masoud shams

To assess the variability of Macrophomina phaseolina (Tassi) Goid, the causal agent of charcoal rot of Sesame, sixty isolates recovered from ten geographic regions, were analyzed using inter simple sequence repeat (ISSR) and universal rice primer (URP) markers. Isolates were grouped into eight clusters at 78% genetic similarity level. Our results showed that the five ISSR primers produced 105 bands of which 77.11% were polymorphic and eight URP-PCR primers generated 135 bands of which 66.84% were polymorphic. These methods showed a considerable genetic diversity among Iranian isolates, but no correlation was found between genetic diversity and geographical origins of the isolates. Analysis of molecular variance revealed that a large proportion of genetic variability resulted from the differences among isolates within regions. The findings of this study demonstrated that the low-genetic differentiation (GST) and high gene flow (Nm) among populations had a significant effect on the emergence and evolutionary development of M. phaseolina.

Turnip mosaic virus (TuMV) has frequently been reported from canola fields. Cultivation of resistant varieties is the best method to control viral diseases. However, plants that are resistant to a virus may lose their resistance in co-infection with other viruses. Therefore, in the present study the effect of mixed infection of TuMV with Cucumber mosaic virus (CMV) and Cauliflower mosaic virus (CaMV) on the resistance of a tolerant canola cultivar (Karaj 3) and a transgenic canola of RGS003spring variety with hairpin construct and high resistance to TuMV was investigated under greenhouse conditions. Canola plants were inoculated mechanically and then, relative TuMV content was assayed by semi-quantitative RT-PCR. Results demonstrated that mixed infection of TuMV and CaMV did not affect the resistance of transgenic canola plants against TuMV. Also, there was not any significant difference in TuMV content in the presence of CaMV. However, synergistic interaction between TuMV and CMV could overcome the resistance of canola plants to TuMV. The result of semi-quantitative RT-PCR indicated that TuMV accumulation was enhanced in the presence of CMV in transgenic plants, whereas TuMV levels were not affected by co-infection with CMV in Karaj 3. Therefore, it is concluded that for the introduction of TuMV-resistant transgenic plants, it is necessary to design a construct that the resulting transgenic plants would also be resistant to CMVsimultaneously.

Crown gall is one of the most destructive bacterial diseases of plants. Nowadays, special attention has been focused on the biological control of plant diseases as an alternative to chemical control. This study was conducted to better understanding the molecular mechanism of Surfactin on Agrobacterium tumefaciens as a prerequisite for the use of Bacillus as a biocontrol agent of this pathogen. In this study, Tobacco plants (Nicotiana tabacum), IBRC-M10701 strains of A. tumefaciens, and pure commercial Surfactin (25 µm) were used. miRNAs and lox genes have an important effect in the auxin signalling pathway. Using Real-time PCR, the expression level of miR167 and lox gene measured 1, 3, and 6 days after inoculations in the interaction of A. tumifaciens IBRC-M10701 and Surfactin. The expression level of lox gene in Agrobacterium treatment showed 0.47, 22.9, and 61.8 increased. In Surfactin treatment it was 4.6, 3.6 and, 11.6 fold and in Surfactin-Agrobacterium treatment this amount was 4.2, 38.8, and 20.3 fold. In addition, the expression level of nta-miRNA167 in Agrobacterium treatment indicated an increase of 3.4 and 214.4 fold (in the first and third day after inoculation), then decreased to 2.5 fold (in the sixth day), in treatment with Surfactin the amount of expression level was detected as 3.2, 13.2 and 4.5 fold and in Surfactin-Agrobacterium treatment this amount was 1.6, 2.2 and 9.6 fold compared with the control. All of the results indicated the positive effect of Surfactin in suppression the strain IBRC-M10701 of Agrobacterium. The results indicated the key role of Surfactin in biocontrol which reflected the possible use of Bacillus strains producing Surfactin in biological control.

Turnip mosaic virus (TuMV) a species of the genus Potyvirus in the family Potyviridae, is one of the most prevalent viruses of canola fields in Iran. So far, commercial resistant lines or varieties against TuMV have not been known in Iran. In this research, the possibility of inducing resistant to TuMV in transgenic canola lines carrying a short fragment of the coat protein gene of the virus was evaluated. Therefore, a 130 nucleotide fragment of TuMV coat protein gene, as sense or antisense orientations, were cloned in the pFGC5941 vector independently and then were transformed to canola cotyledonary (Hayola R-Line 401 variety) explants using Agrobacterium tumefaciens strain LBA4404. Selection and confirmation of transgenic T0 and T1 lines were carried out via PCR and glufosinate ammonium herbicide (selective marker) resistance assay under greenhouse conditions. After virus inoculation to T1 lines, resistance assay was done by the scoring system. The obtained results confirmed that transgenic lines with sense construction did not show any resistant. However, several transgenic lines with antisense construction showed a different level of resistant to TuMV including delay in symptoms appearance and a decrease in symptoms severity (0-30% of T1 progeny) or symptoms recovery (10-66% of T1 progeny). This is the first report of increased resistant against TuMV via transformation of a short viral fragment sequence with the antisense orientation in canola.

The expanding cultivation of canola (Brassica napus L.) in the world indicates the importance of this plant. Out of 12 reported viruses in canola, Cauliflower mosaic virus (CaMV) and Turnip mosaic virus (TuMV) are of particular importance. Use of resistant cultivars would be the best approach to control losses caused by viral diseases. So far, the reaction of commercial canola cultivars in Iran to CaMV has not been assessed. In this study, 13 commercial varieties of canola were evaluated against a CaMV isolate under greenhouse condition. Analysis of symptom severity index and enzyme-linked immunosorbent assay (ELISA) of inoculated plants showed that Karaj3 cultivar was tolerant to this virus. In addition, due to co-infection of canola fields with CaMV and TuMV, the reaction of commercial canola cultivars to mixed infections with both viruses was evaluated. The obtained results showed that commercial canola cultivars in Iran were susceptible to both CaMV and TuMV. However, Karaj3 cultivar was considered to be moderately susceptible to CaMV and TuMV co-infection compared to other tested cultivars.

Tomato yellow leaf curl disease (TYLCD) is one of the most important diseases of tomato (Solanum lycopersicum L.) plants in tropical, subtropical regions and greenhouses in temperate regions of the world. To produce an antibody against the viral agent of the disease for use in a diagnostic kits, the TYLCV cloned viral movement protein gene was expressed in an expression vector (pET26). The resulting recombinant protein was then injected into a rabbit. The prepared antiserum was used in western blot and ELISA analyses in order to assess its efficiency in virus detection. A 13KD protein band related to viral movement protein was detected in infected plants by using Western blot analysis at 1:1500 antiserum dilutions, while in ELISA tests at 1:1000 dilutions of antiserum, denaturation of the protein could increase the differences between the absorbance values of the infected plants and non-infected plants. However, these differences were not statistically significant. The results showed that the prepared antiserum against Tomato yellow leaf curl virus- movement protein was suitable for western blot analyses, but to be used in ELISA test the antibodies need to be extraction and purification of antibodies.

Serological methods are commonly used methods for detection of viruses. Preparation of pure viral antigens is a crucial step in production of antibodies required for serological studies. In this research the gene encoding coat protein of a Beet western yellows virus (BWYV) isolate from Iran was amplified by PCR and was ligated into a bacterial expression vector (pET26b) to obtain pET-BWYV-CP clone. Escherichia coli BL21 was transformed with pET-BWYV-CP and expression of the recombinant coat protein was induced by IPTG. The expressed recombinant coat proteins were purified and used as an antigen for rabbit immunization. The antiserum was able to detect recombinant coat protein in total protein extracts of induced E. coli BL21 cells in western blot analysis.

Culture filtrates (CF) of two species of the nematophagous fungi, Arthrobotrys oligospora and Arthrobotrys conoidesat three concentrations (25%, 50% and 100%) of stock, were tested on the mortality of second stage juveniles (J2) and egg hatching rate of Meloidogyne incognita and Meloidogyne javanica. Results showed that the percent juvenile mortality was directly proportional to concentration of the filtrates. Egg hatching rate of these nematodes was inversely affected by increasing concentrations. Also CFs had various impacts on the mortality of J2 and egg hatching rate. In case of M. incognita maximum J2 mortality (28.98%) occurred after 24 hours of exposure to A. conoides filtrate at concentration of 100%. The minimum toxicity (12.5% J2 mortality) was recorded for A. oligospora at 25% filtrate concentration. At the same time, the highest rate of J2 mortality of M. javanica (19.18%) belonged to the 100% concentration of A. conoides, while minimum toxicity belonged to 25% concentration of A. oligospora causing 9.09% mortality. Maximum egg hatching rate for M. incognita (30.75%) belonged to control and minimum hatching rate (1.25%) belonged to 100% concentration of A. conoides. The highest hatching rate of M. javanica (36.25%)belonged to control and minimum hatching rate (1.25%) occurred at 100% concentration of A. conoides.

Citrus tristeza virus (CTV) is among the most destructive pathogens of citrus and causes substantial economic losses in citrus-growing industry worldwide. Considering recent distribution of this pathogen and its capability of transmission by existing aphid vectors in Iran, detection of this virus is enforceable for controlling the damage caused by this pathogen in Iran, as one of the major citrus producing countries. Toward this aim, developing a reliable and sensitive detection method such as enzyme- linked immunosorbant assay (ELISA) would be the first step to detect CTV in large scale screenings of field samples. As the serological method requires great amounts of specific antibody, the consequent preparation of a large scale antigen source for immunization process is necessary. In this study the coat protein gene of CTV (CP25) was amplified by polymerase chain reaction from a cloned CP25 gene in pTZ57R/T and subcloned in pET26b expression vector and named pET-CP25. Two Escherichia coli strains of BL21 and Rosetta Gami (DE3) were transformed by pET-CP25. Expression of recombinant protein was induced by IPTG. The authenticity of recombinant protein was confirmed by western immunoblot analysis using a polyclonal antiserum against CTV particles. The results indicated that CTV coat protein gene was expressed in E.coli. This recombinant protein could be used as a source of antigen for immunization process.

The witches’ broom disease of lime caused by Candidatus Phytoplasma aurantifolia, is the most devastating disease of acidian lime in the southern parts of Iran.. At present, no efficient method has been developed for controlling the disease, therefore quarantine approaches such as early detection and subsequent eradication of infected trees is very important. Toward this aim, developing a reliable and sensitive detection method would be the first step to prevent transportation of infected plant materials to other places.. In this study, Immunodominant membrane protein (IMP) of the pathogen was selected as a target for detection and preparation of polyclonal antibody. The IMP is the major protein present on the surface of phytoplasma cells. For this purpose, the DNA region encoding IMP gene was isolated and cloned into pET28a bacterial expression vector. The recombinant protein was expressed in a large scale in Escherichia coli. Purification was performed under native conditions and the purity and integrity of produced recombinant protein were confirmed by western immuno blot analysis using anti His-tag and anti-IMP polyclonal antibodies. The purified recombinant IMP was used for immunization of rabbit. Purification of immunoglobulin was performed by affinity chromatography using protein A column. The purified immunoglobulin was conjugated with the alkaline phosphatase enzyme.. The purified antibodies and conjugates were applied for efficient detection of infected plants in double antibody sandwich enzyme-linked immunosorbent assay (DAS-ELISA) and dot immunosorbent assay (DIBA).. These antibodies were proven to be very powerful tools to detect the Candidatus Phytoplasma aurantifolia in plants..

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.

Beet western yellows virus (BWYV)، a species of the genus Polerovirus in the family Luteoviridae، is an agriculturally important virus infecting over 150 plant species in 23 dicotyledonous families worldwide. A survey of BWYV in canola fields in Golestan and Tehran provinces of Iran using indirect triple antibody sandwich enzyme-linked immunosorbent assay (TAS-ELISA) indicated 8. 3 % infection. The presence of BWYV was confirmed by amplification of the coat protein (CP) gene of the virus via running a reverse transcription-polymerase chain reaction (RT-PCR) on total RNA extracted from ELISA positive leaf tissues. DNA sequences of the BWYV coat protein (CP) gene of seven Iranian isolates were determined and compared at the nucleotide (nt) and amino acid (aa) levels with those of twelve BWYV isolates from different countries deposited in GenBank. Sequence analysis data showed that the identity of BWYV-CP at nt and aa levels among the Iranian isolates were 93. 4 % to 100 % and 93. 2 % to 100 %، respectively. The maximum similarity of isolates at nt and aa levels were 97. 2 and 96. 6 %، which occurred among two Iranian isolates (Ir 8 and Ir 100) and four isolates from France (L39967 and X13063) and England (L39973 and L39970). The recombination analysis among the nineteen isolates including seven Iranian isolates revealed that there was no distinct intra-specific recombination event among BWYV isolates. This is the first report of sequencing and analyzing of the BWYV CP gene of Iranian BWYV isolates.

Tomato yellow leaf curl virus (TYLCV) is a major tomato virus in tropical and subtropical regions. In this study, 134 accessions of Solanum lycopersicum and six accessions of Solanum peruvianum were assessed for resistance to an Iranian isolate of TYLCV. Plants were inoculated using whiteflies (Bemisia tabaci) and the reaction of plants was evaluated based on either disease symptoms or viral DNA amplification. All accessions of S. lycopersicum had demonstrated various degrees of disease symptoms. However, all six accessions of S. peruvianum were resistant and remained symptomless. Phenotypic evaluation was confirmed by amplification of a 670bp TYLCV DNA fragment in all tested accessions of S. lycopersicum. Based on both phenotypic and molecular evaluations, no accession provided complete resistance to TYLCV, whereas nine accessions were assessed as tolerant. The high level of resistance noted in whitefly inoculated accessions of S. peruvianum was not observed in graft inoculated plants of these accessions. The TYLCV DNA fragment was detected five weeks post-inoculation when plants were inoculated by grafting. These results suggested that accessions of S. peruvianum may be merely resistant to vector inoculation of TYLCV.

Due to the restriction of Barley yellow dwarf virus (BYDV)-PAV particles to the phloem tissue and very low virus titers, purification of the virus is difficult. The aim of this study was to prepare antibody against viral coat protein without purifying the virus. To produce recombinant coat protein, the coding sequence was first amplified from a PAV full-length cDNA clone by polymerase chain reaction (PCR), ligated into a vector (pBluescript SK+) to check the sequence, and sub-cloned into an expression vector (pGEX-2T). It was then transformed into Escherichia coli DH5a by electroporation. The open reading frame 3 (ORF3) was linked in-frame to the gene encoding glutathione-S-transferase (GST; 26 kDa) and expression induced by IPTG. The expressed coat protein was purified by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) for use as an immunogen. The antisera to BYDV-PAV recombinant coat protein reacted in Western blot analysis with partially purified BYDV-PAV. These antisera were also used to detect BYDV-PAV by immunogold electron microscopy of thin section of barley tissues. The results indicated that BYDV-PAV coat protein can be produced in high yields by E. coli, which provides the ability of simple purification, and because of proper antigencity, can be exploited for diagnostic applications.