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The beet cyst nematode (BCN) (Heterodera schachtii), and beet curly top virus-severe (BCTV-Svr) (Curtovirus betae) are two important pathogens of sugar beet fields worldwide. Therefore, the reaction of 14 genotypes was separately assessed, using Jolgeh and Sanetta cultivars as susceptible and resistant controls, respectively, in completely randomized design experiments for BCN and BCTV-Svr. Reactions were based on the cyst and egg counts and symptoms severity index. Experiments were performed in the greenhouse of Tarbiat Modares University, Tehran, Iran, and were repeated twice independently. Based on the results of initial experiments, the S1-960090, S1-940324, S1-960294, and S1-960284 genotypes resistant to the BCN were selected for further investigation. Furthermore, the reaction of the four selected genotypes to BCN, BCTV-Svr, and the combination (mixture) of the two pathogens was evaluated by analyzing their growth, physiological, and biochemical characteristics, and virus accumulation. Resistant genotypes showed higher levels of defense-related enzymes such as catalase, guaiacol peroxidase, and polyphenol oxidase, whereas susceptible genotypes exhibited significant reductions in photosynthesis, greenness, and chlorophyll a, b, and carotenoid content compared to non-inoculated and resistant plants. This is the first study conducted to search for dual-resistance sources against two devastating pathogens that frequently occur in the sugar beet-growing regions of Iran. Based on the results of this experiment, genotypes S1-960090 and S1-940324 were identified as resistant to both pathogens and are recommended for breeding purposes.
 

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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.

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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 A^w, 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.    

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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.

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Macrophomina phaseolina is one of the major yield limiting factors of melons in tropical and subtropical regions. For eco-friendly and effective management of the disease, 24 gamma induced mutants from Trichoderma harzianum were evaluated against three isolates of the pathogen representing three geographically different regions viz. Khorasan (isolate 1), Garmsar (isolate 2) and Khuzestan (isolate 3). The isolates of Trichoderma (mutants and wild type) were evaluated against the pathogen in dual culture and through production of volatile and non-volatile inhibitors. Maximum growth inhibition was observed in Th1, Th4, Th15, Th9 and Th22 mutants after three days. In greenhouse evaluation against M. phaseolina (isolate 1) among the inoculated treatments minimum plant infection was observed in Th9 treatment (28% disease reduction) as compared to infected control and among the uninoculated treatments Th1and Th9 mutants resulted in maximum growth of roots and shoots of melon plants as compared to uninfected control. These mutants are introduced as potential candidates against M. Phaseolina. The results proved that gamma-mutagenesis by enhancing the antagonistic properties of T. harzianum 65 can be useful for the biocontrol of soil borne plant pathogens such as Macrophomina phaseolina.

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Stem rust caused by Puccinia graminis f. sp. tritici (Pgt) is one of the destructive diseases of wheat in the world. The fungal pathogen can infect 365 different grass and more than 70 Berberis species. DNA sequences for the ribosomal RNA internal transcribed spacer (ITS) have proven suitable to explore relationships at the species and subspecies levels. An isolate of Pgt which was collected from Iran and designated as TTSSK was used in this study. Three samples of the isolate were used. ITS region of the samples was amplified and sequenced. Consensus tree based on Maximum Parsimony clustering method was produced by Mega 6.0. Iranian isolate of TTSSK was placed in a clade with P. graminis which was isolated from Berberis sp. and Pgt isolate from Tajikistan. The results showed that more than one conserved genomic regions would be informative to identify phylogenetic relationship of Iranian Pgt isolates and samples from different parts of the world. Complementary studies with more sequence data from other genome loci are in progress.

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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.  

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One of the best strategies to control bacterial wilt caused by Ralstonia solanacearum (Smith) is generally based on breeding resistant cultivars. The information obtained from the expression of plant defense genes will provide new insight for improving plant resistance against pathogens. This study was to identify inducible genes under defense no death (DND) reaction of tobacco (Nicotiana tabacum)-R. solanacearum interaction using cDNA-AFLP technique. In this assay five different primer combinations were used. Out of 1320 Transcript derived fragments (TDF) that were detected, 101 fragments were identified as differentially expressed genes in 0, 24, 48 and 72 hours post inoculation. Most of the differentially expressed genes were obtained 48 hours post inoculation. Following sequencing, most of sequenced TDFs showed homology to known genes interfering in signaling, regulation and defense functions. DND phenotype in tobacco has some similarities specially in signaling process with mechanism associated with induction of the hypersensitive reaction and it is distinct from general defense mechanisms.
 

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Surfactin is one of the most efficient biosurfactants excreted by Bacillus subtilis which displays the highest potential as induced systemic resistance elicitor among all metabolites produced by B. subtilis. Environmental factors have considerable effect on surfactin production. In this study surfactin production of two Bacillus subtilis strains were analyzed using high performance liquid chromatography (HPLC). C14 and C15 surfactins were detected in the ethanol extract from acid-precipitated supernatant. HPLC analyses of different media including Nutrient Broth (NB) medium, NB plus 40g/l glucose, NB plus 10% soil extract and NB plus 10% plant extract medium, clearly showed that these bacteria produced different amounts of surfactins C14 and C15 in these media. Surfactin production in NB/plant medium was relatively the highest in quantity. Microelements analysis of media containing plant and soil extract with atomic absorption spectrometry showed high amounts of Fe, Mn and Zn in medium containing plant extract compared with that of soil extract. Since these elements play an important role in surfactin production, high amounts of Fe, Mn and Zn in NB/plant extract medium compared to the NB/soil extract medium could be the possible reason for relatively higher amounts of surfactins C14 and C15 produced in NB/plant medium. So adding these important elements to soil may boost biocontrol effect of B. subtilis against plant pathogens.

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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 (G_ST) and high gene flow (N_m) among populations had a significant effect on the emergence and evolutionary development of M. phaseolina.
 
 

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Curly top is one of the most important viral diseases of sugar beet. Use of resistance sources is a promising strategy for control of this disease. In the present study, the efficiency of four gene silencing constructs (OUT-hp، IN-hp، sense and antisense) against two major causes of curly top disease in Iran, beet curly top virus-Svr (BCTV) and beet curly top Iran virus (BCTIV), were evaluated in transgenic plants. Selection of transgenic plant seeds was carried out on selective medium 1/2MS containing glufosinate-ammonium (Basta) and the results showed that the pBCTV-IN-hp construct resulted in the highest germinated seeds. Selected plants were transferred to greenhouse and evaluated for resistance to basta and detection of silencing constructs in the transgenic plants. Afterwards, resistance of the selected transgenic plants to beet curly top viruses and resistance stability against cucumber mosaic virus (CMV) was evaluated in a completely randomized design with six treatments in a factorial experiment. The results showed that the transformed lines with each of four constructs were significantly different in severity of symptoms, plant height and number of flowering stems compared to their respective controls. Although these transgenic plants were resistant to BCTV-Svr and BCTIV, in their challenge inoculation experiments it was shown that this resistance was suppressed by CMV infection. 
 

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The effect of various pesticides (diflubenzuro, malathion, mancozeb and carbendazim), disinfectants (calcium hypochlorite and formaldehyde) and oil cakes (sunflower and soy-bean oil cakes) commonly used as supplements in mushroom cultivation on the growth of the nematophagous fungus, Arthrobotrys oligospora, was studied under in vitro conditions. Carbenazim caused 99% inhibition of radial mycelial growth in Petri dishes at all concen-trations tested (10-40 µg a. i. ml-1) in comparison to non treated dishes. Mancozeb caused 43% and 23% inhibition at 250 and 500 µg a. i. ml-1 respectively and 99% inhibition at concentration of 1000 µg a. i. ml-1 and above. Diflubenzuro and malathion at 10-40 µg a. i. ml-1 caused 30-41% and 24-54% inhibition, respectively. Formalin (0.5-2.0% v/v) inhib-ited growth of A. oligospora completely. However, calcium hypochlorite, sunflower and soybean oil cake at concentrations of up to 2.0% w/v caused less than 3.5% inhibition.

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In an in silico investigation, genome sequences of three RNA viruses were identified in the crown imperial Fritillaria imperialis L. transcriptome dataset. Sequence comparison and phylogenetic analyses revealed that these three novel viruses belong to the genus Amalgavirus. They were tentatively named Crown imperial amalgavirus (CIAV), Iranian amalgavirus (IrAV), and Koohrang amalgavirus (KAV). The RNA-dependent RNA polymerases (RdRps) of CIAV, IrAV, and KAV showed 69.53%, 42.26%, and 37.46% amino acid sequence identities with the homologous RdRp of the most closely related virus, respectively, suggesting that they are novel viruses. Also, the conserved motifs of RdRp were detected in the RdRp of each CIAV, IrAV, and KAV. Genomes of both CIAV and IrAV were complete and contained two overlapping Open Reading Frames (ORFs). A + 1 programmed ribosomal frameshifting (PRF) motif, which matches the conserved amalgaviruses consensus sequence UUU_CGN was found at the ORF1/ORF2 boundary of CIAV and IrAV. The current study reports three novel viruses for the first time from crown imperial, and these findings enrich our understanding of new plant dsRNA virus species, which may also be helpful for the study of amalgaviruses.


 

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The incidence of grapevine virus A (GVA) is reported from almost all of the major grapevine growing regions in Iran. Grapevine is vegetatively propagated by rooting of cuttings or grafting. In such plants, viral diseases are transmitted from stock plants to the progeny. Therefore, the control of grapevine viruses can be achieved primarily through production of healthy stock plants. In the present research, cryotherapy and electrotherapy were employed for elimination of GVA from naturally infected vine (Vitis vinifera L. cv Black) and their efficiency was compared. In cryotherapy, 59% of the shoot tips survived and regenerated into whole plants, of which 42% were free of GVA detected by RT-PCR. In the electrotherapy, the effects of electric current value and treatment duration were investigated on plant survival and virus elimination. The best results were obtained by using 30 milliamps (mA) for 15 minutes. With this treatment, survival and virus-free frequencies were about 62% and 40%, respectively. This is the first report of electrotherapy of grapevine shoot tips as a potential tool for GVA elimination. The results showed that cryotherapy was a more efficient and convenient protocol than electrotherapy for elimination of GVA from infected grapevine.

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Genetic structure and pathogenic diversity of Sclerotinia sclerotiorum, the causal agent of canola white stem rot, were assessed through Mycalial Compatibility Groupings (MCGs), a comparison and comparing of isolate virulence. Fifty-seven isolates from three different regions in Golestan Province were selected for mycelial compatibility and as well for virulence tests. Within the 57 tested isolates, 35 MCGs were identified, 42.86% of which being constituted of single isolate specimens, were all collected from Ali Abad region. The observed MCGs differed within the three regions. From among the 35 MCGs, 25.71%, 28.57% and 45.72% belonged to Kalaleh, Hashem Abad and Ali Abad, respectively. In Kalaleh, nine MCGs were identified all of which fell into two isolates. Ten MCGs were identified within the Hashem Abad region, eight of which represented two isolates and the remaining were constituted from three isolates. Sixteen MCGs were detected in Ali Abad for which, except one MCG which was constituted of two isolates, the rest belonged to one isolate. Moreover, no MCG was identified as common among these regions. Shannon diversity index (Ho) of MCGs for the whole regions found to be was 0.86 (Htot). Partition of total diversity (Htot) showed that 95.45% corresponded to a variation in diversity within S. sclerotiorum populations. Variation in isolate virulence was tested using a petiole inoculation technique under greenhouse conditions. Isolate virulence varied within the three regions. Moreover, in most cases the differences in virulence of isolates within MCGs were significant. The data indicated that populations of S. sclerotiorum obtained from the studied regions were composed of a heterogeneous mix of MCGs, therefore the population structure of this pathogen as well as variations in virulence of isolates must be considered in disease management systems in these regions.

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To study molecular evolutionary characteristics and genetics of beet necrotic yellow vein virus (BNYVV) isolates population from Iran, nucleotide sequences of p25 and coat protein (CP) were determined and the amino acids sequences thus deduced were analyzed using phylogenetic and population genetics methods. A survey of BNYVV in Iran indicated the infection of 288 collected samples out of 392 samples in most beet growing areas and that most of the isolates (92%) were of the A-type and the rest of isolates (8%) were P-type. Our molecular evolutionary analysis showed that CP was highly conserved but allowed to assign all isolates to three distinct groups. Different parts of p25 coding regions were under different evolutionary constraints. The most positive selection was detected at the position 68, the second amino acid of the tetrad motif. Iranian isolates were found to cluster with European isolates into three distinct clusters based on p25 sequences. Population genetics analysis revealed that BNYVV populations have low differentiation (Kt= 3.97145) and low diversity (πT= 0.006, Hd= 0.860) with frequent gene flow indicating lack of phylogeographic structure between populations.

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Bacterial leaf streak (BLS) caused by Xanthomonas translucens pv. cerealis (Xtc) is an important disease of wheat (Triticum aestivum L.) worldwide. The management methods presently in practice are insufficient to meet current safety and/or efficacy standards. Therefore, use of resistant genotypes is the best approach to manage BLS. The present study was undertaken to identify possible sources of resistance to Xtc in cereal cultivars and germplasm. Twelve strains of Xtc were isolated from symptomatic leaves in several regions in Kerman province. Out of twelve, nine strains produced the expected Xtc-specific 120 bp fragment using PCR and the primer pairs PABr/PBf. Six strains produced water-soaked streaks covered with exudates on wheat cultivars, whereas the three remaining strains incited only chlorotic streaks with no water-soaking on leaves. A highly virulent strain that caused conspicuous water-soaking and necrosis was used for inoculation of 645 winter and spring wheat, barley, and rye accessions to identify possible sources of resistance to BLS. The fourth leaves of test plants were infiltrated with bacterial suspension and scored after seven to ten days. Among all the accessions evaluated, only two rye accessions, namely, 4538 and 4794, were resistant to BLS. These two rye accessions can potentially be used in breeding rye and triticale cultivars for resistance to BLS.

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Macrophomina phaseolina is an important soil-borne pathogen causing charcoal rot in many important crop plants including sesame, in Iran. A total of 60 isolates of M. phaseolina were collected from the main sesame producing regions in ten provinces of Iran. The genetic diversity among M. phaseolina populations was estimated using Inter-Simple Sequence Repeat (ISSR), focusing particularly on geographic differentiation. Five ISSR primers generated 105 discernible DNA bands, of which 85 (77.11%) were polymorphic. The greatest value of variability (PPB: 60.00%; H: 0.185; I: 0.284) was estimated for Fars population, whereas the least variability (PPB: 9.52%; H: 0.042; I: 0.060) was estimated for Kerman population. Total gene diversity exhibited high levels of variability (H_T = 0.186). Analysis of molecular variance indicated a large proportion of genetic variability within populations.

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Apple Stem Pitting Virus (ASPV) is one of the most common viruses of apple and pear trees. During 2012 to 2013, a total of 1053 symptomatic and asymptomatic leaf samples were collected from orchards in nine provinces of Iran. ASPV infection was detected by DAS-ELISA and PCR in 54 samples (5.12%) from seven provinces. Based on the geographical origin, 22 representative isolates were selected for phylogenetic analysis. Twenty-two amplicons with a size of about 370 base pair (bp) comprising partial sequence of the Coat Protein (CP) coding regions of the viral genome were sequenced. Sequence data analysis, showed that the identities of 3'-terminal region of CP gene at both nucleotide and amino acid levels among the Iranian isolates were 95–100% and these isolates were closer to the Asian ASPV isolates than to the other isolates. Constructed phylogenetic tree by Neighbor-joining on the basis of the 3'-terminal region of CP gene sequences showed that the Iranian isolates were categorized into two major groups. Furthermore, phylogenetic and population genetic analysis were carried out on the basis of 3'-terminal region of CP gene which revealed that ASPV isolates were not geographically resolved. Also, all values in the GABranch analysis showed a ratio of substitution rates at Non-synonymous and Synonymous sites (dN/dS) below one, suggesting strong negative selection forces during C-terminal region of the ASPV CP gene history. To the best of our knowledge, this is the first report of distribution and partial genome sequence analysis of the ASPV in Iran.

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Trichoderma species are known as effective agents used for biological control of plant pathogenic fungi. The Trichoderma harzianum and its mutant isolates were cultured and their traits including, mycelial growth, antagonistic activity and extracellular proteins and enzymes production (Chitinase and Cellulase) were investigated to select the most effective mutant isolates against plant pathogenic fungus Rhizoctonia solani. Also, the purity and composition of enzyme-rich protein samples were evaluated under denaturing gel electrophoresis. This study clearly showed the possibility of improving mycelia growth rate (from 1.18 to 1.33 cm d^-1), the antagonistic capability of Trichoderma (from 54.9% growth inhibition of R. solani to 66%), extracellular proteins and enzymes production for biological control of plant diseases through mutation with γ-radiation. Also, compared to wild type strain, protein production in the mutant isolates increased. Moreover, the highest specific chitinase enzyme activities were observed in mutant isolates T. h M8 (42.48 U mg^-1) and T. h M15 (38.25 U mg^-1). Trichoderma mutant of T. h M8 maintained higher mycelia growth rate and higher ability to inhibit growth of R. solani. The SDS-PAGE profiles had several enzyme protein bands such as CelloBioHydrolases (CBHs), EndoGlucanases (EGs), β-Glucosidases (BGLs), endochitinases, and β-(1, 4)-N-acetyl glucoaminidases. SDS-PAGE analysis indicated the presence of different protein bands in the range of 10.5 to 245 KDa. Interestingly, expression of chitinase in 95 percent of mutants was higher than wild type of T. harzianum. The results showed that gamma mutation could increase the efficiency and amount of enzymes in T. harzianum, while these enzymes are involved in antagonistic properties of T. harzianum.