Developing of specific monoclonal recombinant antibody fused to alkaline phosphatase (AP) for one-step detection of fig mosaic virus.

Present study was performed to develop a fusion recombinant monoclonal antibody for one-step and accurate detection of FMV with a specific single-chain variable fragment (scFv) fused to alkaline phosphatase (AP) named as scFv(FMV-NP)-AP. The gene encoding-specific scFv recombinant antibody binding to nucleocapsid protein of Fig Mosaic Virus (FMV-NP) was fused to upstream of AP gene and integrated in pET26b bacterial expression vector. As vector contain pelB signal peptide, the expressed protein is secreted into periplasmic compartment. Recombinant fusion protein was produced in transformed E. coli following induction by IPTG. Extraction and purification of fusion protein was performed under denatured condition. The results of SDS-PAGE and western blot analysis indicated high integrity and purity with a single band protein with expected size of 72 kDa. The total yield of purified scFv(FMV-NP)-AP fusion protein estimated around 0.5-1 mg/l cultured medium. Subsequent colorimetric analysis confirmed presence of alkaline phosphatase activity in prepared scFv-AP fusion protein. Specificity of generated recombinant fusion antibody against cognate antigen and the native virus presented in infected plant extracts was assessed by ELISA, western blot and dot blot assays. Results revealed that scFv(FMV-NP)-AP is able to detect the presence of FMV in infected fig plants. The novel approach, implementing specific recombinant fusion antibody developed in this research, leads to one-step detection of FMV in plants by avoiding the use of chemical enzyme-labeled secondary antibodies.

Tissue-specific synergistic bio-priming of pepper by two Streptomyces species against Phytophthora capsici.

Among several studied strains, Streptomyces rochei IT20 and S. vinaceusdrappus SS14 showed a high level of inhibitory effect against Phytophthora capsici, the causal agent of pepper blight. The effect of two mentioned superior antagonists, as single or combination treatments, on suppression of stem and fruit blight diseases and reproductive growth promotion was investigated in pepper. To explore the induced plant defense reactions, ROS generation and transcriptional changes of selected genes in leaf and fruit tissues of the plant were evaluated. The plants exposed to the combination of two species responded differently in terms of H2O2 accumulation and expression ratio of GST gene compared to single treatments upon pathogen inoculation. Besides, the increment of shoot length, flowering, and fruit weight were observed in healthy plants compared to control. Likely, these changes depended on the coordinated relationships between PR1, ACCO genes and transcription factors WRKY40 enhanced after pathogen challenge. Our findings indicate that appropriate tissue of the host plant is required for inducing Streptomyces-based priming and relied on the up-regulation of SUS and differential regulation of ethylene-dependent genes.

Streptomyces Strains Induce Resistance to Fusarium oxysporum f. sp. lycopersici Race 3 in Tomato Through Different Molecular Mechanisms.

Plant growth promoting rhizobacteria (PGPR) are potential natural alternatives to chemical fungicides in greenhouse production via inducing plant immune system against biotic stresses. In this research, 126 Streptomyces isolates were recovered from rhizosphere soils of 13 different commercial vegetable greenhouses in Iran. Streptomyces isolates were screened for in vitro Plant growth promoting (PGP) traits and ability to antagonize Fusarium oxysporum f. sp. lycopersici race 3 (FOL), the causal agent of Fusarium wilt of tomato (FWT). Six isolates with the highest antagonistic activity and at least three PGP traits were selected and compared with chemical fungicide Carbendazim® in a greenhouse experiment. All bacterial treatments mitigated FWT disease symptoms like chlorosis, stunting and wilting at the same level or better than Carbendazim®. Strains IC10 and Y28 increased shoot length and shoot fresh and dry weight compared to not inoculated control plants. Phenotypic characterization and 16S rRNA gene sequencing showed, strains IC10 and Y28 were closely related to S. enissocaesilis and S. rochei, respectively. The ability of the superior biocontrol strains to induce antioxidant enzymes activity and systemic resistance (ISR) was investigated. Increased activity of catalase (CAT) in plant treated with both strains as well as an increase in peroxidase (POX) activity in plants treated with Y28 pointed to a strain specific-induced systemic resistance (ss-ISR) in tomato against FOL. The differential induced expression of WRKY70 and ERF1 (two transcription factors involved in plant defense) and LOX and TPX by the analyzed Streptomyces strains, especially after inoculation with FOL, suggests that ss-ISR is triggered at the molecular level.

Localized surface plasmon resonance biosensing of tomato yellow leaf curl virus.

Current techniques for plant virus detection, such as RT- PCR and ELISA, require multistep procedures and rely on sophisticated equipment. Due to the global spread of plant viruses, the development of simpler, faster and cheaper assay methods is inevitable. Gold nanoparticles (AuNPs) had raised much interest during recent years due to their novel optical properties or diagnostic purposes. The localized surface plasmon resonance (LSPR1) of AuNPs had been used in the development of novel colorimetric nano-biosensing systems. The frequency and intensity of the LSPR peak generally depend on the shape, size and the surrounding medium of the AuNPs. In this study, unmodified AuNPs had been used to detect the unamplified Tomato yellow leaf curl virus (TYLCV) genome in infected plants. A specific DNA probe complementary to the coat protein region of virus genome was designed. The extracted total DNA of uninfected and infected plants was mixed with hybridization buffer and the designed probe. The mixture was denatured, annealed and then cooled to room temperature and was followed by AuNPs addition. The color changes in the samples indicating the presence of target virus infections were assessed visually after the addition of salt and confirmed by UV-Vis spectroscopy. The results showed that this strategy allowed for fast and sensitive detection of TYLCV genome and eliminated the need for PCR amplification and detection equipment.