Three new discovery effector proteins from Candidatus Liberibacter asiaticus psy62 inhibit plant defense through interaction with AtCAT3 and AtGAPA.

KEY MESSAGE: We identify three SDEs that inhibiting host defence from Candidatus Liberibacter asiaticus psy62, which is an important supplement to the pathogenesis of HLB. Candidatus Liberibacter asiaticus (CLas) is the main pathogen of citrus Huanglongbing (HLB). 38 new possible sec-dependent effectors (SDEs) of CLas psy62 were predicted by updated predictor SignalP 5.0, which 12 new SDEs were found using alkaline phosphate assay. Among them, SDE4310, SDE4435 and SDE4955 inhibited hypersensitivity reactions (HR) in Arabidopsis thaliana (Arabidopsis, At) and Nicotiana benthamiana leaves induced by pathogens, which lead to a decrease in cell death and reactive oxygen species (ROS) accumulation. And the expression levels of SDE4310, SDE4435, and SDE4955 genes elevated significantly in mild symptom citrus leaves. When SDE4310, SDE4435 and SDE4955 were overexpressed in Arabidopsis, HR pathway key genes pathogenesis-related 2 (PR2), PR5, nonexpressor of pathogenesis-related 1 (NPR1) and isochorismate synthase 1 (ICS1) expression significantly decreased and the growth of pathogen was greatly increased relative to control with Pst DC3000/AvrRps4 treatment. Our findings also indicated that SDE4310, SDE4435 and SDE4955 interacted with AtCAT3 (catalase 3) and AtGAPA (glyceraldehyde-3-phosphate dehydrogenase A). In conclusion, our results suggest that SDE4310, SDE4435 and SDE4955 are CLas psy62 effector proteins that may have redundant functions. They inhibit ROS burst and cell death by interacting with AtCAT3 and AtGAPA to negatively regulate host defense.

Transient expression of anti-HrpE scFv antibody reduces the hypersensitive response in non-host plant against bacterial phytopathogen Xanthomonas citri subsp. citri.

Citrus canker is a bacterial disease caused by Xanthomonas citri subsp. citri (Xcc) that affects the citrus industry worldwide. Hrp pili subunits (HrpE), an essential component of Type III secretion system (T3SS) bacteria, play a crucial role in the pathogenesis of Xcc by transporting effector proteins into the host cell and causing canker symptoms. Therefore, development of antibodies that block HrpE can suppress disease progression. In this study, a specific scFv detecting HrpE was developed using phage display technique and characterized using sequencing, ELISA, Western blotting, and molecular docking. In addition, a plant expression vector of pCAMBIA-scFvH6 was constructed and agroinfiltrated into Nicotiana tabacum cv. Samson leaves. The hypersensitive response (HR) in the leaves of transformed and non-transformed plants was evaluated by inoculating leaves with Xcc. After three rounds of biopanning of the phage library, a specific human scFv antibody, named scFvH6, was identified that showed high binding activity against HrpE in ELISA and Western blotting. Molecular docking results showed that five intermolecular hydrogen bonds are involved in HrpE-scFvH6 interaction, confirming the specificity and high binding activity of scFvH6. Successful transient expression of pCAMBIA-scFvH6 in tobacco leaves was verified using immunoassay tests. The binding activity of plant-produced scFvH6 to detect HrpE in Western blotting and ELISA was similar to that of bacterial-produced scFvH6 antibody. Interestingly, tobacco plants expressing scFvH6 showed a remarkable reduction in HR induced by Xcc compared with control plants, so that incidence of necrotic lesions was significantly higher in non-transformed controls (≥ 1.5 lesions/cm2) than in the plants producing scFvH6 (≤ 0.5 lesions/cm2) after infiltration with Xcc inoculum. Our results revealed that the expression of scFvH6 in tobacco leaves can confer resistance to Xcc, indicating that this approach could be considered to provide resistance to citrus bacterial canker disease.

Visible-light-driven water-soluble zinc oxide quantum dots for efficient control of citrus canker.

BACKGROUND: Citrus canker caused by Xanthomonas citri subsp. citri (Xcc) is a devastating bacterial disease that reduces citrus yield and quality, posing a serious threat to the citrus industry. Several conventional chemicals have been used to control citrus canker. However, this approach often leads to the excessive use of chemical agents, can exacerbate environmental pollution and promotes the development of resistant Xcc. Therefore, there is significant interest in the development of efficient and environmentally friendly technologies to control citrus canker. RESULTS: In this study, water-soluble ZnO quantum dots (ZnO QDs) were synthesised as an efficient nanopesticide against Xcc. The results showed that the antibacterial activity of ZnO QDs irradiated with visible light [half-maximal effective concentration (EC50) = 33.18 µg mL-1] was ~3.5 times higher than that of the dark-treated group (EC50 = 114.80 µg mL-1). ZnO QDs induced the generation of reactive oxygen species (•OH, •O- 2 and 1O2) under light irradiation, resulting in DNA damage, cytoplasmic destruction, and decreased catalase and superoxide dismutase activities. Transcription analysis showed downregulation of Xcc genes related to 'biofilms, virulence, adhesion' and 'DNA transfer' exposure to ZnO QDs. More importantly, ZnO QDs also promoted the growth of citrus. CONCLUSION: This research provides new insights into the photocatalytic antibacterial mechanisms of ZnO QDs and supports the development of more efficient and safer ZnO QDs-based nanopesticides to control citrus canker. © 2024 Society of Chemical Industry.

A combinatorial approach of Monarda citriodora essential oil (MEO) and linalool vapors to control fruit rot of Citrus limon caused by a new pathogen, Aspergillus foetidus, and its underlying mode of action.

AIMS: Citrus limon (lemon) is a widely cultivated citrus fruit. Significant postharvest losses due to fungi plague its production. Environmental and human health hazards have made the application of synthetic fungicides unsuitable. Despite the previous reports of antifungal activities of essential oil (EO) vapors, their synergistic combinations are understudied. Synergistic vapor combinations are advantageous due to less concentration of active components. This study aimed to isolate and identify postharvest fungal pathogens lemon and to evaluate the antifungal effects of synergistic Monarda citriodora EO (MEO)-constituent vapor combinations in vivo and in vitro. METHODS AND RESULTS: Postharvest fungal pathogens of lemon (C. limon) were isolated from various infected samples. The most pathogenic isolate was identified through morphology and its ITS-based rRNA gene sequencing as Aspergillus foetidus (O4). This is the first report of A. foetidus as a postharvest pathogen of lemon. The minimum fungicidal concentrations (MFCs) of MEO vapors treatment against O4 were 1346.15 µL/L air. For carvacrol, hexanal, and linalool, MFC was same (96.16 µL/L air). Checkerboard assays demonstrated that 1/4 MFC of MEO (336.54 µL/L air) and 1/4 MFC of linalool (24.04 µL/L air) (M + L) were synergistic against O4. M + L vapors reduced the O4 growth on lemons during storage by 64% ± 1.50% and preserved their quality (low weight loss %, unchanged pH, increased ascorbic acid content). Propidium iodide staining, ergosterol content analysis, calcofluor white staining and chitin content analysis revealed the integrity loss of the O4 plasma membrane and cell wall. 2',7'-Dichlorofluorescin diacetate staining revealed accumulation of intracellular reactive oxygen species (ROS), and scanning electron microscopy (SEM) analysis exposed the M + L treated mycelia with malformations. CONCLUSIONS: M + L vapors offer protection for lemons from A. foetidus and preserve their quality during storage.

'Candidatus Liberibacter asiaticus' secretory protein SDE3 inhibits host autophagy to promote Huanglongbing disease in citrus.

Antimicrobial acroautophagy/autophagy plays a vital role in degrading intracellular pathogens or microbial molecules in host-microbe interactions. However, microbes evolved various mechanisms to hijack or modulate autophagy to escape elimination. Vector-transmitted phloem-limited bacteria, Candidatus Liberibacter (Ca. Liberibacter) species, cause Huanglongbing (HLB), one of the most catastrophic citrus diseases worldwide, yet contributions of autophagy to HLB disease proliferation remain poorly defined. Here, we report the identification of a virulence effector in "Ca. Liberibacter asiaticus" (Las), SDE3, which is highly conserved among the "Ca. Liberibacter". SDE3 expression not only promotes the disease development of HLB and canker in sweet orange (Citrus sinensis) plants but also facilitates Phytophthora and viral infections in Arabidopsis, and Nicotiana benthamiana (N. benthamiana). SDE3 directly associates with citrus cytosolic glyceraldehyde-3-phosphate dehydrogenases (CsGAPCs), which negatively regulates plant immunity. Overexpression of CsGAPCs and SDE3 significantly inhibits autophagy in citrus, Arabidopsis, and N. benthamiana. Intriguingly, SDE3 undermines autophagy-mediated immunity by the specific degradation of CsATG8 family proteins in a CsGAPC1-dependent manner. CsATG8 degradation is largely rescued by treatment with an inhibitor of the late autophagic pathway, E64d. Furthermore, ectopic expression of CsATG8s enhances Phytophthora resistance. Collectively, these results suggest that SDE3-CsGAPC interactions modulate CsATG8-mediated autophagy to enhance Las progression in citrus.Abbreviations: ACP: asian citrus psyllid; ACD2: ACCELERATED CELL DEATH 2; ATG: autophagy related; Ca. Liberibacter: Candidatus Liberibacter; CaMV: cauliflower mosaic virus; CMV: cucumber mosaic virus; Cs: Citrus sinensis; EV: empty vector; GAPC: cytosolic glyceraldehyde-3-phosphate dehydrogenase; HLB: huanglongbing; H2O2: hydrogen peroxide; Las: liberibacter asiaticus; Laf: liberibacter africanus; Lam: liberibacter americanus; Pst: Pseudomonas syringae pv. tomato; PVX: potato virus X; ROS: reactive oxygen species; SDE3: sec-delivered effector 3; TEM: transmission electron microscopy; VIVE : virus-induced virulence effector; WT: wild-type; Xcc: Xanthomonas citri subsp. citri.

Discovery and Mechanism of Azatryptanthrin Derivatives as Novel Anti-Phytopathogenic Bacterial Agents for Potent Bactericide Candidates.

The natural alkaloids of tryptanthrin and their derivatives have a wide range of biological activities. In this research, four series of azatryptanthrin derivatives containing 4-aza/3-aza/2-aza/1-aza tryptanthrin were prepared by condensation cyclization reaction against plant pathogens to develop a new natural product-based bacterial pesticide. Compound 4Aza-8 displayed a remarkable growth inhibitory effect on pathogenic bacteria of Xanthomonas axonopodis pv. citri (Xac), Xanthomonas oryzae pv. Oryzae (Xoo), and Pseudomonas syringae pv. actinidiae (Psa) with the final corrected EC50 values of 0.312, 1.91, and 18.0 µg/mL, respectively, which were greatly superior than that of tryptanthrin (Tryp). Moreover, 4Aza-8 also showed effective therapeutic and protective activities in vivo on citrus canker. Further mechanism studies on Xac elucidated that compound 4Aza-8 was able to affect the growth curve of Xac and the formation of biofilm, cause severe shrinkage in bacterial morphology, increase reactive oxygen species levels, and induce apoptosis in bacterial cells. Quantitative analysis of differential protein profiles found that the major differences were mainly concentrated on the endometrial protein in the bacterial secretion system pathway, which blocked the membrane transport and affected the transfer of DNA to the host cell. In summary, these research results suggest that 4Aza-8 represents a promising anti-phytopathogenic-bacteria agent, which is worth being further investigated as a bactericide candidate.

GAR-transferase contributes to purine synthesis and mitochondrion function to maintain fungal development and full virulence of Penicillium digitatum.

Penicillium digitatum is one of the most critical phytopathogens during the citrus postharvest period. However, the molecular mechanism of pathogenesis remains to be further explored. Purine is a multiple functional substance in organisms. To verify the role of the de novo purine biosynthesis (DNPB) pathway in P. digitatum, we investigated the third gene Pdgart, glycinamide ribonucleotide (GAR)-transferase, of this pathway in this study. The deletion mutant ΔPdgart was generated in the principle of homologous recombination via Agrobacterium tumefaciens-mediated transformation (ATMT). The phenotypic assay indicated that the ΔPdgart mutant displayed severe defects in hyphae growth, conidiation and germination, which can be rescued by the addition of exogenous ATP and AMP. Compared with wild-type strain N1, the ATP level of strain ΔPdgart was detected to be sharply declined during conidial germination, and this was resulted from the damage to purine synthesis and aerobic respiration. The pathogenicity assay suggested that mutant ΔPdgart infected citrus fruit but attenuated disease, which was owing to its reduced production of organic acids and activities of cell wall degradation enzymes. Additionally, the ΔPdgart mutant showed altered sensitivity to stress agents and fungicides. Taken together, the present study provides insights into the essential functions of Pdgart, and paves the way for further study and novel fungicide development.

Suppression of citrus canker disease mediated by flagellin perception.

Citrus cancer, caused by strains of Xanthomonas citri (Xc) and Xanthomonas aurantifolii (Xa), is one of the most economically important citrus diseases. Although our understanding of the molecular mechanisms underlying citrus canker development has advanced remarkably in recent years, exactly how citrus plants fight against these pathogens remains largely unclear. Using a Xa pathotype C strain that infects Mexican lime only and sweet oranges as a pathosystem to study the immune response triggered by this bacterium in these hosts, we herein report that the Xa flagellin C protein (XaFliC) acts as a potent defence elicitor in sweet oranges. Just as Xa blocked canker formation when coinfiltrated with Xc in sweet orange leaves, two polymorphic XaFliC peptides designated flgIII-20 and flgIII-27, not related to flg22 or flgII-28 but found in many Xanthomonas species, were sufficient to protect sweet orange plants from Xc infection. Accordingly, ectopic expression of XaFliC in a Xc FliC-defective mutant completely abolished the ability of this mutant to grow and cause canker in sweet orange but not Mexican lime plants. Because XaFliC and flgIII-27 also specifically induced the expression of several defence-related genes, our data suggest that XaFliC acts as a main immune response determinant in sweet orange plants.

A prophage-encoded effector from "Candidatus Liberibacter asiaticus" targets ASCORBATE PEROXIDASE6 in citrus to facilitate bacterial infection.

Citrus huanglongbing (HLB), associated with the unculturable phloem-limited bacterium "Candidatus Liberibacter asiaticus" (CLas), is the most devastating disease in the citrus industry worldwide. However, the pathogenicity of CLas remains poorly understood. In this study, we show that AGH17488, a secreted protein encoded by the prophage region of the CLas genome, suppresses plant immunity via targeting the host ASCORBATE PEROXIDASE6 (APX6) protein in Nicotiana benthamiana and Citrus sinensis. The transient expression of AGH17488 reduced the chloroplast localization of APX6 and its enzyme activity, inhibited the accumulation of reactive oxygen species (H2 O2 and O2 - ) and the lipid oxidation endproduct malondialdehyde in plants, and promoted the proliferation of Pseudomonas syringae pv. tomato DC3000 and Xanthomonas citri subsp. citri. This study reveals a novel mechanism underlying how CLas uses a prophage-encoded effector, AGH17488, to target a reactive oxygen species accumulation-related gene, APX6, in the host to facilitate its infection.

Transgenic Sweet Orange Expressing the Sarcotoxin IA Gene Produces High-Quality Fruit and Shows Tolerance to 'Candidatus Liberibacter asiaticus'.

Huanglongbing (otherwise known as HLB or greening) is currently the most devastating citrus disease worldwide. HLB is primarily associated with the phloem-inhabiting bacterium 'Candidatus Liberibacter asiaticus' (CLas). Currently, there are no citrus species resistant to CLas. Genetic transformation is one of the most effective approaches used to induce resistance against plant diseases. Antimicrobial peptides (AMPs) have shown potential breakthroughs to improve resistance to bacterial diseases in plants. In this paper, we confirm the Agrobacterium-mediated transformation of Pera sweet orange expressing the AMP sarcotoxin IA (stx IA) gene isolated from the flesh fly Sarcophaga peregrina and its reaction to CLas, involving plant performance and fruit quality assessments. Four independent transgenic lines, STX-5, STX-11, STX-12, and STX-13, and a non-transgenic control, were graft-inoculated with CLas. Based on our findings, none of the transgenic plants were immune to CLas. However, the STX-5 and STX-11 lines showed reduced susceptibility to HLB with mild disease symptoms and low incidence of plants with the presence of CLas. Fruit and juice quality were not affected by the genetic transformation. Further, no residues of the sarcotoxin IA protein were found in the juice of the STX-11 and STX-12 fruits, though detected in the juice of the STX-5 and STX-13 lines, as revealed by the immunoblotting test. However, juices from all transgenic lines showed low traces of sarcotoxin IA peptide in its composition. The accumulation of this peptide did not cause any deleterious effects on plants or in fruit/juice. Our findings reinforce the challenges of identifying novel approaches to managing HLB.

'Candidatus Liberibacter asiaticus'-Encoded BCP Peroxiredoxin Suppresses Lipopolysaccharide-Mediated Defense Signaling and Nitrosative Stress In Planta.

The lipopolysaccharides (LPS) of gram-negative bacteria trigger a nitrosative and oxidative burst in both animals and plants during pathogen invasion. Liberibacter crescens strain BT-1 is a surrogate for functional genomic studies of the uncultured pathogenic 'Candidatus Liberibacter' spp. that are associated with severe diseases such as citrus greening and potato zebra chip. Structural determination of L. crescens LPS revealed the presence of a very long chain fatty acid modification. L. crescens LPS pretreatment suppressed growth of Xanthomonas perforans on nonhost tobacco (Nicotiana benthamiana) and X. citri subsp. citri on host orange (Citrus sinensis), confirming bioactivity of L. crescens LPS in activation of systemic acquired resistance (SAR). L. crescens LPS elicited a rapid burst of nitric oxide (NO) in suspension cultured tobacco cells. Pharmacological inhibitor assays confirmed that arginine-utilizing NO synthase (NOS) activity was the primary source of NO generation elicited by L. crescens LPS. LPS treatment also resulted in biological markers of NO-mediated SAR activation, including an increase in the glutathione pool, callose deposition, and activation of the salicylic acid and azelaic acid (AzA) signaling networks. Transient expression of 'Ca. L. asiaticus' bacterioferritin comigratory protein (BCP) peroxiredoxin in tobacco compromised AzA signaling, a prerequisite for LPS-triggered SAR. Western blot analyses revealed that 'Ca. L. asiaticus' BCP peroxiredoxin prevented peroxynitrite-mediated tyrosine nitration in tobacco. 'Ca. L. asiaticus' BCP peroxiredoxin (i) attenuates NO-mediated SAR signaling and (ii) scavenges peroxynitrite radicals, which would facilitate repetitive cycles of 'Ca. L. asiaticus' acquisition and transmission by fecund psyllids throughout the limited flush period in citrus.[Formula: see text] Copyright © 2022 The Author(s). This is an open access article distributed under the CC BY-NC-ND 4.0 International license.

Tad pilus-mediated twitching motility is essential for DNA uptake and survival of Liberibacters.

Axenically cultured Liberibacter crescens (Lcr) is a closely related surrogate for uncultured plant pathogenic species of the genus Liberibacter, including 'Candidatus L. asiaticus' (CLas) and 'Ca. L. solanacearum' (CLso). All Liberibacters encode a completely conserved gene repertoire for both flagella and Tad (Tight Adherence) pili and all are missing genes critical for nucleotide biosynthesis. Both flagellar swimming and Tad pilus-mediated twitching motility in Lcr were demonstrated for the first time. A role for Tad pili in the uptake of extracellular dsDNA for food in Liberibacters was suspected because both twitching and DNA uptake are impossible without repetitive pilus extension and retraction, and no genes encoding other pilus assemblages or mechanisms for DNA uptake were predicted to be even partially present in any of the 35 fully sequenced Liberibacter genomes. Insertional mutations of the Lcr Tad pilus genes cpaA, cpaB, cpaE, cpaF and tadC all displayed such severely reduced growth and viability that none could be complemented. A mutation affecting cpaF (motor ATPase) was further characterized and the strain displayed concomitant loss of twitching, viability and reduced periplasmic uptake of extracellular dsDNA. Mutations of comEC, encoding the inner membrane competence channel, had no effect on either motility or growth but completely abolished natural transformation in Lcr. The comEC mutation was restored by complementation using comEC from Lcr but not from CLas strain psy62 or CLso strain RS100, indicating that unlike Lcr, these pathogens were not naturally competent for transformation. This report provides the first evidence that the Liberibacter Tad pili are dynamic and essential for both motility and DNA uptake, thus extending their role beyond surface adherence.

Single- or double-membrane-bound vesicles and P, Ca, and Fe-containing granules in Xanthomonas citri cultured on a solid medium.

The organelle-like structures of Xanthomonas citri, a bacterial pathogen that causes citrus canker, were investigated using an analytical transmission electron microscope. After high-pressure freezing, the bacteria were then freeze-substituted for imaging and element analysis. Miniscule electron-dense structures of varying shapes without a membrane enclosure were frequently observed near the cell poles in a 3-day culture. The bacteria formed cytoplasmic electron-dense spherical structures measuring approximately 50 nm in diameter. Furthermore, X. citri produced electron-dense or translucent ellipsoidal intracellular or extracellular granules. Single- or double-membrane-bound vesicles, including outer-inner membrane vesicles, were observed both inside and outside the cells. Most cells had been lysed in the 3-week X. citri culture, but they harbored one or two electron-dense spherical structures. Contrast-inverted scanning transmission electron microscopy images revealed distinct white spherical structures within the cytoplasm of X. citri. Likewise, energy-dispersive X-ray spectrometry showed the spatial heterogeneity and co-localization of phosphorus, oxygen, calcium, and iron only in the cytoplasmic electron-dense spherical structures, thus corroborating the nature of polyphosphate granules.

Control of postharvest diseases caused by Penicillium spp. with myrtle leaf phenolic extracts: in vitro and in vivo study on mandarin fruit during storage.

BACKGROUND: In the postharvest handling of horticultural commodities, plant extracts with fungicidal activity are a valid alternative to synthetic fungicides. The fungicidal activity of myrtle leaf extracts from eight cultivars was studied in vitro against Penicillium digitatum, Penicillium italicum, and Penicillium expansum and on artificially inoculated mandarins with green and blue molds during storage for 12 days at 20 °C and 90% RH. RESULTS: Hydroxybenzoic acids, hydrolysable tannins, and flavonols were identified by high-performance liquid chromatography (HPLC). Despite sharing the same phenolic profile, extracts of eight myrtle cultivars significantly differed in the concentration of phenolics. Hydrolysable tannins are the principal subclass representing nearly 44.9% of the total polyphenols, whereas myricitrin was the most abundant flavonol in all cultivars. Myrtle extracts strongly inhibited conidial germination of the pathogens tested, although the greatest efficacy was observed against P. digitatum. At a concentration of 20 g L-1 , all the extracts completely inhibited fungi growth; only 'Angela', 'Tonina' and 'Grazia' extracts were effective at lower concentrations (15 g L-1 ). On inoculated fruit, myrtle extracts significantly controlled rot development. As a preventive treatment, 'Ilaria' and 'Maria Rita' extracts significantly reduced the rate of fruit with green mold decay lesions. When applied as a curative treatment, all the exacts decreased the incidence of decay. Against P. italicum, all the extracts applied as preventive treatments controlled decay effectively, while as curative treatment some of the extracts were not effective. All the extracts reduced the size of the infected areas. CONCLUSION: The results propose myrtle extracts as a possible natural alternative to synthetic fungicides. © 2021 Society of Chemical Industry.

Novel Chitosan-Riboflavin Conjugate with Visible Light-Enhanced Antifungal Properties against Penicillium digitatum.

A novel chemical conjugate between chitosan (CH) and riboflavin (RF) has been synthesized and characterized via Fourier transform infrared, NMR, and other spectroscopic methods. Photophysical and photochemical properties such as absorption spectra, fluorescence emission, fluorescence anisotropy, and singlet oxygen generation were characterized as well. This new biopolymer-based conjugate was designed to have an antifungal effect enhanced through antimicrobial photodynamic therapy. The antifungal effect of this conjugate (CH-RF) was compared with CH and RF against Penicillium digitatum in vitro. The conjugate showed the highest fungal growth inhibition of all systems tested at a dose of 0.5% w/v. This new biopolymer-based compound could be a promising alternative to fungicides used in citrus fruits postharvest.

Effect of red thyme oil (Thymus vulgaris L.) vapours on fungal decay, quality parameters and shelf-life of oranges during cold storage.

This work has been aimed at studying the effect of red thyme oil (RTO, Thymus vulgaris L.) on the shelf-life and Penicillium decay of oranges during cold storage. RTO vapours significantly reduced (P ≤ 0.05) the percentage of infected wounds, the external growth area and the production of spores in inoculated orange fruit stored for 12 days at 7 °C in a polypropylene film selected for its appropriate permeability. Among the RTO compounds, p-cymene and thymol were the most abundant in packed boxes at the end of cold storage. The RTO vapours did not affect the main quality parameters of the oranges, or the taste and odour of the juice. The results have shown that an active packaging, using RTO vapours, could be employed, by the citrus industry, to extend the shelf-life of oranges for fresh market use and juice processing.

Overexpression of Citrus reticulata SAMT in Nicotiana tabacum increases MeSA volatilization and decreases Xylella fastidiosa symptoms.

MAIN CONCLUSION: Nicotiana tabacum overexpressing CrSAMT from Citrus reticulata increased production of MeSA, which works as an airborne signal in neighboring wild-type plants, inducing PR1 and increasing resistance to the pathogen Xylella fastidiosa. Xylella fastidiosa is one of the major threats to plant health worldwide, affecting yield in many crops. Despite many efforts, the development of highly productive resistant varieties has been challenging. In studying host plant resistance, the S-adenosyl-L-methionine: salicylic acid carboxyl methyltransferase gene (SAMT) from Citrus reticulata, a X. fastidiosa resistant species, was upregulated in response to pathogen infection. SAMT is involved with the catalysis and production of methyl salicylate (MeSA), an airborne signal responsible for triggering systemic acquired resistance. Here we used tobacco as a model system and generated transgenic plants overexpressing C. reticulata SAMT (CrSAMT). We performed an in silico structural characterization of CrSAMT and investigated its biotechnological potential in modulating the immune system in transgenic plants. The increase of MeSA production in transgenic lines was confirmed by gas chromatography (GC-MS). The transgenic lines showed upregulation of PR1, and their incubation with neighboring wild-type plants activated PR1 expression, indicating that MeSA worked as an airborne signal. In addition, transgenic plants showed significantly fewer symptoms when challenged with X. fastidiosa. Altogether, these data suggest that CrSAMT plays a role in host defense response and can be used in biotechnology approaches to confer resistance against X. fastidiosa.

Antibacterial Functions and Proposed Modes of Action of Novel 1,2,3,4-Tetrahydro-ß-carboline Derivatives that Possess an Attractive 1,3-Diaminopropan-2-ol Pattern against Rice Bacterial Blight, Kiwifruit Bacterial Canker, and Citrus Bacterial Canker.

In recent years, naturally occurring tetrahydro-ß-carboline (THC) alkaloids and their derivatives have been of biological interest. However, few studies and developments have reported the use of such structures in managing plant bacterial diseases. Herein, an array of novel THC derivatives containing an attractive 1,3-diaminopropan-2-ol pattern were prepared to evaluate the antiphytopathogen activity in vitro and in vivo and explore innovative antibacterial frameworks. Notably, target compounds exhibited excellent activities against three rebellious phytopathogens, namely, Pseudomonas syringae pv. actinidiae (Psa), Xanthomonas axonopodis pv. citri, and Xanthomonas oryzae pv. oryzae, at related optimal EC50 values of 2.39 (II9), 2.06 (I23), and 1.69 (II9) µg/mL, respectively. These effects were superior to those of the parent structure 1,2,3,4-THC and positive controls. In vivo assays showed that II9 exhibited excellent control efficiencies of 51.89 and 65.45% at 200 µg/mL against rice bacterial blight and kiwifruit bacterial canker, respectively, and I23 substantially relieved the citrus canker on the leaves. Antibacterial mechanisms indicated that these THC compounds could induce the increment of reactive oxygen species and subsequently endow the tested bacteria with distinct apoptotic behavior. In addition, II9 could alleviate the hypersensitive response and pathogenicity of Psa. Overall, these simple THC derivatives can be further developed as versatile antibacterial agents.

Complete genome sequence and analysis of Alcaligenes faecalis strain Mc250, a new potential plant bioinoculant.

Here we present and analyze the complete genome of Alcaligenes faecalis strain Mc250 (Mc250), a bacterium isolated from the roots of Mimosa calodendron, an endemic plant growing in ferruginous rupestrian grasslands in Minas Gerais State, Brazil. The genome has 4,159,911 bp and 3,719 predicted protein-coding genes, in a single chromosome. Comparison of the Mc250 genome with 36 other Alcaligenes faecalis genomes revealed that there is considerable gene content variation among these strains, with the core genome representing only 39% of the protein-coding gene repertoire of Mc250. Mc250 encodes a complete denitrification pathway, a network of pathways associated with phenolic compounds degradation, and genes associated with HCN and siderophores synthesis; we also found a repertoire of genes associated with metal internalization and metabolism, sulfate/sulfonate and cysteine metabolism, oxidative stress and DNA repair. These findings reveal the genomic basis for the adaptation of this bacterium to the harsh environmental conditions from where it was isolated. Gene clusters associated with ectoine, terpene, resorcinol, and emulsan biosynthesis that can confer some competitive advantage were also found. Experimental results showed that Mc250 was able to reduce (~60%) the virulence phenotype of the plant pathogen Xanthomonas citri subsp. citri when co-inoculated in Citrus sinensis, and was able to eradicate 98% of juveniles and stabilize the hatching rate of eggs to 4% in two species of agricultural nematodes. These results reveal biotechnological potential for the Mc250 strain and warrant its further investigation as a biocontrol and plant growth-promoting bacterium.

'Candidatus Liberibacter Asiaticus' SDE1 Effector Induces Huanglongbing Chlorosis by Downregulating Host DDX3 Gene.

'Candidatus Liberibacter asiaticus' (CLas) is the pathogenic bacterium that causes the disease Huanglongbing (HLB) in citrus and some model plants, such as Nicotiana benthamiana. After infection, CLas releases a set of effectors to modulate host responses. One of these critical effectors is Sec-delivered effector 1 (SDE1), which induces chlorosis and cell death in N. benthamiana. In this study, we revealed the DEAD-box RNA helicase (DDX3) interacts with SDE1. Gene silencing study revealed that knockdown of the NbDDX3 gene triggers leaf chlorosis, mimicking the primary symptom of CLas infection in N. benthamiana. The interactions between SDE1 and NbDDX3 were localized in the cell membrane. Overexpression of SDE1 resulted in suppression of NbDDX3 gene expression in N. benthamiana, which suggests a critical role of SDE1 in modulating NbDDX3 expression. Furthermore, we verified the interaction of SDE1 with citrus DDX3 (CsDDX3), and demonstrated that the expression of the CsDDX3 gene was significantly reduced in HLB-affected yellowing and mottled leaves of citrus. Thus, we provide molecular evidence that the downregulation of the host DDX3 gene is a crucial mechanism of leaf chlorosis in HLB-affected plants. The identification of CsDDX3 as a critical target of SDE1 and its association with HLB symptom development indicates that the DDX3 gene is an important target for gene editing, to interrupt the interaction between DDX3 and SDE1, and therefore interfere host susceptibility.