Search

Abstract The fabrication of the first label-free electrochemical DNA probe biosensor for highly sensitive detection of Candidatus Liberibacter asiaticus (CLas), as the causal agent of citrus huanglongbing disease, is conducted here. An OMP probe was designed based on the hybridization with its target-specific sequence in the outer membrane protein (OMP) gene of CLas. The characterization of the steps of biosensor fabrication and hybridization process between the immobilized OMP-DNA probe and the target ssDNA oligonucleotides (OMP-complementary and three mismatches OMP or OMP-mutation) was monitored using cyclic voltammetry and electrochemical impedance spectroscopy based on increasing or decreasing in the electron transfer in [Fe (CN)6]3−/4− on the modified gold electrode surface. The biosensor sensitivity indicated that the peak currents were linear over ranges from 20 to 100 nM for OMP-complementary with the detection limit of 0.026 nM (S/N = 3). The absence of any cross-interference with other biological DNA sequences confirmed a high selectivity of fabricated biosensor. Likewise, it showed good specificity in discriminating the mutation oligonucleotides from complementary target DNAs. The functional performance of optimized biosensor was achieved via the hybridization of OMP-DNA probe with extracted DNA from citrus plant infected with CLas. Therefore, fabricated biosensor indicates promise for sensitivity and early detection of citrus huanglongbing disease.

Abstract 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.

Glycerol dry reforming presents an intriguing approach for converting CO2, a greenhouse gas, and glycerol, a renewable resource, into syngas. This study delves into the impact of introducing 5 wt% of ZrO2, CeO2, La2O3, and Y2O3 to the Al2O3 support. Additionally, the study examines the influence of the optimal metal oxide ratio within the support formula on both structural properties and catalytic performance in the glycerol dry reforming process. To investigate these aspects, various catalyst supports were synthesized using a mechanochemical method. Subsequently, nickel-based catalysts were prepared through a wet impregnation technique. The catalysts underwent comprehensive characterization employing techniques such as XRD, BET, H2-TPR, CO2-TPD, TPO, and FESEM analyses to determine their physicochemical properties. The results demonstrated that the sample enhanced with 5 wt% ZrO2 exhibited remarkable performance metrics. Notably, it achieved a catalytic activity of 85.4% glycerol conversion at 650 ℃, while also displaying better stability and resistance to carbon deposition compared to the unpromoted catalyst throughout a 600 minutes reaction period. These enhanced attributes were attributed to the heightened specific surface area of the support (241.95 m2/gr), the effective dispersion of Ni on the support and the increase in basic sites.

Considering the adaptability and importance of the safflower for the production of oilseeds in Iran, this research was conducted to evaluate and investigate of main agronomic traits in 12 mutant lines, produced in the National Institute of Genetic Engineering and Biotechnology (NIGEB) along with Sina, Faraman, Omid, Sofeh, Goldasht, Golmehr, Parnian and Padideh cultivars. The project was carried out as a complete randomized block design in three replications under rainfed conditions during the crop season 2021-2022 in the Dryland Agricultural Research Institute-Sararood, Kermanshah, Iran. The results of variance analysis for all measured traits showed significant differences between cultivars and mutants at the probability level of 1%. The highest and lowest weight of 1000 seeds in this research was related to the Faraman cultivar (40.65 g) and Safeh (23.47 g), respectively. The first to third highest seed yield was observed in the Omid and Sina and Mutant No. 8 respectively with an average of 267.3, 242.2 and 233.3 kg/ha. The lowest yield belonged to Padideh and Golmehr cultivars (65.3 and 73.4 kg/ha, respectively).

This work is concentrated on designing of CO2 methanation process over nickel-based catalysts doped by different promoters (CuO, Cr2O3, Co3O4, Fe2O3, and MnO2) supported on CaO.2Al2O3 powder. The CaO-Al2O3 powder was fabricated by mechanochemical method, and catalysts were synthesized via a wet impregnation procedure. The characterization properties of all samples were tested by XRD, BET, TPR, FESEM, and EDS-mapping analyses. The optimum amount of NiO was selected at 10 wt% along with CaO:Al2O3 as support with the molar ratio of 1:2. The Co-doped catalyst showed the lowest pore size (9.4 nm) and the highest surface area (106 m2.g−1) among the promoted catalysts. The results exhibited that the Co3O4(3)-NiO(10)/CaO.2Al2O3 catalyst possessed superior catalytic performance in the CO2 methanation reaction. The results illustrated that the increase in cobalt oxide percentage up to 3 wt% improved catalytic performance. However, further augmentation in the Co3O4 from 3 to 7 wt% diminished the specific surface area. According to the performance tests, carbon dioxide conversion and methane selectivity were 83.1% and 99.5% at 450 °C under operating conditions (GHSV = 18,000 ml.gcat−1.h−1 and H2:CO2 = 4:1) over this catalyst. The processing conditions including calcination temperature, reduction temperature, GHSV, feed ratio, and long-term stability test were also examined over the selected catalyst. Outcomes revealed that CO2 conversion increased with a decrement in calcination temperature from 650° to 450°C. Also, chosen catalyst reduced at 650 °C showed the highest performance. Besides, catalytic performance enhanced by raising feed ratio (H2:CO2) from 2:1 to 5:1 and decreased with augmentation of GHSV from 12,000 to 30,000 ml.gcat−1.h−1. Moreover, Co3O4(3)-NiO(10)/CaO.2Al2O3 catalyst possessed high stability during 14 h time on stream.

A series of Bi2WO6/TiO2 composite photocatalysts with different ratios of Bi2WO6 nanosheets to TiO2 nanobelts (1, ½, ⅓ and ¼) were synthesized by hydrothermal method and evaluated for CO2 photoreduction. The structural and optical properties of these photocatalysts were investigated by XRD, N2-physisorption, EDX, FESEM, Raman, TPD, DRS and PL. Bi2WO6/TiO₂ composite photocatalysts show enhanced CO2 adsorption capacity and coversion performance compared to both pure TiO₂ and bulk Bi2WO6. Loading Bi2WO6 nanosheets on TiO2 nanobelts leads to formation a heterojunction, which enhances the photocatalytic visible light performance by decreasing the recombination rate of photoinduced electron-hole pairs. The maximum CH4 production of 18.95 μmol/gcat in 8 h has been reported for composite with Bi2WO6/TiO₂ ratio of ½, which is 3 times higher than that of bulk Bi2WO6 and pure TiO2.

ObjectiveThe omp (Outer membrane protein) protein located at outer surface of the bacterial membrane body agent citrus greening disease (Candidatus Liberibacter asiaticus). The mentioned-protein can be used as antigen for generating polyclonal and monoclonal antibodies. Therefore, the purification of membrane proteins is exposed to many problems due to presence of hydrophilic structure. The purpose of this study was optimizing recovery and purification of the recombinant omp protein.Materials and MethodsThe pET28a bacterial expression construct containing omp gene was used to recombinant production of the mentioned-protein. The omp gene expression was conducted in E. coli strain Rosetta-Gami2. With considering to be hydrophilic of omp protein, its purification was investigated under denature condition. To attain it, the different concentrations of NaCL, urea and guanidine at the different acidity pH condition was applied. To obtain the pure omp recombinant protein, the protein recovery from polyacrylamide gel was performed by methods based on elution by diffusion, sonication, and electroelution. The evaluation of quality and quantity purified protein was analyzed by SDS-PAGE and the protein concentration was calculated by ImageJ software. Also, to validate the purified proteins, Western blot was applied by specific antibody.ResultsPrimary results showed that using the 500 mM NaCL was detected as an essential share in purification buffers. Also, using urea as denature agent had twice the efficiency on the quantity of purified protein more than guanidine hydrocholoride and thus, when 8 m urea supplemented in purification buffer, the highest purified protein was attained (450 µl/ml). Furthermore, the results related to recovery the single band of protein from gel indicated that the electroelution has highest efficient on recovery of the target protein from gel matrix compared to sonication and elution by diffusion methods. The results of SDS-PAGE confirmed to appear a protein with 34 kDa related to molecular weight of target protein. Western blot validated the recombinant protein in membrane. ConclusionsThe current study showed that using urea in 8 M, NaCL in 500 mM concentration in purification buffers following adjusting the elution buffer on pH (4) in elution purification step can be apply as an optimized condition to attain the highest target protein concentration. Furthermore, electroelution method introduced as efficient method in order to recovery band of the target protein from polyacrylamide gel matrix.

Since the Persian Gulf is a semi-closed water ecosystem, climate change has more severe impacts on organisms, especially on non-motile ones. The harmful effects on organisms can be studied at all biological levels of morphology, anatomy, physiology, to cellular and molecular levels. The collapse of the symbiotic relationship between coral and its symbiont algae can result in bleaching and it is the first effect of changing favorable conditions on hard coral. The cells produce Heat Shock Proteins (hereinafter referred to as HSP) in response to heat stress. The rate and way of HSP production in corals depend on the expression of the genes encoding these proteins. In this study, we investigated the expression of heat shock gene HSP70 in response to elevated temperature in Acropora downingi (Wallace, 1999) coral reef. After sampling hard corals in March 2018 and adapting them to laboratory conditions, their ambient water temperature increased to 9 °C (from 25 °C to 34 °C). The samples were adapted to laboratory conditions for 2 weeks after being transferred to the laboratory. The water temperature was 25 °C under control conditions, which was increased to 34 °C to create thermal stress. The expression of the gene Hsp70 was evaluated at control time and also 24 h and 48 h after temperature rise. The study showed that gene expression was significantly increased 24 h after stress and it decreased at 48. However, the corals were bleached, which could be due to the deterioration and reduced resistance to heat shock.

Climate change due to global warming is one of the worst environmental disasters in the world, which affects all ecosystems and has led to increasing degradation of coral reefs. The increase of sea surface temperature is inversely related to the resistance of corals and is directly associated with their bleaching. High temperature disrupts the symbiotic relationship between coral and algal symbiont and results in coral bleaching. To evaluate the adaptation of corals to heat stress, in this study, we investigated the thermal stress effect on the expression of genes involved in programmed cell death (PCD), cysteinyl aspartate proteases 3 (will be mentioned as Caspas3 hereafter) and anti-apoptotic pathway, B-cell lymphoma 2 (will be mentioned as Bcl2 hereafter) in Porites lobata (Dana, 1846). Corals were incubated at 25°C for 2 weeks (adaptation period) and then exposed to 34°C (heat shock) for 24 and 48 hours. Then, the expression of genes was measured using real-time PCR. The results revealed that both genes were up-regulated at 24 hours after heat induction. Bcl-2 expression (anti-apoptotic gene) was induced at 24 hours and was down-regulated at 48 hours. In contrast, Caspase3 (apoptotic gene) continued to be expressed up to 48 hours. These results might indicate that coral cells are headed towards bleaching and death with increased temperature. The results of this study, regarding the observed expression patterns, can clarify the response of different genes to a thermal stress in coral reefs. The exposure of corals to acute conditions with high temperatures presented the behavior of the desired genes in the studied conditions.

Introduction: Human Serum Albumin (HSA) is one of the most important proteins in blood that can bind a wide range of components and different drugs such as Warfarin and is also circulated in the body by HSA. Therefore, studying HSA is very significant in pharmacology. In this research, dynamic behavior of residues of Warfain binding site of HSA has been investigated. Methods: Firstly, PDB format of HSA was downloaded from RCSB (PDB ID:2BX8) which was in complex with Azapropazone. After that, molecular dynamics simulation was during 30ns by GROMACS package. CHIMERA software was used to discover residues of binding site. Results: After RMSD analysis was done on residues of binding site, it was seen that Arginines 186 and 218 have a wide fluctuations in their RMSD plot. Also, two Lysines 185 & 190 have nearly wide fluctuations of RMSD, this fluctuations were less than Arginine's, however. Other residues such as Glycine 189 have few fluctuations. Fluctuations in RMSD have direct relationship with accessible surface area (ASA) of the residues. Conclusions: The results show that the Warfarin binding site in HSA can have various conformational situations because of the dynamics of its residues. This problem can be important to drug design. Overall, according to the results in this study, residues have been divided into 3 categories. Among them, Tryptophan 214 according to different articles is one of the most important residues site and based on our result, it stays on category of small mobility.

The Xanthomonas citri pv. citri (Xcc) is causal agent of bacterial citrus canker which is major disease of citrus throughout the world. The pthA bacterial effector protein is presented within the infected plants and indispensable of canker. The scFv antibodies are valuable tools for diagnosis and suppression of pathogens within plants. The present article describes developing and characterization of specific recombinant monoclonal scFv antibodies against pthA effector protein. For this aim, the gene encoding pthA protein was heterologously expressed in Escherichia coli and used for screening of Tomlinson phage display antibody library to pinpoint specific single chain variable fragment (scFv). In each round of panning, the affinity of phage towards pthA was checked by enzyme linked immunosorbent assay (ELISA). The data was indicative of about 50% of the monoclonal phages to be reactive strongly against pthA protein. Among the positive clones, 5 samples (A12, B8, C1, H8 and G8) were capable of detecting Xcc-infected plant samples and recombinant pthA protein. Restriction fragment length polymorphism showed similar banding pattern for all 5 scFvs as renamed to pthA-scFG8. HB2151 E. coli cells were infected by the phage bearing pthA-scFG8, and the expression of the peptide was induced by IPTG to produce a 30 kDa recombinant molecule. I-TASSER was used for homology modeling of both scFv and pthA and docking was carried out by Hex program. The latter demonstrated binding energy of −784 kcal/mol in scFv-pthA.

Introduction: Xanthan is an extracellular polysaccharide produced by Xanthomonas genus. Because of high viscosity and other properties, this gum is used in various industries. Hence, the use of cheap carbon sources such as whey can be economically feasible to produce Xanthan gum with positive lactose strains of Xanthomonas. Materials and Methods: In this study, the native strain of Xanthmonas citri 386 was used to produce xanthan gum in cheese whey medium. The strain was cultured in a yeast extract lactose broth (YL) and then inoculated into production medium, the fermentation process was investigated in 5 days’ time in terms of production variables such as growth, consumption of lactose, viscosity and amount of xanthan. The content of pyruvate and acetate of product were compared with standard xanthan and the FTIR method was used to determine the functional groups. Results: The strain used was the ability to use lactose in cheese whey. At the end of the fermentation process, the amount and viscosity of xanthan were estimated to be 20.3 g/L and 2066.5 centipoise respectively. The acetate and pyruvate content was acceptable in comparison with standard xanthan, and the FTIR study confirmed the position of the functional groups of the xanthan structure. Conclusion: In this study, for the first time, the native isolate of Xanthomonas citri 386 was evaluated for the production of xanthan in whey. This strain showed high potential for lactose consumption in whey and produced a significant amount of xanthan with an optimum viscosity. Therefore, at an industrial scale, the use of this native isolate, can be suitable for the production of xanthan by low-priced whey medium. Keywords: Xanthan gum, Xanthomonas citri, cheese whey, acetate and Pyruvate

Huanglongbing (HLB) also known as citrus greening, is a destructive disease of citrus and now, is considered as a new emergence and spread out threat to the Middle East and North Africa (MENA) citrus production. In a survey conducted in southern Iran in 2013-2014, 77 citrus samples exhibiting symptoms of HLB were collected. Single-step and nested polymerase chain reactions (PCR) were employed to determine the presence of the phloem-limited bacterial pathogens ‘Candidatus Liberibacter asiaticus’(CLas), phytoplasma and Spiroplasma citri. Both CLas and phytoplasma were detected in HLB-affected citrus trees as co-infection (7.79%) and single infection (10.38% for phytoplasma and 42.85% for CLas). According to the proposed 16S rDNA-based phytoplasma classification scheme, the HLB-associated phytoplasma from this study was a member of peanut witches’broom (16SrII) phytoplasma group. This is the first report of association of a phytoplasma with HLB in sweet lime in the world and first record of association of CLas with sour orange (Citrus aurantium L.) and sweet lime in Iran.

Asiatic citrus canker caused by Xanthomonas citri subsp. citri is becoming a disease of high economic impact, affecting all types of important citrus crops. In this study, the potential antibacterial activity of ten organic and inorganic salts on X. citri subsp. citri and on citrus canker disease development was evaluated. Among the salt compounds, copper, iron and zinc inorganic salts particularly zinc (with the highest diameter of inhibition, the lowest MIC and MBC values and the highest bacterial growth inhibitory effect) had direct antibacterial activity and strongly reduced the development of canker disease and bacterial population of lime plants.

Introduction: Xanthan is a heteropolysaccharide that is produced by the group of plant pathogen bacteria from Xanthomonas genus. Usually, the media containing glucose, sucrose and starch is used for xanthan production. Because their preparation and transferring is expensive, the final cost of xanthan production by these carbon sources is high. On the other hand, many Xanthomonas species such as X.campestris are not able to use cheap media rich of lactose such as whey to produce xanthan due to the low expression and sometimes the defect of their β-galactosidase enzyme. The access to bacterial strains capable of decomposing lactose will provide a possibility for producing a valuable commercial product (xanthan) from an inexpensive carbon source (whey).Materials and methods: In the present study, a collection containing 210 isolated Xanthomonas citrisubsp.citri Iranian strains from citrus orchards in south Iran was studied. Then, the genus of these bacteria was determined by using molecular techniques and sequencing of 16S-rDNA gene. Also, their growth in lactose – based medium was investigated.Results: Among 210 strains, 27 strains were able to grow on lactose rich medium. Then, the genus of these bacteria was proved by sequencing of 16S-rDNA gene and comparison with another 16S-rDNA gene sequences existing in NCBI. Also, these bacteria had considerable growth in lactose-based medium. Discussion and conclusion: At last, we can say that separated lactose-positive Xanthomonas strains from southern citrus orchards have good ability to utilize lactose and in the near future, it would be possible to apply these native strains for xanthan production in cheaper lactose media such as whey.

Asiatic citrus canker is a devastating disease resulting in drastic economic losses in citriculture worldwide. Amongst three different types of the disease, i.e. A, A* and Aw, the A* type is genetically less known. In order to comprehend the behavior of the Asiatic citrus canker A*-type strain (Xanthomonas citri subsp. citri) in the vicinity of the host cells, a targeted semi-quantitative transcript analysis approach via RT-PCR was carried out. A subset of sixteen genes, as representative of different steps involved in phytopathogencity, was analyzed on the culture medium (as uninduced) and compared with the subset isolated from the infected Mexican lime (Citrus auarntifolia L.) plants (as induced). The results showed that certain genes were up-regulated in induced condition, suggesting a putative role in bacteria-host interaction. Furthermore, the transcripts in induced condition could be classified into constitutive, early- and late-responsive genes, demonstrating their functional relevance during the host-pathogen interaction.