening disease, 'Candidatus Liberibacter asiaticus' (CLas). Grounded in a design of experiments framework, we evaluated the peptides in a screening pipeline involving three distinct assays: a bacterial culture assay with Liberibacter crescens, a CLas-infected excised citrus leaf assay, and an assay to evaluate effects on bacterial acquisition by the nymphal stage of hemipteran vector Diaphorina citri. A subset of the 20-mer NCR peptide fragments inhibits both CLas growth in citrus leaves and CLas acquisition by D. citri. Two peptides induced higher levels of D. citri mortality. These findings reveal 20-mer NCR peptides as a new class of plant-derived biopesticide molecules to control citrus greening disease.
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Citrus , Medicago truncatula , Péptidos , Enfermedades de las Plantas , Enfermedades de las Plantas/microbiología , Enfermedades de las Plantas/prevención & control , Citrus/microbiología , Péptidos/química , Péptidos/metabolismo , Medicago truncatula/microbiología , Cisteína , Hemípteros/microbiología , Agentes de Control Biológico , Hojas de la Planta/microbiología , Proteínas de Plantas/genética , Proteínas de Plantas/metabolismo , Liberibacter/genética , Animales , Rhizobiaceae/genéticaRESUMEN
The Asian citrus psyllid (ACP) Diaphorina citri transmits the causative agent of huanglongbing, or citrus greening disease, that has decimated global citrus production. Pesticidal proteins derived from bacteria such as Bacillus thuringiensis (Bt) can provide effective and environmentally friendly alternatives for management of D. citri, but few with sufficient toxicity to D. citri have been identified. Here, we report on the toxicity of 14 Bt-derived pesticidal proteins from five different structural groups against D. citri. These proteins were selected based on previously reported toxicity to other hemipteran species and on pesticidal protein availability. Most of the proteins were expressed in Escherichia coli and purified from inclusion bodies or His-tag affinity purification, while App6Aa2 was expressed in Bt and purified from spore/crystal mixtures. Pesticidal proteins were initially screened by feeding psyllids on a single dose, and lethal concentration (LC50) then determined for proteins with significantly greater mortality than the buffer control. The impact of CLas infection of D. citri on toxicity was assessed for selected proteins via topical feeding. The Bt protein Tpp78Aa1 was toxic to D. citri adults with an LC50 of approximately 204 µg/mL. Nymphs were more susceptible to Tpp78Aa1 than adults but no significant difference in susceptibility was observed between healthy and CLas-infected nymphs or adults. Tpp78Aa1 and other reported D. citri-active proteins may provide valuable tools for suppression of D. citri populations.
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Proteínas Bacterianas , Hemípteros , Control Biológico de Vectores , Animales , Hemípteros/microbiología , Citrus/microbiología , Insectos Vectores , Bacillus thuringiensis/química , Enfermedades de las Plantas/microbiología , InsecticidasRESUMEN
Citrus Huanglongbing (HLB), caused by a vector-transmitted phloem-limited bacterium Candidatus Liberibacter asiaticus (CLas), is the most devastating citrus disease worldwide. Currently, there are no effective strategies to prevent infection or to cure HLB-positive trees. Here, using comparative analysis between HLB-sensitive citrus cultivars and HLB-tolerant citrus hybrids and relatives, we identified a novel class of stable antimicrobial peptides (SAMPs). The SAMP from Microcitrusaustraliasica can rapidly kill Liberibacter crescens (Lcr), a culturable Liberibacter strain, and inhibit infections of CLas and CL. solanacearum in plants. In controlled greenhouse trials, SAMP not only effectively reduced CLas titer and disease symptoms in HLB-positive trees but also induced innate immunity to prevent and inhibit infections. Importantly, unlike antibiotics, SAMP is heat stable, making it better suited for field applications. Spray-applied SAMP was taken up by citrus leaves, stayed stable inside the plants for at least a week, and moved systemically through the vascular system where CLas is located. We further demonstrate that SAMP is most effective on α-proteobacteria and causes rapid cytosol leakage and cell lysis. The α-helix-2 domain of SAMP is sufficient to kill Lcr Future field trials will help determine the efficacy of SAMP in controlling HLB and the ideal mode of application.
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Citrus/efectos de los fármacos , Enfermedades de las Plantas/prevención & control , Proteínas Citotóxicas Formadoras de Poros/farmacología , Rutaceae/química , Citrus/microbiología , Resistencia a la Enfermedad/genética , Liberibacter/efectos de los fármacos , Liberibacter/patogenicidad , Enfermedades de las Plantas/genética , Enfermedades de las Plantas/microbiología , Hojas de la Planta/microbiología , Proteínas Citotóxicas Formadoras de Poros/química , Proteínas Citotóxicas Formadoras de Poros/genéticaRESUMEN
Huanglongbing (HLB) is a citrus infectious disease caused by 'Candidatus Liberibacter' spp. Recently, it has begun to spread rapidly worldwide, causing significant losses to the citrus industry. Early diagnosis of HLB relies on quantitative real-time PCR assays. However, the PCR inhibitors found in the nucleic acid extracted from plant materials pose challenges for PCR assays because they may result in false-negative results. Internal standard (IS) can be introduced to establish a single-tube duplex PCR for monitoring the influence of the PCR inhibitor, but it also brings the risk of false-negative results because the amplification of IS may compete with the target. To solve this problem, we proposed a mutation-enhanced single-tube duplex PCR (mSTD-PCR) containing IS with mutant-type primers. By introducing the 3'-terminal mutation in the primer of IS to weaken its amplification reaction and its inhibition of 'Candidatus Liberibacter asiaticus' (CLas) detection, the sensitivity and quantitative accuracy of CLas detection will not be affected by IS. In evaluating the sensitivity of CLas detection using simulation samples, the mSTD-PCR showed consistent sensitivity at 25 copies per test compared with the single-plex CLas assay. The detection result of 30 leaves and 30 root samples showed that the mSTD-PCR could recognize false-negative results caused by the PCR inhibitors and reduce workload by 48% compared with the single-plex CLas assay. Generally, the proposed mSTD-PCR provides a reliable, efficient, inhibitor-monitorable, quantitative screening method for accurately controlling HLB and a universal method for establishing a PCR assay for various pathogens.
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Citrus , Enfermedades de las Plantas , Reacción en Cadena en Tiempo Real de la Polimerasa , Rhizobiaceae , Reacción en Cadena en Tiempo Real de la Polimerasa/métodos , Enfermedades de las Plantas/microbiología , Citrus/microbiología , Rhizobiaceae/genética , Rhizobiaceae/aislamiento & purificación , Cartilla de ADN/genética , Sensibilidad y Especificidad , Mutación , ADN Bacteriano/genética , Liberibacter/genéticaRESUMEN
Citrus Huanglongbing (HLB) is caused by the phloem-limited α-proteobacterium "Candidatus Liberibacter spp.", among which "Ca. Liberibacter africanus" (CLaf) have posed a significant threat to citrus production in Africa near a century. CLaf is closely related to the globally prevalent "Ca. Liberibacter asiaticus" (CLas), whereas little is known about the virulence of CLaf, primarily due to limited genome resources. In this study, we completed the whole-genome assembly and annotation of CLaf strain Zim (from Zimbabwe). Compared to CLas, a total of 102 CLaf unique genes were identified, including 14 potential Sec-dependent effectors (SDEs) genes, 29 phage-associated genes, and 59 genes with hypothetical function. Among 14 SDEs, V9J15_03810 was able to induce a significant hypersensitive response (HR) in Nicotiana benthamiana, indicating its potential as a virulence factor for CLaf. Genome analysis showed that CLaf strain Zim genome harbored a complete prophage region (named P-Zim-1, 42,208 bp). P-Zim-1 retained two immunosuppressive peroxidase genes (V9J15_02125 and V9J15_02130) homologous to CLas prophage SC1/SC2, whereas the lysogen-associated genes encoding integrase (V9J15_01970) and repressor (V9J15_02080) were homologous to the prophage of "Ca. Liberibacter solanacearum", the causal agent of potato zebra chip disease. In addition, P-Zim-1 carried a novel CRISPR/Cas system, including a CRISPR array (located within V9J15_02040, ranging from 443,643 to 443,897) and five CRISPR-related Cas proteins (V9J15_02005, 02010, 02015, 02025 and 02035). This study first characterized the unique genomic feature of CLaf related to virulence and prophage, which will facilitate future research on CLaf biology and African HLB management.
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The Asian citrus psyllid (ACP) is the main vector of Citrus Huanglongbing, the most damaging citrus disease, causing significant financial losses in the citrus industry. Global warming has expanded the habitat of this pest, allowing it to continue its northward migration to China. Population genetic information of ACP is fundamentally essential for species management. This study investigated the genetic diversity and population structure of Chinese ACP using the mitochondrial cytochrome oxidase subunit I gene by dataset comprised 721 sequences from 27 geographic sites in China. Low haplotype diversity (0.323â ±â 0.022) and low nucleotide diversity (0.00071â ±â 0.00007) were observed in the entire population, which may indicate recent founder events. Twenty-three haplotypes were identified and clustered into 2 haplogroups: haplogroup I and haplogroup II. Haplogroup II included only 2 unique haplotypes, which occurred exclusively in the Southwest China ACP population. Genetic differentiation analyses were also indicative of Southwest China population was significantly differentiated from the remaining populations. Demographic history analysis showed that ACP population in China has experienced demographic expansion. Our results provided a better understanding of the genetic distribution patterns and structures of ACP populations in China.
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Citrus , Hemípteros , Animales , Hemípteros/genética , China , Citrus/genética , Variación GenéticaRESUMEN
Given the severe impact of Citrus Huanglongbing on orchard production, accurate detection of the disease is crucial in orchard management. In the natural environments, due to factors such as varying light intensities, mutual occlusion of citrus leaves, the extremely small size of Huanglongbing leaves, and the high similarity between Huanglongbing and other citrus diseases, there remains an issue of low detection accuracy when using existing mainstream object detection models for the detection of citrus Huanglongbing. To address this issue, we propose YOLO-EAF (You Only Look Once-Efficient Asymptotic Fusion), an improved model based on YOLOv8n. Firstly, the Efficient Multi-Scale Attention Module with cross-spatial learning (EMA) is integrated into the backbone feature extraction network to enhance the feature extraction and integration capabilities of the model. Secondly, the adaptive spatial feature fusion (ASFF) module is used to enhance the feature fusion ability of different levels of the model so as to improve the generalization ability of the model. Finally, the focal and efficient intersection over union (Focal-EIOU) is utilized as the loss function, which accelerates the convergence process of the model and improves the regression precision and robustness of the model. In order to verify the performance of the YOLO-EAF method, we tested it on the self-built citrus Huanglongbing image dataset. The experimental results showed that YOLO-EAF achieved an 8.4% higher precision than YOLOv8n on the self-built dataset, reaching 82.7%. The F1-score increased by 3.33% to 77.83%, and the mAP (0.5) increased by 3.3% to 84.7%. Through experimental comparisons, the YOLO-EAF model proposed in this paper offers a new technical route for the monitoring and management of Huanglongbing in smart orange orchards.
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Algoritmos , Citrus , Enfermedades de las Plantas , Citrus/microbiología , Enfermedades de las Plantas/microbiología , Hojas de la Planta/microbiologíaRESUMEN
Extensive microbial interactions occur within insect hosts. However, the interactions between the Huanglongbing (HLB) pathogen and endosymbiotic bacteria within the Asian citrus psyllid (ACP, Diaphorina citri Kuwayama) in wild populations remain elusive. Thus, this study aimed to detect the infection rates of HLB in the ACP across five localities in China, with a widespread prevalence in Ruijin (RJ, 58%), Huidong (HD, 28%), and Lingui (LG, 15%) populations. Next, microbial communities of RJ and LG populations collected from citrus were analyzed via 16S rRNA amplicon sequencing. The results revealed a markedly higher microbial diversity in the RJ population compared to the LG population. Moreover, the PCoA analysis identified significant differences in microbial communities between the two populations. Considering that the inter-population differences of Bray-Curtis dissimilarity in the RJ population exceeded those between populations, separate analyses were performed. Our findings indicated an increased abundance of Enterobacteriaceae in individuals infected with HLB in both populations. Random forest analysis also identified Enterobacteriaceae as a crucial indicator of HLB infection. Furthermore, the phylogenetic analysis suggested a potential regulatory role of ASV4017 in Enterobacteriaceae for ACP, suggesting its possible attractant activity. This research contributes to expanding the understanding of microbial communities associated with HLB infection, holding significant implications for HLB prevention and treatment.
Asunto(s)
Enterobacteriaceae , Hemípteros , Filogenia , Enfermedades de las Plantas , ARN Ribosómico 16S , Animales , Hemípteros/microbiología , Enterobacteriaceae/genética , Enterobacteriaceae/clasificación , Enterobacteriaceae/patogenicidad , ARN Ribosómico 16S/genética , Enfermedades de las Plantas/microbiología , China/epidemiología , Citrus/microbiología , MicrobiotaRESUMEN
Of the two putative amino acid binding periplasmic receptors of ABC transporter family in Candidatus Liberibacter asiaticus (CLas), cystine binding receptor (CLasTcyA) has been shown to mainly express in phloem of citrus plant and is a target for inhibitor development. The crystal structure of CLasTcyA in complex with substrates has been reported earlier. The present work reports the identification and evaluation of potential candidates for their inhibitory potential against CLasTcyA. Among many compounds, selected through virtual screening, and MD simulation, pimozide, clidinium, sulfasalazine and folic acid showed significantly higher affinities and stability in complex with CLasTcyA. The SPR studies with CLasTcyA revealed significantly higher binding affinities for pimozide and clidinium (Kd, 2.73 nM and 70 nM, respectively) as compared to cystine (Kd, 1.26 µM). The higher binding affinities could be attributed to significantly increased number of interactions in the binding pocket as evident from the crystal structures of CLasTcyA in complex with pimozide and clidinium as compared to cystine. The CLasTcyA possess relatively large binding pocket where bulkier inhibitors fit quite well. In planta studies, carried out to assess the effect of inhibitors on HLB infected Mosambi plants, showed significant reduction in CLas titre in plants treated with inhibitors as compared to control plants. The results showed that pimozide exhibited higher efficiency as compared to clidinium in reducing CLas titre in treated plants. Our results showed that the inhibitor development against critical proteins like CLasTcyA can be an important strategy in management of HLB.
Asunto(s)
Rhizobiaceae , Cistina/farmacología , Pimozida/farmacología , Enfermedades de las PlantasRESUMEN
BACKGROUND: Salicylic Acid (SA) is a pivotal phytohormone in plant innate immunity enhancement of triggered by various pathogens, such as Candidatus Liberibacter asiaticus (CLas), the causal agent of Huanglongbing (HLB). WRKY is a plant specific transcription factor (TF) family, which plays crucial roles in plant response to biotic stresses. So far, the evolutionary history, functions, and expression patterns under SA treatment and CLas infection of WRKY family are poorly understood in Citrus, despite the release of the genome of several Citrus species. A comprehensive genomic and expressional analysis is worth to conduct for this family. RESULTS: Here, a genome-wide identification of WRKY TFs was performed in two Citrus species: Citrus sinensis (HLB-sensitive) and Poncirus trifoliata (HLB-tolerant). In total, 52 CsWRKYs and 51 PtrWRKYs were identified, whose physical and chemical properties, chromosome locations, phylogenetic relationships and structural characteristics were comparatively analyzed. Especially, expression patterns of these WRKY genes before and after SA treatment and CLas infection were compared. Based on this result, seven pairs of orthologous WRKY genes showing opposite expression patterns in two Citrus species were screened out. Moreover, two pairs of orthologous WRKY genes with significant differences in the number or type of stress-responsive cis-elements in the promoter regions were discovered. Subcellular localization and transcriptional activation activity assays revealed that these two pairs of orthologous genes are classic WRKY TFs localize in the nucleus and could function as transcriptional activators. CONCLUSION: In this study, we systematically analyzed the genomic characterization of WRKY family in two Citrus species, together with the analyses of expression patterns under SA signaling and CLas infection. Our study laid a foundation for further study on the function of WRKY TFs in HLB response and SA signaling of Citrus.
Asunto(s)
Citrus , Rhizobiaceae , Citrus/genética , Liberibacter , Rhizobiaceae/genética , Factores de Transcripción/genética , Filogenia , Enfermedades de las Plantas/genéticaRESUMEN
Autophagy functions in plant host immunity responses to pathogen infection. The molecular mechanisms and functions used by the citrus Huanglongbing (HLB)-associated intracellular bacterium 'Candidatus Liberibacter asiaticus' (CLas) to manipulate autophagy are unknown. We identified a CLas effector, SDE4405 (CLIBASIA_04405), which contributes to HLB progression. 'Wanjincheng' orange (Citrus sinensis) transgenic plants expressing SDE4405 promotes CLas proliferation and symptom expression via suppressing host immunity responses. SDE4405 interacts with the ATG8-family of proteins (ATG8s), and their interactions activate autophagy in Nicotiana benthamiana. The occurrence of autophagy is also significantly enhanced in SDE4405-transgenic citrus plants. Interrupting NbATG8s-SDE4405 interaction by silencing of NbATG8c reduces Pseudomonas syringae pv. tomato strain DC3000ΔhopQ1-1 (Pst DC3000ΔhopQ1-1) proliferation in N. benthamiana, and transient overexpression of CsATG8c and SDE4405 in citrus promotes Xanthomonas citri subsp. citri (Xcc) multiplication, suggesting that SDE4405-ATG8s interaction negatively regulates plant defense. These results demonstrate the role of the CLas effector protein in manipulating autophagy, and provide new molecular insights into the interaction between CLas and citrus hosts.