Design, Synthesis, and Antibacterial Evaluation of Novel Isoindolin-1-ones Derivatives Containing Piperidine Fragments.

A series of novel isoindoline-1-one derivatives containing piperidine moiety were designed and synthesized using natural compounds as raw materials, and their biological activities were tested for three bacterial and three fungal pathogens. These derivatives exhibited good against phytopathogenic bacteria activities against Pseudomonas syringae pv actinidiae (Psa) and Xanthomonas axonopodis pv.citri (Xac). Some compounds exhibited excellent antibacterial activities against Xanthomonas oryzae pv oryzae (Xoo). The dose of Y8 against Xoo (the maximum half lethal effective concentration (EC50) = 21.3 µg/mL) was better than that of the thiediazole copper dose (EC50 = 53.3 µg/mL). Excitingly, further studies have shown that the molecular docking of Y8 with 2FBW indicates that it can fully locate the interior of the binding pocket through hydrogen bonding and hydrophobic interactions, thereby enhancing its anti-Xoo activity. Scanning electron microscopy (SEM) studies revealed that Y8 induced the Xoo cell membrane collapse. Moreover, the proteomic results also indicate that Y8 may be a multifunctional candidate as it affects the formation of bacterial Xoo biofilms, thereby exerting antibacterial effects.

Screening Plant Growth-Promoting Bacteria with Antimicrobial Properties for Upland Rice.

This study explores beneficial bacteria isolated from the roots and rhizosphere soil of Khao Rai Leum Pua Phetchabun rice plants. A total of 315 bacterial isolates (KK001 to KK315) were obtained. Plant growth-promoting traits (phosphate solubilization and indole-3-acetic acid (IAA) production), and antimicrobial activity against three rice pathogens (Curvularia lunata NUF001, Bipolaris oryzae 2464, and Xanthomonas oryzae pv. oryzae) were assessed. KK074 was the most prolific in IAA production, generating 362.6 ± 28.0 µg/ml, and KK007 excelled in tricalcium phosphate solubilization, achieving 714.2 ± 12.1 µg/ml. In antimicrobial assays using the dual culture method, KK024 and KK281 exhibited strong inhibitory activity against C. lunata, and KK269 was particularly effective against B. oryzae. In the evaluation of antimicrobial metabolite production, KK281 and KK288 exhibited strong antifungal activities in cell-free supernatants. Given the superior performance of KK281, taxonomically identified as Bacillus sp. KK281, it was investigated further. Lipopeptide extracts from KK281 had significant antimicrobial activity against C. lunata and a minimum inhibitory concentration (MIC) of 3.1 mg/ml against X. oryzae pv. oryzae. LC-ESI-MS/MS analysis revealed the presence of surfactin in the lipopeptide extract. The crude extract was non-cytotoxic to the L-929 cell line at tested concentrations. In conclusion, the in vitro plant growth-promoting and disease-controlling attributes of Bacillus sp. KK281 make it a strong candidate for field evaluation to boost plant growth and manage disease in upland rice.

Evaluation of Phoma sp. Biomass as an Endophytic Fungus for Synthesis of Extracellular Gold Nanoparticles with Antibacterial and Antifungal Properties.

The aim of our study was to examine the different concentrations of AuNPs as a new antimicrobial substance to control the pathogenic activity. The extracellular synthesis of AuNPs performed by using Phoma sp. as an endophytic fungus. Endophytic fungus was isolated from vascular tissue of peach trees (Prunus persica) from Baft, located in Kerman province, Iran. The UltraViolet-Visible Spectroscopy (UV-Vis spectroscopy) and Fourier transform infrared spectroscopy provided the absorbance peak at 526 nm, while the X-ray diffraction and transmission electron microscopy images released the formation of spherical AuNPs with sizes in the range of 10-100 nm. The findings of inhibition zone test of Au nanoparticles (AuNPs) showed a desirable antifungal and antibacterial activity against phytopathogens including Rhizoctonia solani AG1-IA (AG1-IA has been identified as the dominant anastomosis group) and Xanthomonas oryzae pv. oryzae. The highest inhibition level against sclerotia formation was 93% for AuNPs at a concentration of 80 µg/mL. Application of endophytic fungus biomass for synthesis of AuNPs is relatively inexpensive, single step and environmentally friendly. In vitro study of the antifungal activity of AuNPs at concentrations of 10, 20, 40 and 80 µg/mL was conducted against rice fungal pathogen R. solani to reduce sclerotia formation. The experimental data revealed that the Inhibition rate (RH) for sclerotia formation was (15, 33, 74 and 93%), respectively, for their corresponding AuNPs concentrations (10, 20, 40 and 80 µg/mL). Our findings obviously indicated that the RH strongly depend on AuNPs rates, and enhance upon an increase in AuNPs rates. The application of endophytic fungi biomass for green synthesis is our future goal.

An atypical class of non-coding small RNAs is produced in rice leaves upon bacterial infection.

Non-coding small RNAs (sRNA) act as mediators of gene silencing and regulate plant growth, development and stress responses. Early insights into plant sRNAs established a role in antiviral defense and they are now extensively studied across plant-microbe interactions. Here, sRNA sequencing discovered a class of sRNA in rice (Oryza sativa) specifically associated with foliar diseases caused by Xanthomonas oryzae bacteria. Xanthomonas-induced small RNAs (xisRNAs) loci were distinctively upregulated in response to diverse virulent strains at an early stage of infection producing a single duplex of 20-22 nt sRNAs. xisRNAs production was dependent on the Type III secretion system, a major bacterial virulence factor for host colonization. xisRNA loci overlap with annotated transcripts sequences, with about half of them encoding protein kinase domain proteins. A number of the corresponding rice cis-genes have documented functions in immune signaling and xisRNA loci predominantly coincide with the coding sequence of a conserved kinase motif. xisRNAs exhibit features of small interfering RNAs and their biosynthesis depend on canonical components OsDCL1 and OsHEN1. xisRNA induction possibly mediates post-transcriptional gene silencing but they do not broadly suppress cis-genes expression on the basis of mRNA-seq data. Overall, our results identify a group of unusual sRNAs with a potential role in plant-microbe interactions.

High-throughput screening alternative to crystal violet biofilm assay combining fluorescence quantification and imaging.

The crystal violet assay is widely used for biofilm quantitation despite its toxicity and variability. Here, we instead combine fluorescence labelling with the Cytation 5 multi-mode plate reader, to enable simultaneous acquisition of both quantitative and imaging biofilm data. This high-throughput method produces more robust data and provides information about morphology and spatial species organization within the biofilm.

Identification of a Chitooligosaccharide Mechanism against Bacterial Leaf Blight on Rice by In Vitro and In Silico Studies.

This study focuses on a commercial plant elicitor based on chitooligosaccharides (BIG®), which aids in rice plant growth and disease resistance to bacterial leaf blight (BLB). When the pathogen (Xoo) vigorously attacks rice that has suffered yield losses, it can cause damage in up to 20% of the plant. Furthermore, Xoo is a seed-borne pathogen that can survive in rice seeds for an extended period. In this study, when rice seeds were soaked and sprayed with BIG®, there was a significant increase in shoot and root length, as well as plant biomass. Furthermore, BIG®-treated rice plants showed a significant reduction in BLB severity of more than 33%. Synchrotron radiation-based Fourier transform infrared (SR-FTIR) analysis was used to characterize BIG®'s mechanism in the chemical structure of rice leaves. The SR-FTIR results at 1650, 1735, and 1114 cm-1 indicated changes in biochemical components such as pectins, lignins, proteins, and celluloses. These findings demonstrated that commercial BIG® not only increased rice growth but also induced resistance to BLB. The drug's target enzyme, Xoo 1075 from Xanthomonas oryzae (PDB ID: 5CY8), was analyzed for its interactions with polymer ingredients, specifically chitooligosaccharides, to gain molecular insights down to the atomic level. The results are intriguing, with a strong binding of the chitooligosaccharide polymer with the drug target, revealing 10 hydrogen bonds between the protein and polymer. Overall, the computational analysis supported the experimentally demonstrated strong binding of chitooligosaccharides to the drug target.

Design, Synthesis, and Bioactivity Evaluation of New Thiochromanone Derivatives Containing a Carboxamide Moiety.

In this study, using the botanical active component thiochromanone as the lead compound, a total of 32 new thiochromanone derivatives containing a carboxamide moiety were designed and synthesized and their in vitro antibacterial activities against Xanthomonas oryzae pv. oryzae (Xoo), Xanthomonas oryzae pv. oryzicolaby (Xoc), and Xanthomonas axonopodis pv. citri (Xac) were determined, as well as their in vitro antifungal activities against Botryosphaeria dothidea (B. dothidea), Phomopsis sp., and Botrytis cinerea (B. cinerea). Bioassay results demonstrated that some of the target compounds exhibited moderate to good in vitro antibacterial and antifungal activities. In particular, compound 4e revealed excellent in vitro antibacterial activity against Xoo, Xoc, and Xac, and its EC50 values of 15, 19, and 23 µg/mL, respectively, were superior to those of Bismerthiazol and Thiodiazole copper. Meanwhile, compound 3b revealed moderate in vitro antifungal activity against B. dothidea at 50 µg/mL, and the inhibition rate reached 88%, which was even better than that of Pyrimethanil, however, lower than that of Carbendazim. To the best of our knowledge, this is the first report on the antibacterial and antifungal activities of this series of novel thiochromanone derivatives containing a carboxamide moiety.

The PilB-PilZ-FimX regulatory complex of the Type IV pilus from Xanthomonas citri.

Type IV pili (T4P) are thin and flexible filaments found on the surface of a wide range of Gram-negative bacteria that undergo cycles of extension and retraction and participate in a variety of important functions related to lifestyle, defense and pathogenesis. During pilus extensions, the PilB ATPase energizes the polymerization of pilin monomers from the inner membrane. In Xanthomonas citri, two cytosolic proteins, PilZ and the c-di-GMP receptor FimX, are involved in the regulation of T4P biogenesis through interactions with PilB. In vivo fluorescence microscopy studies show that PilB, PilZ and FimX all colocalize to the leading poles of X. citri cells during twitching motility and that this colocalization is dependent on the presence of all three proteins. We demonstrate that full-length PilB, PilZ and FimX can interact to form a stable complex as can PilB N-terminal, PilZ and FimX C-terminal fragments. We present the crystal structures of two binary complexes: i) that of the PilB N-terminal domain, encompassing sub-domains ND0 and ND1, bound to PilZ and ii) PilZ bound to the FimX EAL domain within a larger fragment containing both GGDEF and EAL domains. Evaluation of PilZ interactions with PilB and the FimX EAL domain in these and previously published structures, in conjunction with mutagenesis studies and functional assays, allow us to propose an internally consistent model for the PilB-PilZ-FimX complex and its interactions with the PilM-PilN complex in the context of the inner membrane platform of the X. citri Type IV pilus.

Ginger Essential Oils-Loaded Nanoemulsions: Potential Strategy to Manage Bacterial Leaf Blight Disease and Enhanced Rice Yield.

The bacterial leaf blight (BLB) caused by Xanthomonas oryzae pv. oryzae (Xoo) is one of the most serious rice diseases, causing huge yield losses worldwide. Several technologies and approaches have been opted to reduce the damage; however, these have had limited success. Recently, scientists have been focusing their efforts on developing efficient and environmentally friendly nanobactericides for controlling bacterial diseases in rice fields. In the present study, a scanning electron microscope (SEM), transmission electron microscope (TEM), and a confocal laser scanning microscope (CLSM) were utilized to investigate the mode of actions of ginger EOs on the cell structure of Xoo. The ginger EOs caused the cells to grow abnormally, resulting in an irregular form with hollow layers, whereas the dimethylsulfoxide (DMSO) treatment showed a typical rod shape for the Xoo cell. Ginger EOs restricted the growth and production of biofilms by reducing the number of biofilms generated as indicated by CLSM. Due to the instability, poor solubility, and durability of ginger EOs, a nanoemulsions approach was used, and a glasshouse trial was performed to assess their efficacy on BLB disease control. The in vitro antibacterial activity of the developed nanobactericides was promising at different concentration (50-125 µL/mL) tested. The efficacy was concentration-dependent. There was significant antibacterial activity recorded at higher concentrations. A glasshouse trial revealed that developed nanobactericides managed to suppress BLB disease severity effectively. Treatment at a concentration of 125 µL/mL was the best based on the suppression of disease severity index, AUDPC value, disease reduction (DR), and protection index (PI). Furthermore, findings on plant growth, physiological features, and yield parameters were significantly enhanced compared to the positive control treatment. In conclusion, the results indicated that ginger essential oils loaded-nanoemulsions are a promising alternative to synthetic antibiotics in suppressing Xoo growth, regulating the BLB disease, and enhancing rice yield under a glasshouse trial.

Silicon enhancement for endorsement of Xanthomonas albilineans infection in sugarcane.

Silicon (Si) is considered to be a plant growth and development regulator element as well as provide the regulatory response against various biotic stressors. However, the potential mechanism of Si enhancement to regulate plant disease resistance remains to be studied. Therefore, the current study evaluated the effects of Si application on the performance of sugarcane against Xanthomonas albilineans (Xa) infection. Si was applied exogenously (0, 3.85 and 7.70 g Si/kg soil) and the results show that plant height, stem circumference and leaf width of siliconized sugarcane have been improved, which effectively reduced the disease index (0.17-0.21) and incidence (58.2%-69.1%) after Xa infection. Lowest values of MDA (348.5 nmol g-1 FW) and H2O2 (3539.4 mmol/L) were observed in 7.70 g Si/kg soil followed by in 3.85 g Si/kg soil (MDA: 392.6 nmol g-1 FW and H2O2: 3134.6 mmol/L) than that of the control. Whereas, PAL enzyme activity (50.8 mmol/L), JA (230.2 mmol/L) and SA (2.7 ug mL-1) contents were significantly higher in 7.70 g Si/kg soil followed by in 3.85 g Si/kg soil (PAL: 46.3 mmol/L, JA: 182.7 mmol/L and SA: 2.4 ug mL-1) as compared to control. The lower MDA, H2O2 level and higher enzymatic activities were associated with the highest expression levels of their metabolic pathway associated genes i.e., ShMAPK1, ShLOX, ShPAL, ShAOS, ShAOC, ShC4H, ShCAT, Sh4CL and ShNPR1 (22.08, 15.56, 10.42, 3.35, 2.54, 2.14, 1.82, 1.67 and 1.22 folds, respectively) in 7.70 g Si/kg soil as compared to other experimental units and control. Overall, the results of current study indicates that siliconized sugarcane more actively regulates disease resistance through modulation of growth and MDA, H2O2, SA and JA associated metabolic pathways.

Biogenic silver nanoparticles as an antibacterial agent against bacterial leaf blight causing rice phytopathogen Xanthomonas oryzae pv. oryzae.

Silver nanoparticles (Ag NP) were produced utilizing leaf extract of rice cultivar Taichung native-1. Various factors like leaf extract, silver nitrate concentrations, and duration of autoclaving were standardized during synthesis. Nanoparticles were analyzed with UV-visible absorption spectroscopy (UV-vis), dynamic light scattering, zeta potential, X-ray diffraction and transmission electron microscopy techniques. The synthesis was noted at 0.4% extract, 0.6 mM silver nitrate, 30 min of autoclaving and NP formation was confirmed from 424 nm peak in UV-vis. NP showed zeta potential of - 27 mV, face-centered cubic (fcc) crystal nature and sized around 16.5 ± 5.9 nm. Biogenic NP synthesized from susceptible rice variety were used as an antibacterial agent against phytopathogen Xanthomonas oryzae pv. oryzae (Xoo), the causative agent of bacterial leaf blight (BLB) disease in rice. Antibacterial effect of Ag NP was evaluated using in vitro assays and in vivo efficacy under greenhouse conditions. Results confirmed effective inhibition of Xoo growth and colony formation by Ag NP and found to be the more powerful antibacterial agent. Besides, Ag NP treatment (10 µg/mL) caused an enhancement in seedling vigor index. Pots treated with Ag NP (15 µg/mL) in vivo in greenhouse showed disease severity of 26.6% and disease decrease over control of 49.2%, at a much lower NP concentration than earlier reported studies. Thus, the current report implies using the leaf extract synthesized Ag NP to control and BLB disease management in field conditions.

Design of novel peptide inhibitors against the conserved bacterial transcription terminator, Rho.

The transcription terminator Rho regulates many physiological processes in bacteria, such as antibiotic sensitivity, DNA repair, RNA remodeling, and so forth, and hence, is a potential antimicrobial target, which is unexplored. The bacteriophage P4 capsid protein, Psu, moonlights as a natural Rho antagonist. Here, we report the design of novel peptides based on the C-terminal region of Psu using phenotypic screening methods. The resultant 38-mer peptides, in addition to containing mutagenized Psu sequences, also contained plasmid sequences, fused to their C termini. Expression of these peptides inhibited the growth of Escherichia coli and specifically inhibited Rho-dependent termination in vivo. Peptides 16 and 33 exhibited the best Rho-inhibitory properties in vivo. Direct high-affinity binding of these two peptides to Rho also inhibited the latter's RNA-dependent ATPase and transcription termination functions in vitro. These two peptides remained functional even if eight to ten amino acids were deleted from their C termini. In silico modeling and genetic and biochemical evidence revealed that these two peptides bind to the primary RNA-binding site of the Rho hexamer near its subunit interfaces. In addition, the gene expression profiles of these peptides and Psu overlapped significantly. These peptides also inhibited the growth of Mycobacteria and inhibited the activities of Rho proteins from Mycobacterium tuberculosis, Xanthomonas, Vibrio cholerae, and Salmonella enterica. Our results showed that these novel anti-Rho peptides mimic the Rho-inhibition function of the ∼42-kDa dimeric bacteriophage P4 capsid protein, Psu. We conclude that these peptides and their C-terminal deletion derivatives could provide a basis on which to design novel antimicrobial peptides.

Genome sequencing of the bacterial blight pathogen DY89031 reveals its diverse virulence and origins of Xanthomonas oryzae pv. oryzae strains.

The bacterial pathogen Xanthomonas oryzae pv. oryzae (Xoo), belonging to Xanthomonas sp., causes one of the most destructive vascular diseases in rice worldwide, particularly in Asia and Africa. To better understand Xoo pathogenesis, we performed genome sequencing of the Korea race 1 strain DY89031 (J18) and analyzed the phylogenetic tree of 63 Xoo strains. We found that the rich diversity of evolutionary features is likely associated with the rice cultivation regions. Further, virulence effector proteins secreted by the type III secretion system (T3SS) of Xoo showed pathogenesis divergence. The genome of DY89031 shows a remarkable difference from that of the widely prevailed Philippines race 6 strain PXO99A, which is avirulent to rice Xa21, a well-known disease resistance (R) gene that can be broken down by DY89031. Interestingly, plant inoculation experiments with the PXO99A transformants expressing the DY89031 genes enabled us to identify additional TAL (transcription activator-like) and non-TAL effectors that may support DY89031-specific virulence. Characterization of DY89031 genome and identification of new effectors will facilitate the investigation of the rice-Xoo interaction and new mechanisms involved.

Alternative tehcniques for Xanthomonas arboricola pv pruni preservation at room temperature.

The efficiency of alternative preservation techniques for Xanthomonas arboricola pv pruni was studied. The preservation methods in sunflower seeds, glass beads and sterile soil were suitable for maintaining viability and productive capacity of xanthan pruni.

Design, synthesis, crystal structure and in vitro antimicrobial activity of novel 1,2,4-triazolo[1,5-a]pyrimidine-containing quinazolinone derivatives.

A series of novel 1,2,4-triazolo[1,5-a]pyrimidine-containing quinazolin-4(3H)-one derivatives (8a-8o) were designed, synthesized and assessed for their in vitro antibacterial and antifungal activities in agriculture. All the title compounds were completely characterized via 1H NMR, 13C NMR, HRMS and IR spectroscopic data. In particular, the molecular structure of compound 8f was further corroborated through a single-crystal X-ray diffraction measurement. The turbidimetric method revealed that some of the compounds displayed noticeable bactericidal potencies against the tested plant pathogenic bacteria. For example, compounds 8m, 8n and 8o possessed higher antibacterial efficacies in vitro against Xanthomonas oryzae pv. oryzae with EC50 values of 69.0, 53.3 and 58.9 µg/mL, respectively, as compared with commercialized agrobactericide bismerthiazol (EC50 = 91.4 µg/mL). Additionally, compound 8m displayed an EC50 value of 71.5 µg/mL toward Xanthomonas axonopodis pv. citri, comparable to control bismerthiazol (EC50 = 60.5 µg/mL). A preliminary structure-activity relationship (SAR) analysis was also conducted, based on the antibacterial results. Finally, some compounds were also found to have a certain antifungal efficacy in vitro at the concentration of 50 µg/mL.

Evaluating the potential of electrolysed water for the disinfection of citrus fruit in packinghouses.

BACKGROUND: The largest and most profitable market for citrus is the production of fresh fruit. Xanthomonas citri subsp. citri is a Gram-negative plant pathogen and the etiological agent of citrus canker, one of the major threats to citrus production worldwide. In the early stages of infection, X. citri can attach to plant surfaces by means of biofilms. Biofilm is considered an essential virulence factor, which helps tissue colonization in plants. Thus, sanitization of citrus fruit is mandatory in packinghouses before any logistic operation as packing and shipment to the market. The aim of this study was to evaluate electrolysed water (EW) as a sanitizer for the disinfection of citrus fruit in packinghouses. RESULTS: Using a protocol to monitor cell respiration we show that EW, obtained after 8 and 9 min of electrolysis, sufficed to kill X. citri when applied at a concentration of 500 µL mL-1 . Furthermore, microscopy analysis, combined with time-response growth curves, confirmed that EW affects the bacterial cytoplasmatic membrane and it leads to cell death in the first few minutes of contact. Pathogenicity tests using limes to simulate packinghouse treatment showed that EW, produced with 9 min of electrolysis, was a very effective sanitizer capable of eliminating X. citri from contaminated fruit. CONCLUSION: It was possible to conclude that EW is significantly effective as sodium hypochlorite (NaClO) at 200 ppm. Therefore, EW could be an alternative for citrus sanitization in packinghouses. © 2020 Society of Chemical Industry.

Periplasm-enriched fractions from Xanthomonas citri subsp. citri type A and X. fuscans subsp. aurantifolii type B present distinct proteomic profiles under in vitro pathogenicity induction.

The causative agent of Asiatic citrus canker, the Gram-negative bacterium Xanthomonas citri subsp. citri (XAC), produces more severe symptoms and attacks a larger number of citric hosts than Xanthomonas fuscans subsp. aurantifolii XauB and XauC, the causative agents of cancrosis, a milder form of the disease. Here we report a comparative proteomic analysis of periplasmic-enriched fractions of XAC and XauB in XAM-M, a pathogenicity- inducing culture medium, for identification of differential proteins. Proteins were resolved by two-dimensional electrophoresis combined with liquid chromatography-mass spectrometry. Among the 12 proteins identified from the 4 unique spots from XAC in XAM-M (p<0.05) were phosphoglucomutase (PGM), enolase, xylose isomerase (XI), transglycosylase, NAD(P)H-dependent glycerol 3-phosphate dehydrogenase, succinyl-CoA synthetase ß subunit, 6-phosphogluconate dehydrogenase, and conserved hypothetical proteins XAC0901 and XAC0223; most of them were not detected as differential for XAC when both bacteria were grown in NB medium, a pathogenicity non-inducing medium. XauB showed a very different profile from XAC in XAM-M, presenting 29 unique spots containing proteins related to a great diversity of metabolic pathways. Preponderant expression of PGM and XI in XAC was validated by Western Blot analysis in the periplasmic-enriched fractions of both bacteria. This work shows remarkable differences between the periplasmic-enriched proteomes of XAC and XauB, bacteria that cause symptoms with distinct degrees of severity during citrus infection. The results suggest that some proteins identified in XAC can have an important role in XAC pathogenicity.

Enantiomeric Dibenzo-α-Pyrone Derivatives from Alternaria alternata ZHJG5 and Their Potential as Agrochemicals.

Three pairs of enantiomeric dibenzo-α-pyrone derivatives (1-3) including two pairs of new racemates (±)-alternaone A (1) and (±)-alternaone B (2) and one new enantiomer (-)-alternatiol (3), together with five known compounds (4-8) were isolated from the fungus Alternaria alternata ZHJG5. Their structures were confirmed by spectroscopic data and single-crystal X-ray diffraction analysis. All enantiomers were separated via chiral high-performance liquid chromatography, with their configurations determined by electronic circular dichroism calculation. Biogenetically, a key epoxy-rearrangement step was proposed for the formation of skeletons in 1-3; (+) 1, (-)-1, and 5 presented moderate antibacterial inhibition on phytopathogenic bacteria Xanthomonas oryzae pv. oryzae and Xanthomonas oryzae pv. oryzicola. In the antifungal test, compounds 7 and 8 showed a moderate protective effect against Botrytis cinerea in vivo.

Whole Genome Sequencing and Tn5-Insertion Mutagenesis of Pseudomonas taiwanensis CMS to Probe Its Antagonistic Activity Against Rice Bacterial Blight Disease.

The Gram-negative bacterium Pseudomonas taiwanensis is a novel bacterium that uses shrimp shell waste as its sole sources of carbon and nitrogen. It is a versatile bacterium with potential for use in biological control, with activities including toxicity toward insects, fungi, and the rice pathogen Xanthomonas oryzae pv.oryzae (Xoo). In this study, the complete 5.08-Mb genome sequence of P. taiwanensis CMS was determined by a combination of NGS/Sanger sequencing and optical mapping. Comparison of optical maps of seven Pseudomonas species showed that P. taiwanensis is most closely related to P. putida KT 2400. We screened a total of 11,646 individual Tn5-transponson tagged strains to identify genes that are involved in the production and regulation of the iron-chelator pyoverdine in P. taiwanensis, which is a key anti-Xoo factor. Our results indicated that the two-component system (TCS) EnvZ/OmpR plays a positive regulatory role in the production of pyoverdine, whereas the sigma factor RpoS functions as a repressor. The knowledge of the molecular basis of the regulation of pyoverdine by P. taiwanensis provided herein will be useful for its development for use in biological control, including as an anti-Xoo agent.

Photodynamic inactivation of the phytopathogenic bacterium Xanthomonas citri subsp. citri.

The present work intended to evaluate the applicability of photodynamic inactivation (PDI) of Xanthomonas citri subsp. citri with toluidine blue O (TBO), a commercial photosensitizer, as a strategy to control citrus canker. Assays were conducted with cell suspensions and biofilms, constructed either on polypropylene microtubes (in vitro assays) or on the surface of orange leaves (ex vivo assays), in the presence of TBO and under irradiation with artificial white light or natural sunlight. PDI assays using TBO alone caused a maximum 5·8 log10 reduction of X. citri viable cells in suspensions, and a much smaller inactivation (1·5 log10) in biofilms. However, concomitant use of KI potentiated the TBO photosensitization. Biofilms were inactivated down to the detection limit (>6 log10 reduction) with 5·0 µmol l-1 TBO + 10 mmol l-1 KI (in vitro) or 5·0 µmol l-1 TBO + 100 mmol l-1 KI (ex vivo) after artificial white light irradiation. Under natural sunlight, a reduction down to the detection limit of the Miles-Misra method was achieved with 50 µmol l-1 TBO and 100 mmol l-1 KI. PDI has potential to be applied in the control of citrus canker in field conditions although further studies are needed to show that there are no risks to plant physiology or fruit quality. SIGNIFICANCE AND IMPACT OF THE STUDY: Xanthomonas citri subsp. citri is a major cause of disease in citrus orchards. Because of the low efficacy and high environmental toxicity of copper-based treatments, there is growing interest on more sustainable phytosanitary approaches. Photodynamic inactivation (PDI) is being successfully used to control infectious agents and literature reports indicate that it is effective against some fungi and bacteria attacking fruit crops. The results of the present work open the perspective of using a low-cost photosensitizer and sunlight, as energy source, to control of the causative agent of citrus canker.