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Plant pathogens can alter the behavior of their insect vectors as well as their survival and reproduction. The African psyllid, Trioza erytreae, is one of the vectors of Huanglongbing, a citrus disease caused mainly by "Candidatus Liberibacter asiaticus" (CLas). The purpose of this study was to characterize the effects of CLas on the psyllid, T. erytreae using Citrus volkamerina plants as the study system. The study focused more specifically on the CLas effects prior to and after its acquisition by the psyllid T. erytreae. Our results did not support the hypothesis that CLas effects psyllid probing behavior prior to acquisition; few differences were observed between uninfected T. erytrea feeding on CLas‐infected versus control plants. On the other hand, compared to psyllids that had completed their development on control plants, the ones that had completed their development on a CLas‐infected plant exhibited changes in their behavior (greater velocity), physiology (smaller mass) and biochemistry (lower water and lipid content). Altogether, our results confirm the existence of a marked postacquisition effect on the vector locomotor behavior and a minor preacquisition effect of CLas on the vector behavior, which can be partially explained by physiological and biochemical changes. [ABSTRACT FROM AUTHOR]

Wolbachia, a group of Gram-negative symbiotic bacteria, infects nematodes and a wide range of arthropods. Diaphorina citri Kuwayama, the vector of Candidatus Liberibacter asiaticus (CLas) that causes citrus greening disease, is naturally infected with Wolbachia (wDi). However, the interaction between wDi and D. citri remains poorly understood. In this study, we performed a pan-genome analysis using 65 wDi genomes to gain a comprehensive understanding of wDi. Based on average nucleotide identity (ANI) analysis, we classified the wDi strains into Asia and North America strains. The ANI analysis, principal coordinates analysis (PCoA), and phylogenetic tree analysis supported that the D. citri in Florida did not originate from China. Furthermore, we found that a significant number of core genes were associated with metabolic pathways. Pathways such as thiamine metabolism, type I secretion system, biotin transport, and phospholipid transport were highly conserved across all analyzed wDi genomes. The variation analysis between Asia and North America wDi showed that there were 39,625 single-nucleotide polymorphisms (SNPs), 2153 indels, 10 inversions, 29 translocations, 65 duplications, 10 SV-based insertions, and 4 SV-based deletions. The SV-based insertions and deletions involved genes encoding transposase, phage tail tube protein, ankyrin repeat (ANK) protein, and group II intron-encoded protein. Pan-genome analysis of wDi contributes to our understanding of the geographical population of wDi, the origin of hosts of D. citri, and the interaction between wDi and its host, thus facilitating the development of strategies to control the insects and huanglongbing (HLB). [ABSTRACT FROM AUTHOR]

Citrus canker is a disease caused by the gram-negative bacterium Xanthomonas citri subp. citri (X. citri), which affects all commercially important varieties of citrus and can lead to significant losses. Fruit sanitization with products such as chlorine-based ones can reduce the spread of the disease. While effective, their use raises concerns about safety of the workers. This work proposes essential oils (EOs) as viable alternatives for fruit sanitization. EOs from Cymbopogon species were evaluated as to their antibacterial activity, their effect on the bacterial membrane, and their ability to sanitize citrus fruit. The in vitro assays revealed that the EOs from C. schoenanthus and C. citratus had a lower bactericidal concentration at 312 mg L−1, followed by 625 mg L−1 for C. martini and C. winterianus. Microscopy assay revealed that the bacterial cell membranes were disrupted after 15 min of contact with all EOs tested. Regarding the sanitizing potential, the EOs with higher proportions of geraniol were more effective in sanitizing acid limes. Fruit treated with C. shoenanthus and C. martini showed a reduction of ∼68% in the recovery of viable bacterial cells. Therefore, these EOs can be used as viable natural alternatives in citrus fruit disinfection. [ABSTRACT FROM AUTHOR]

SUMMARY: Citrus bacterial canker (CBC) is a serious bacterial disease caused by Xanthomonas citri subsp. citri (Xcc) that adversely impacts the global citrus industry. In a previous study, we demonstrated that overexpression of an Xcc‐inducible apetala 2/ethylene response factor encoded by Citrus sinensis, CsAP2‐09, enhances CBC resistance. The mechanism responsible for this effect, however, is not known. In the present study, we showed that CsAP2‐09 targeted the promoter of the Xcc‐inducible WRKY transcription factor coding gene CsWRKY25 directly, activating its transcription. CsWRKY25 was found to localize to the nucleus and to activate transcriptional activity. Plants overexpressing CsWRKY25 were more resistant to CBC and showed higher expression of the respiratory burst oxidase homolog (RBOH) CsRBOH2, in addition to exhibiting increased RBOH activity. Transient overexpression assays in citrus confirmed that CsWRKY25 and CsRBOH2 participated in the generation of reactive oxygen species (ROS) bursts, which were able to restore the ROS degradation caused by CsAP2‐09 knockdown. Moreover, CsWRKY25 was found to bind directly to W‐box elements within the CsRBOH2 promoter. Notably, CsRBOH2 knockdown had been reported previously to reduce the CBC resistance, while demonstrated in this study, CsRBOH2 transient overexpression can enhance the CBC resistance. Overall, our results outline a pathway through which CsAP2‐09‐CsWRKY25 transcriptionally reprograms CsRBOH2‐mediated ROS homeostasis in a manner conducive to CBC resistance. These data offer new insight into the mechanisms and regulatory pathways through which CsAP2‐09 regulates CBC resistance, highlighting its potential utility as a target for the breeding of CBC‐resistant citrus varieties. Significant Statement: In the present study, we identify a pathway in which CsAP2‐09 and CsWRKY25 form a transcription factor cascade, transcriptionally reprogramming CsRBOH2‐mediated reactive oxygen species homeostasis and citrus bacterial canker (CBC) resistance. Our study explores new insights into the mechanistic role of CsAP2‐09 and the associated regulatory network, extending the potential value of CsAP2‐09 in the breeding of CBC‐tolerant citrus plants. [ABSTRACT FROM AUTHOR]

Aims Citrus canker caused by Xanthomonas citri subsp. citri (X. citri) is a disease of economic importance. Control of this disease includes the use of metallic copper, which is harmful to the environment and human health. Previous studies showed that the crude extract from the fungus Pseudogymnoascus sp. LAMAI 2784 isolated from Antarctic soil had in vitro antibacterial action against X. citri. The aim of the present study was to expand the applications of this extract. Methods and results In greenhouse assays, the crude extract was able to reduce bacterial infection on citrus leaves from 1.55 lesions/cm2 (untreated plants) to 0.04 lesions/cm2. Bisdechlorogeodin was identified as the main compound of the bioactive fraction produced by Pseudogymnoascus sp. LAMAI 2784, which inhibited bacterial growth in vitro (IC90 ≈ 156 µg ml−1) and permeated 80% of X. citri cells, indicating that the membrane is the primary target. Conclusion The present results showed that the bioactive fraction of the extract is mainly composed of the compound bisdechlorogeodin, which is likely responsible for the biological activity against X. citri , and the main mechanism of action is the targeting of the cell membrane. This study indicates that bisdechlorogeodin has valuable potential for the control of X. citri. [ABSTRACT FROM AUTHOR]

Virus-induced gene silencing (VIGS) is a powerful technology for molecular characterization of gene functions in plants and has the potential to prevent and control plant diseases. Unfortunately, VIGS in many woody perennials such as citrus are severely hindered because they have a long juvenile period and are recalcitrant to infection compared to herbaceous plants. Here, we report the development of a simple and effective VIGS system based on citrus leaf blotch virus (CLBV), a virus endemic in most citrus-producing countries but showing no symptoms in most citrus varieties. By Agrobacterium-mediated vacuum-infiltration (AVI), pCLBV201-su245 and pCLBV201-pds391 triggered efficient gene silencing (85%) and silencing phenotypes began to appear at 30 days post infiltration/inoculation (dpi), a similar period for achieving that on herbaceous plants. Moreover, the silencing phenotypes could still be observed four years post inoculation. Further, pCLBV201-lob369 was constructed to silence the gene lateral organ boundaries1 (lob1), encoding a key factor for susceptibility to citrus canker that caused by Xanthomonas citri ssp. citri (Xcc). The resistance evaluation results showed that the lesion area, disease index, and bacterial content in the pCLBV201-lob369 treatment group were decreased by 64%, 14%, and 67%, respectively. This work provides a simple and effective VIGS system for citrus which has the potential to be used for diseases control. [ABSTRACT FROM AUTHOR]

Citrus canker is an infectious bacterial disease and one of the major threats to the orange juice industry, a multibillion-dollar market that generates hundreds of thousands of jobs worldwide. This disease is caused by the Gram-negative bacterium Xanthomonas citri subsp. citri. In Brazil, the largest producer and exporter of concentrate orange juice, the control of citrus canker is exerted by integrated management practices, in which cupric solutions are intensively used in the orchards to refrain bacterial spreading. Copper ions accumulate and are as heavy metals toxic to the environment. Therefore, the aim of the present work was to evaluate bifunctional fusion proteins (BiFuProts) as novel and bio-/peptide-based alternatives to copper formulations to control citrus canker. BiFuProts are composed of an anchor peptide able to bind to citrus leaves, and an antimicrobial "killer" peptide to protect against bacterial infections of plants. The selected BiFuProt (Mel-CgDEF) was bactericidal against X. citri at 125 μg mL−1, targeting the bacterial cytoplasmic membrane within the first minutes of contact. The results in the greenhouse assays proved that Mel-CgDEF at 250 μg mL−1 provided protection against X. citri infection on the leaves, significantly reducing the number of lesions by area when compared with the controls. Overall, the present work showed that the BiFuProt Mel-CgDEF is a biobased and biodegradable possible alternative for substitute cupric formulations. Key points: • The bifunctional fusion protein Mel-CgDEF was effective against Xanthomonas citri. • Mel-CgDEF action mechanism was the disruption of the cytoplasmic membrane. • Mel-CgDEF protected citrus leaves against citrus canker disease. [ABSTRACT FROM AUTHOR]

In Mexico, Huanglongbing infection caused 42 and 33% decreases in orange and lemon production, respectively. Asian citrus psyllid, Diaphorina citri Kuwayama (Hemiptera: Liviidae), is a vector of Candidatus Liberibacter bacterium that causes the disease. Commercial orchards were searched to identify entomopathogenic fungi of D. citri in the citrus zone of Huasteca Potosina, San Luis Potosi, and Llera, Tamaulipas, Mexico. Entomopathogenic fungi associated with Asian citrus psyllid adults are in three genera: Hirsutella citriformis (88%), Beauveria bassiana (2%), and Sporothrix insectorum (10%). This is the first report of Sporothrix insectorum parasitizing H. citriformis in México. [ABSTRACT FROM AUTHOR]

Summary: Citrus bacterial canker (CBC), caused by Xanthomonas citri subsp. citri (Xcc), causes dramatic losses to the citrus industry worldwide. Transcription activator‐like effectors (TALEs), which bind to effector binding elements (EBEs) in host promoters and activate transcription of downstream host genes, contribute significantly to Xcc virulence. The discovery of the biochemical context for the binding of TALEs to matching EBE motifs, an interaction commonly referred to as the TALE code, enabled the in silico prediction of EBEs for each TALE protein. Using the TALE code, we engineered a synthetic resistance (R) gene, called the Xcc‐TALE‐trap, in which 14 tandemly arranged EBEs, each capable of autonomously recognizing a particular Xcc TALE, drive the expression of Xanthomonas avrGf2, which encodes a bacterial effector that induces plant cell death. Analysis of a corresponding transgenic Duncan grapefruit showed that transcription of the cell death‐inducing executor gene, avrGf2, was strictly TALE‐dependent and could be activated by several different Xcc TALE proteins. Evaluation of Xcc strains from different continents showed that the Xcc‐TALE‐trap mediates resistance to this global panel of Xcc isolates. We also studied in planta‐evolved TALEs (eTALEs) with novel DNA‐binding domains and found that these eTALEs also activate the Xcc‐TALE‐trap, suggesting that the Xcc‐TALE‐trap is likely to confer durable resistance to Xcc. Finally, we show that the Xcc‐TALE‐trap confers resistance not only in laboratory infection assays but also in more agriculturally relevant field studies. In conclusion, transgenic plants containing the Xcc‐TALE‐trap offer a promising sustainable approach to control CBC. [ABSTRACT FROM AUTHOR]

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' (C Las) to manipulate autophagy are unknown. We identified a C Las effector, SDE4405 (CLIBASIA_04405), which contributes to HLB progression. 'Wanjincheng' orange (Citrus sinensi s) transgenic plants expressing SDE4405 promotes C Las 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 DC3000 ΔhopQ1-1 (Pst DC3000 ΔhopQ1-1) 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 C Las effector protein in manipulating autophagy, and provide new molecular insights into the interaction between C Las and citrus hosts. [ABSTRACT FROM AUTHOR]

Plant–pathogen interaction is influenced by multiple environmental factors, including temperature and light. Recent works have shown that light modulates not only the defense response of plants but also the pathogens virulence. Xanthomonas citri subsp. citri (Xcc) is the bacterium responsible for citrus canker, an important plant disease worldwide. The Xcc genome presents four genes encoding putative photoreceptors: one bacteriophytochrome and three blue light photoreceptors, one LOV and two BLUFs (bluf1: XAC2120 and bluf2: XAC3278). The presence of two BLUFs proteins is an outstanding feature of Xcc. In this work we show that the bluf2 gene is functional. The mutant strain, XccΔbluf2, was constructed demonstrating that BLUF2 regulates swimming-type motility, adhesion to leaves, exopolysaccharide production and biofilm formation, features involved in the Xcc virulence processes. An important aspect during the plant–pathogen interaction is the oxidative response of the host and the consequent reaction of the pathogen. We observed that ROS detoxification is regulated by Xcc bluf2 gene. The phenotypes of disease in orange plants produced by WT and XccΔbluf2 strains were evaluated, observing different phenotypes. Altogether, these results show that BLUF2 negatively regulates virulence during citrus canker. This work constitutes the first report on BLUF-like receptors in plant pathogenic bacteria. [ABSTRACT FROM AUTHOR]

Citrus canker, caused by the bacterium Xanthomonas citri (Xcc), is one of the most devastating diseases for the citrus industry. Xylose is a constituent of the cell wall of plants, and the ability of Xcc to use this carbohydrate may play a role in virulence. Xcc has two genes codifying for xylose isomerase (XI), a bifunctional enzyme that interconverts D-xylose into D-xylulose and D-glucose into D-fructose. The aim of this work was to investigate the functional role of the two putative XI ORFs, XAC1776 (xylA1) and XAC4225 (xylA2), in Xcc pathogenicity. XI-coding genes of Xcc were deleted, and the single mutants (XccΔxylA1 or XccΔxylA2) or the double mutant (XccΔxylA1ΔxylA2) remained viable. The deletion of one or both XI genes (xylA1 and/or xylA2) increased the aggressiveness of the mutants, causing disease symptoms. RT-qPCR analysis of wild strain and xylA deletion mutants grown in vivo and in vitro revealed that the highest expression level of hrpX and xylR was observed in vivo for the double mutant. The results indicate that XI depletion increases the expression of the hrp regulatory genes in Xcc. We concluded that the intracellular accumulation of xylose enhances Xcc virulence. [ABSTRACT FROM AUTHOR]

Citrus canker (CC), caused by one of the most destructive subfamilies of the bacterial phytopathogen Xanthomonas citri subsp. Citri (Xcc), poses a serious threat to the significantly important citrus fruit crop grown worldwide. This has been the subject of ongoing epidemiological and disease management research. Currently, five different forms have been identified of CC, in which Canker A (Xanthomonas citri subsp. citri) being the most harmful and infecting the majority of citrus cultivars. Severe infection symptoms include leaf loss, premature fruit drop, dieback, severe fruit blemishing or discoloration, and a decrease in fruit quality. The infection spreads rapidly through wind, rain splash, and warm and humid climates. The study of the chromosomal and plasmid DNA of bacterium has revealed the evolutionary pattern among the pathovars, and research on the Xcc genome has advanced our understanding of how the bacteria specifically recognize and infect plants, spread within the host, and propagates itself. Quarantine or exclusion programs, which prohibit the introduction of infected citrus plant material into existing stock, are still in use. Other measures include eliminating sources of inoculum, using resistant hosts, applying copper spray for protection, and implementing windbreak systems. The main focus of this study is to highlight the most recent developments in the fields of Xcc pathogenesis, epidemiology, symptoms, detection and identification, host range, spread, susceptibility, and management. Additionally, it presents an analysis of the economic impact of this disease on the citrus industry and suggests strategies to reduce its spread, including the need for international collaboration and research to reduce the impact of this disease on the global citrus industry. [ABSTRACT FROM AUTHOR]

Citrus canker, caused by the bacterium Xanthomonas citri subsp. citri (Xcc), is a disease that causes serious problems to the global citrus industry. Matrix-Assisted Laser Desorption/Ionization Time-of-Flight (MALDI-TOF) Mass Spectrometry (MS) has been used in human medicine to diagnose various diseases caused by both fungi and bacteria. In agriculture, this technique has potential for the diagnosis of diseases due to the low cost of large-scale analysis and quickness. This study showed that MALDI-TOF MS combined with chemometric analysis was effective for differentiating the macromolecule profile of orange leaves with canker lesions, healthy leaves, and leaves with phytotoxicity symptoms, proving that this technique may be used for the rapid diagnosis of citrus canker. [ABSTRACT FROM AUTHOR]

Background: As one of the most widely planted fruit trees in southern China, citrus occupies an important position in the agriculture field and forestry economy in China. There are many kinds of citrus diseases. If citrus infected with diseases cannot be controlled in time, it easily seriously affects citrus production and causes large economic losses. Timely monitoring of disease characteristics in the citrus growth process is important for implementing timely control measures. Citrus images are easily disturbed by environmental factors such as dust, low light, clouds or leaf shadows. This makes it easy for some disease spot features in citrus pictures to be obscured. Occluded lesions cannot be effectively extracted and recognized. Second, similar characteristics of different diseases also make it difficult to distinguish the different types of diseases. However, the existing machine vision technology for identifying citrus diseases still has some difficulties in dealing with the above problems. Results: This paper proposes a new citrus disease identification framework. First, a citrus image enhancement algorithm based on the MSR-AMSR algorithm is proposed, which can enhance the image and highlight the disease characteristic information. The AMSR algorithm can also greatly alleviate the interference of clouds and low light on image lesions, making the image features clearer. Second, an MF-RANet network is proposed to recognize citrus disease images. MF-RANet is composed of a main feature frame and a detail feature frame. The main feature frame uses the cross stacking structure of ResNet50 and RAM to extract the main features in the citrus image dataset. RAM is used to extract the attention weight in the feature layer, which enables RAM to give higher weight to disease features. The detailed feature frame path uses AugFPN to extract features from multiple scales and fuse the main feature frame path. AugFPN enables the network to retain more detailed features, so it can effectively distinguish similar features in different diseases. In addition, we use the ELU activation function not only to solve the problem of gradient explosion and gradient disappearance but also to effectively use the negative input of the network. Finally, we use the label smoothing regularization method to prevent overfitting the network in the classification process. Finally, the experimental results show that the highest detection accuracy of the network for Huanglong disease, Corynespora blight of citrus, fat spot macular disease, citrus scab, citrus canker and healthy citrus is 96.77%, 96.22%, 95.96%, 95.93%, 94.04% and 97.55%, respectively. Conclusions: The citrus disease algorithm based on AMSR and MF-RANet can effectively perform the disease detection function. It has a high recognition rate for different kinds of citrus diseases. With the addition of AMSR preprocessing, RAM, AugFPN, ELU activation function and other structures, the MF-RANet network performance improves. [ABSTRACT FROM AUTHOR]

The bacterium Xanthomonas citri ssp. citri (Xcc), causing citrus canker disease, poses a severe threat to citrus production worldwide. New approaches for managing the disease are required because of the limited number of bactericides available, reported pathogen resistance to copper pesticides, and the side effects of such compounds on citrus trees and the environment. In this study, the bacterium was isolated from a Mexican lime tree showing typical disease symptoms and identified as Xcc using conventional biochemical tests and specific PCR. The antibacterial activity of the recombinant peptide CAP18 (rCAP18) against the Xcc bacterium was assessed. Measuring the MIC parameter showed that purified rCAP18 caused bacterial growth inhibition by 90% at a concentration of 4.5 μM. Furthermore, a heterogeneous membrane system consisting of POPE:POPG:TOCL (5:1:3) was created using molecular dynamics simulation. The interaction effect of the oligomeric structure of rCAP18 (rCAP18OLIGO) on the components of a lipid bilayer membrane was determined by measuring the membrane thickness, the order parameter of the acyl chains and the area of each phospholipid. The results obtained from simulation and measurement of order parameters showed that, among the three phospholipids, rCAP18OLIGO more affected the orientation of carbon atoms in POPG. Fluctuations of average area per lipid for each phospholipid showed that rCAP18OLIGO had more interactions with POPG > TOCL > POPE, respectively, in the outer half of the membrane. The results obtained from in silico studies confirmed that the penetration of rCAP18OLIGO into the lipid bilayer membrane reduced the membrane thickness and caused its deformation during the simulation time. This mechanism is possibly the leading factor of Xcc growth inhibition by rCAP18 in vitro. Therefore, the rCAP18 peptide as an antibacterial agent can provide a way to develop a new approach to control the citrus canker disease. [ABSTRACT FROM AUTHOR]

Xanthomonas citri subsp. citri, a causative agent of the citrus canker (CC) disease, belongs to one of the essential groups of the bacterial phytopathogen family, Xanthomonadaceae. It has been a potential threat to the globally significant citrus fruit crop, which has remained under investigation for disease management and epidemiology since the 1980s. In Pakistan, the average yield of citrus is 11 t/ha, which is lower than other countries, including China, Brazil, and India, having average productions of 27, 26, and 22 tons/hectare, respectively. Citrus canker is one of the most devastating diseases, posing a significant threat to crop yield and fruit quality. To date, five distinct types (or forms) of the citrus canker have been recognized; the Asiatic (Canker A) form is most destructive and affects most citrus cultivars. Severe infection outcomes include dieback, defoliation, severely blemished fruit, premature fruit drop, and reduced fruit quality. The infection increases under humid, warm, cloudy climate, wind, and heavy rainfall. The analysis of plasmid and chromosomal DNA of X. citri subsp. citri depicted an evolutionary relationship among pathovars of Xanthomonas. The extensive study on the genome of X. citri subsp. citri has contributed to the current knowledge of plant host recognition of pathogens, host specificities, dissemination, and propagation. Regulatory programs, i.e., quarantine or exclusion, continued to be practiced, prohibiting infected citrus plant material into the existing stock. Other measures include removal of inoculums sources, resistant hosts, protective copper-containing sprays, and windbreak systems. In this review, we explored the latest trends in the areas of epidemiology, pathogenome, detection, host–pathogen interaction, biofilm formation, and management of X. citri subsp. citri. [ABSTRACT FROM AUTHOR]

Xanthomonas citri subsp. citri (Xcc) is the causal agent of citrus bacterial canker (CBC), one of the most devastating citrus diseases. Most commercial citrus varieties are susceptible to CBC. However, some citrus varieties and wild citrus germplasms are CBC resistant and are promising in genetic increases in citrus resistance against CBC. We aimed to evaluate citrus germplasms for resistance against CBC. First, we developed a rapid evaluation method based on enhanced yellow fluorescent protein (eYFP)- labeled Xcc. The results demonstrated that eYFP does not affect the growth and virulence of Xcc. Xcc-eYFP allows measurement of bacterial titers but is more efficient and rapid than the plate colony counting method. Next, we evaluated citrus germplasms collected in China. Based on symptoms and bacterial titers, we identified that two citrus germplasms (Tchang' papeda and 'Huapi' kumquat) are resistant, whereas eight citrus germplasms ('Chongyi' wild mandarin, 'Mangshan' wild mandarin, 'Ledong' kumquat, 'Dali' citron. 'Yiliang' citron, 'Longyan' kumquat, 'Bawang' kumquat, and 'Daoxian' wild mandarin) are tolerant. In summary, we have developed a rapid evaluation method to test the resistance of citrus plants against CBC. This method was successfully used to identify two highly canker-resistant citrus germplasms and eight citrus germplasms with canker tolerance. These results could be leveraged in traditional breeding contexts or be used to identify canker resistance genes to increase the disease resistance of commercial citrus varieties via biotechnological approaches. [ABSTRACT FROM AUTHOR]

Citrus canker caused by Xanthomonas citri subsp. citri is one of the most devastating citrus diseases worldwide. Generating disease-resistant citrus varieties is considered one of the most efficient and environmentally friendly measures for controlling canker. X. citri subsp. citri causes canker symptoms by inducing the expression of canker susceptibility gene LOB1 via PthA4, a transcription activator-like (TAL) effector, by binding to the effector binding element (EBE) in the promoter region. In previous studies, canker-resistant plants were generated by mutating the coding region or the EBE of LOB1. However, homozygous or biallelic canker-resistant plants have not been generated for commercial citrus varieties, such as grapefruit (Citrus paradisi), which usually contain two alleles of LOB1 and thus, have two types of LOB1 promoter sequences: TI LOBP and TII LOBP. Two different sgRNAs were used to target both EBE types. Both 35S promoter and Yao promoter were used to drive the expression of SpCas9p to modify EBEPthA4-LOBP in grapefruit. Using 'Duncan' grapefruit epicotyls as explants, 19 genome-edited grapefruit plants were generated with one biallelic mutant line (#DunYao7). X. citri subsp. citri caused canker symptoms on wild-type and nonbiallelic mutant plants but not on #DunYao7. XccPthA4 mutant containing the designer TAL effector dLOB1.5, which recognizes a conserved sequence in both wild-type and #DunYao7, caused canker symptoms on both wild-type and #DunYao7. No off-target mutations were detected in #DunYao7. This study represents the first time that CRISPR-mediated genome editing has been successfully used to generate disease-resistant plants for 'Duncan' grapefruit, paving the way for using disease-resistant varieties to control canker. [ABSTRACT FROM AUTHOR]

Citrus canker caused by Xanthomonas citri subsp. citri (X. citri) is a devastating disease of citrus. In a previous experiment, bacterial strain GN223, an endophyte with the potential to serve as a biocontrol agent against citrus canker disease, was screened. In this study, strain GN223 was identified as Kosakonia cowanii by observing its morphological features, determining its physiological and biochemical characteristics and analysing the sequence of its 16S rDNA. Shaking flask fermentation conditions to grow strain GN223 were optimised using a single factor method. The results showed that the optimal culture medium for the strongest antagonistic effect on the growth of X. citri was yeast extract peptone dextrose (YPD). The optimal fermentation conditions were as follows: 50 mL of liquid (the best inoculum size was 7%) that was shaken in a 300 mL conical flask at 200 rpm for 60 h at 30°C, with an initial pH of 8. Furthermore, the ability of strain GN223 to control citrus canker disease in navel orange was evaluated. The experimental results showed that strain GN223 could positively control the citrus canker in new shoots of navel orange seedling and adult trees and young fruits in field trials. The treatment with a bacterial suspension diluted 50-fold on citrus canker in seedling and adult navel oranges resulted in control by approximately 40.0% and 50.1%, respectively. And the effect of strain GN223 on the incidence of canker on young fruits of navel orange did not differ significantly from that of copper hydroxide. [ABSTRACT FROM AUTHOR]

Over the past decade, ancient genomics has been used in the study of various pathogens. In this context, herbarium specimens provide a precious source of dated and preserved DNA material, enabling a better understanding of plant disease emergences and pathogen evolutionary history. We report here the first historical genome of a crop bacterial pathogen, Xanthomonas citri pv. citri (Xci), obtained from an infected herbarium specimen dating back to 1937. Comparing the 1937 genome within a large set of modern genomes, we reconstructed their phylogenetic relationships and estimated evolutionary parameters using Bayesian tip-calibration inferences. The arrival of Xci in the South West Indian Ocean islands was dated to the 19th century, probably linked to human migrations following slavery abolishment. We also assessed the metagenomic community of the herbarium specimen, showed its authenticity using DNA damage patterns, and investigated its genomic features including functional SNPs and gene content, with a focus on virulence factors. Author summary: Herbarium collections are a precious resource to plant pathologists, tracking crop diseases on specimens collected in the past centuries. In addition to indicating the presence of a disease at a specific time and locality, recent molecular technologies now allow extraction and microbial DNA sequencing from dead specimens. Despite challenges due to the degraded nature of DNA retrieved from historical samples, we were able to reconstruct the genome of a pathogenic bacterium from a 1937 herbarium specimen collected in Mauritius: Xanthomonas citri pv. citri, responsible for Asiatic citrus canker (ACC, an economically important agricultural disease controlled mostly through prophylactic and quarantine measures). Enhanced knowledge about the epidemiology and evolution of this bacterial pathogen is valuable to improve these measures. We compared the genome of this 1937 bacterial strain to a collection of modern strains, included it in a tree representing their genetic relationships, and calculated both evolutionary mutation rate and divergence times. This "forensic investigation" informs us about how and when the disease developed in the South West Indian Ocean Islands. We hypothesize that there was a single (or a few related) introduction of ACC in Mauritius in the mid-19th century, followed by expansion to the neighbouring islands. [ABSTRACT FROM AUTHOR]

The genus Xanthomonas comprises several cosmopolitan plant‐pathogenic bacteria that affect more than 400 plant species, most of which are of economic interest. Citrus canker is a bacterial disease that affects citrus species, reducing fruit yield and quality, and is caused by the bacterium Xanthomonas citri subsp. citri (Xac). The Xac3819 gene, which has previously been reported to be important for citrus canker infection, encodes an uncharacterized glutathione S‐transferase (GST) of 207 amino‐acid residues in length (XacGST). Bacterial GSTs are implicated in a variety of metabolic processes such as protection against chemical and oxidative stresses. XacGST shares high sequence identity (45%) with the GstB dehalogenase from Escherichia coli O6:H1 strain CFT073 (EcGstB). Here, XacGST is reported to be able to conjugate glutathione (GSH) with bromoacetate with a Km of 6.67 ± 0.77 mM, a kcat of 42.69 ± 0.32 s−1 and a kcat/Km of 6.40 ± 0.72 mM−1 s−1 under a saturated GSH concentration (3.6 mM). These values are comparable to those previously reported for EcGstB. In addition, crystal structures of XacGST were determined in the apo form (PDB entry 6nxv) and in a GSH‐bound complex (PDB entry 6nv6). XacGST has a canonical GST‐like fold with a conserved serine residue (Ser12) at the GSH‐binding site near the N‐terminus, indicating XacGST to be a serine‐type GST that probably belongs to the theta‐class GSTs. GSH binding stabilizes a loop of about 20 residues containing a helix that is disordered in the apo XacGST structure. [ABSTRACT FROM AUTHOR]

Citrus canker is one of the most devastating disease of citrus crop. It is a worldwide disease, caused by bacterium Xanthomonas axonopodis pv. citri. Citrus canker incidence has been observed on most of the citrus varieties. Severe infection of the bacterium produces a variety of effects including defoliation, dieback, severely blemished fruit, reduced fruit quality and premature fruit drop. Present study was conducted at Citrus Research Institute, Sargodha during the years 2016-18 on ten years old kinnow plants. There were ten treatments including control with four replications. Spray was done four times in a year during the months of March, April, August, and September. The efficacy of different treatments was compared to control the disease incidence on leaves and fruits. It was concluded that Bordeaux mixture was the most suitable chemical to control the canker on kinnow plant and it also improved the quality of fruit in terms of fruit weight, fruit size and juice weight. Besides, copper hydroxide and onion extract were also found efficacious to control the disease. In future, these chemicals and plant extracts could be used in different combinations to control canker on different citrus varieties. [ABSTRACT FROM AUTHOR]

Citrus canker type A is a serious disease caused by Xanthomonas citri subsp. citri (X. citri), which is responsible for severe losses to growers and to the citrus industry worldwide. To date, no canker-resistant citrus genotypes are available, and there is limited information regarding the molecular and genetic mechanisms involved in the early stages of the citrus canker development. Here, we present the CitrusKB knowledge base. This is the first in vivo interactome database for different citrus cultivars, and it was produced to provide a valuable resource of information on citrus and their interaction with the citrus canker bacterium X. citri. CitrusKB provides tools for a user-friendly web interface to let users search and analyse a large amount of information regarding eight citrus cultivars with distinct levels of susceptibility to the disease, with controls and infected plants at different stages of infection by the citrus canker bacterium X. citri. Currently, CitrusKB comprises a reference citrus genome and its transcriptome, expressed transcripts, pseudogenes and predicted genomic variations (SNPs and SSRs). The updating process will continue over time by the incorporation of novel annotations and analysis tools. We expect that CitrusKB may substantially contribute to the field of citrus genomics. CitrusKB is accessible at http://bioinfo.deinfo.uepg.br/citrus. Users can download all the generated raw sequences and generated datasets by this study from the CitrusKB website. [ABSTRACT FROM AUTHOR]

Although some plant pathogenic bacteria represent a significant threat to agriculture, the determinants of their ecological success and evolutionary potential are still poorly understood. Refining our understanding of bacterial strain circulation at small spatial scales and the biological significance and evolutionary consequences of co‐infections are key questions. The study of bacterial population biology can be challenging, because it requires high‐resolution markers that can be genotyped with a high throughput. Here, we overcame this difficulty for Xanthomonas citri pv. citri, a genetically monomorphic bacterium causing Asiatic citrus canker (ACC). Using a genotyping method that did not require cultivating the bacterium or purifying DNA, we deciphered the pathogen's spatial genetic structure at several microgeographic scales, down to single lesion, in a situation of ACC endemicity. In a grove where copper was recurrently applied for ACC management, copper‐susceptible and copper‐resistant X. citri pv. citri coexisted and the bacterial population structured as three genetic clusters, suggesting a polyclonal contamination. The range of spatial dependency, estimated for the two largest clusters, was four times greater for the cluster predominantly composed of copper‐resistant bacteria. Consistently, the evenness value calculated for this cluster was indicative of increased transmission. Linkage disequilibrium was high even at a tree scale, probably due to a combination of clonality and admixture. Approximately 1% of samples exhibited within‐lesion multilocus polymorphism, explained at least in part by polyclonal infections. Canker lesions, which are of major biological significance as an inoculum source, may also represent a preferred niche for horizontal gene transfer. This study points out the potential of genotyping data for estimating the range of spatial dependency of plant bacterial pathogens, an important parameter for guiding disease management strategies. [ABSTRACT FROM AUTHOR]

Plant extracts and streptomycin sulfate were evaluated against Xanthomonas axonopodis pv. citri. In first experiment, Azadirachta indica, Allium cepa, Catharanthus roseus, Allium sativum, Zingiber officinale (Roscoe) were investigated in vitro through inhibition zone technique against the growth of X. a. pv. citri. Results indicated that A. indica exhibited statistically significant inhibition (4 cm) zone over control. In second experiment, A. indica and streptomycin sulfate disjointedly and in amalgamation were evaluated in vitro. Streptomycin alone and in permutation with A. indica articulated significant inhibition of the bacterium. In third study, streptomycin sulfate and A. indica (S) and in combination were evaluated against citrus canker disease in green house. Results showed that streptomycin sulfate reduced disease significantly than control. In fourth experiment, streptomycin sulfate, A. indica, in combination and their interaction with days were evaluated under field condition. Streptomycin sulfate proved to be most effective and reduced the disease severity as compared to control. [ABSTRACT FROM AUTHOR]

Control of Asian citrus psyllid Diaphorina citri Kuwayama and citrus leafminer Phyllocnistis citrella Stainton is important to reduce the spread and severity of huanglongbing (HLB) (citrus greening) and citrus canker diseases, respectively. Insecticides are critical for the management of these pests. We therefore conducted two replicated experiments using spray treatments containing single or multiple modes of action (MoA) insecticides to reduce the incidence of these two pests in bearing citrus. Tank mixing in 47 L·ha-1 (5 gal/acre) of waterwith synthetic plant terpenes (Requiem 25 EC,Unknown MoA) or adjuvant petroleum oil (PureSpray Green, Unknown MoA) did not improve the effectiveness of the pyrethroid zeta-cypermethrin (Mustang Max 0.15 EC, MoA 3A) against D. citri. Its control with flupyradifurone (Sivanto 200 SL MoA 4D) and PureSpray Green in 935 L·ha-1 (100 gal/acre) water was similar toMustangMax 0.15 ECand Requiem25EC, but mixtures did not provide better control than Mustang Max 0.15 EC alone. Phyllocnistis citrella was controlled only with Sivanto 200 SL and PureSpray Green and Requiem 25 EC alone. The addition of cyantraniliprole (group 28 MoA in A16971 premixed with thiamethoxam MoA 4A), pymetrozine (Fulfill 50 WDG, MoA 9B), or abamectin (Agri-Mek SC, MoA 6) did not improve and in many cases reduced the performance of thiamethoxam (Actara 25WG,MoA 4A) against D. citri and P. citrella. These results demonstrated no advantage to single applications of multiple MoAs over the most effective active ingredients when applied alone for control of D. citri or P. citrella. Therefore, rotations of these active ingredients would be preferable to mixtures to avoid selection for resistance against multiple MoAs by any one application. [ABSTRACT FROM AUTHOR]

Citrus leprosis is one of the most destructive diseases of Citrus spp. and is associated with two unrelated virus groups that produce particles primarily in either the cytoplasm or nucleus of infected plant cells. Symptoms of leprosis, including chlorotic spots surrounded by yellow haloes on leaves and necrotic spots on twigs and fruit, were observed on leprosis-affected mandarin and navel sweet orange trees in the state of Queretaro, Mexico. Serological and molecular assays showed that the cytoplasmic types of Citrus leprosis virus (CiLV-C) often associated with leprosis symptomatic tissues were absent. However, using transmission electron microscopy, bullet-shaped rhabdovirus-like virions were observed in the nuclei and cytoplasm of the citrus leprosis-infected leaf tissues. An analysis of small RNA populations from symptomatic tissue was carried out to determine the genome sequence of the rhabdovirus-like particles observed in the citrus leprosis samples. The complete genome sequence showed that the nuclear type of CiLV (CiLV-N) present in the samples consisted of two negative-sense RNAs: 6,268-nucleotide (nt)- long RNA1 and 5,847-nt-long RNA2, excluding the poly(A) tails. CiLV-N had a genome organization identical to that of Orchid fleck virus (OFV), with the exception of shorter 5' untranslated regions in RNA1 (53 versus 205 nt) and RNA2 (34 versus 182 nt). Phylogenetic trees constructed with the amino acid sequences of the nucleocapsid (N) and glycoproteins (G) and the RNA polymerase (L protein) showed that CiLV-N clusters with OFV. Furthermore, phylogenetic analyses of N protein established CiLV-N as a member of the proposed genus Dichorhavirus. Reversetranscription polymerase chain reaction primers for the detection of CiLV-N were designed based on the sequence of the N gene and the assay was optimized and tested to detect the presence of CiLV-N in both diseased and symptom-free plants. [ABSTRACT FROM AUTHOR]

A test population consisting of progenies of 92 seed-source genotypes (hereafter called "parent genotypes") of Citrus and Citrus relatives in the field in east-central Florida was assessed after natural freeze events in the winters of 2010 and 2011. Eight seedlings per parent genotype were planted in a randomized complete block design; however, as a result of mortality, the number of plants assessed in some genotype groups was reduced at some or all sampling dates. The citrus diseases huanglongbing and citrus canker were endemic in the planting and may have influenced tree response to cold temperatures. Unusually low temperatures (near -4 °C each winter) for east-central Florida were experienced during the trial period. Defoliation and dieback were significantly greater in the winter of 2011 than in the winter of 2010. The winter in 2011 was preceded by a period of extraordinarily low temperatures in mid-December with no period of cool temperatures to allow trees to acclimate. In 2010 the average defoliation was 53% ± 28% and less than 13% of the trees exhibited any noticeable dieback, whereas in 2011, the average defoliation and dieback were 93% ± 17% and 51% ± 35%, respectively. Within the genus Citrus, several progenies were identified that had 16% to 24% dieback in 2011 and these were from parent genotypes C. reticulata (CRC 2590) (23%), C. sinensis (CRC 3858) (24%), C. maxima (CRC 3945) (16%), C. hassaku (CRC 3907 and 3942) (16% and 17%), C. aurantium (CRC 628 and 2717) (18% and 7%), C. taiwanica (CRC 2588) (21%), and C. neo-aurantium (C. obovoidea + C. unshiu graft chimera) (CRC 3816) (23%). Within other genera in the Aurantiodeae, Poncirus trifoliata (CRCs 301,3957,3549, and 4007), Severinia buxifoiia (CRC 1497), Bergera koenigii (CRC 3165), and Glycosmis pentaphylla (CRC 3285) had the least amount of dieback, all at less than 23%. The two species within the Toddalioideae subfamily of the Rutaceae (Casimiroa edulis and Zanthoxylum ailanthoides) had among the least amount of dieback (1% and 8%, respectively). When considered by groups, the Citrons and Australian natives had the greatest amount of dieback in 2011, 68% and 65%, respectively. The trifoliates (Poncirus and hybrids) had the least dieback, ranging from 4% to 40%. The information from this study may be useful in germplasm enhancement and Citrus breeding targeting greater cold tolerance. [ABSTRACT FROM AUTHOR]

Citrus canker caused by Xanthomonas citri subsp. citri (Xcc) is an economically important disease in most citrus production regions worldwide. Xcc secretes a transcriptional activator like effector (TALE) PthA4 to bind to the effector binding elements (EBEs) in the promoter region of canker susceptibility gene LOB1 to activate its expression, which in turn causes canker symptoms. Editing the EBE region with Cas9/gRNA has been used to generate canker resistant citrus plants. However, most of the EBE-edited lines generated contain indels of 1–2 bp, which has higher possibility to be overcome by PthA4 adaptation. The adaptation capacity of TALEs inversely correlates with the number of mismatches with the EBE. LbCas12a/crRNA is known to generate longer deletion than Cas9. In this study, we used a temperature-tolerant and more efficient LbCas12a variant (ttLbCas12a), harboring the single substitution D156R, to modify the EBE region of LOB1. We first constructed GFP-p1380N-ttLbCas12a:LOBP, which was shown to be functional via Xcc-facilitated agroinfiltration in Pummelo (Citrus maxima) leaves. Subsequently, we stably expressed ttLbCas12a:LOBP in Pummelo. Eight transgenic lines were generated, with seven lines showing 100% mutations of the EBE, among which one line is homozygous. The EBE-edited lines had the ttLbCas12a-mediated deletions of up to 10 bp. Importantly, the seven lines were canker resistant and no off-targets were detected. In summary, ttLbCas12a can be used to efficiently generate biallelic/homozygous citrus mutant lines with short deletions, thus providing a useful tool for the functional study and breeding of citrus. [ABSTRACT FROM AUTHOR]

MicroRNAs (miRNAs) are short noncoding RNA molecules that regulate gene expression at the posttranscriptional level. Reverse transcription-quantitative PCR (RT-qPCR) is one of the most common methods used for quantification of miRNA expression, and the levels of expression are normalized by comparing with reference genes. Thus, the selection of reference genes is critically important for accurate quantification. The present study was intended to identify appropriate miRNA reference genes for normalizing the level of miRNA expression in Citrus sinensis L. Osbeck and Citrus reticulata Blanco infected by Xanthomonas citri subsp. citri, which caused citrus canker disease. Five algorithms (Delta Ct, geNorm, NormFinder, BestKeeper and RefFinder) were used for screening reference genes, and two quantification approaches, poly(A) extension RT-qPCR and stem-loop RT-qPCR, were used to determine the most appropriate method for detecting expression patterns of miRNA. An overall comprehensive ranking output derived from the multi-algorithms showed that poly(A)-tailed miR162-3p/miR472 were the best reference gene combination for miRNA RT-qPCR normalization in citrus canker research. Candidate reference gene expression profiles determined by poly(A) RT-qPCR were more consistent in the two citrus species. To the best of our knowledge, this is the first systematic comparison of two miRNA quantification methods for evaluating reference genes. These results highlight the importance of rigorously assessing candidate reference genes and clarify some contradictory results in miRNA research on citrus. [ABSTRACT FROM AUTHOR]

Citrus bacterial canker (CBC), caused by the plant pathogenic bacterium Xanthomonas citri subsp. citri (Xcc), is a devastating disease in many commercial citrus cultivars. Every year, CBC causes a substantial reduction in fruit quality and quantity that corresponds to significant economic losses worldwide. Endophytic microorganisms produce numerous bioactive secondary metabolites that can control plant pathogens. We investigated the antagonistic activities of 66 endophytic bacteria isolated from nine citrus cultivars to control streptomycin-resistant Xcc. The suspension of Endophytic Bacteria-39 (EB-39), identified as Bacillus velezensis, exhibited the highest antibacterial activity against three wild-type and six streptomycin-resistant Xcc strains, with the inhibition zones between 39.47 ± 1.6 and 45.31 ± 1.6 mm. The ethyl acetate extract of EB-39 also controlled both wild-type and streptomycin-resistant Xcc strains, with the inhibition zones between 29.28 ± 0.6 and 33.88 ± 1.3 mm. Scanning electron microscopy indicated the ethyl acetate extract of EB-39-induced membrane damage and lysis. The experiments using the detached leaves of a susceptible Citrus species showed that EB-39 significantly reduced the incidence of canker on the infected leaves by 38%. These results strongly suggest that our newly isolated EB-39 is a novel biocontrol agent against CBC caused by wild-type and streptomycin-resistant Xcc strains. [ABSTRACT FROM AUTHOR]