Farajollah Shahriari; Abbas Tanhaeian; Mahdi Akhlaghi; Narges Nazifi
Abstract
Introduction: The most serious diseases of cultivated mushrooms are caused by Pseudomonas tolaasii and Trichoderma harzianum pathogens. A common method of pathogen control on farms worldwide is the application of various chemical pesticides. Many of these substances are unsafe for human and environment. ...
Read More
Introduction: The most serious diseases of cultivated mushrooms are caused by Pseudomonas tolaasii and Trichoderma harzianum pathogens. A common method of pathogen control on farms worldwide is the application of various chemical pesticides. Many of these substances are unsafe for human and environment. The interest in discovering and developing natural antimicrobials has significantly increased due to consumer preferences for foods that are free of chemical residues. So, a major challenge for mushroom growers is to control diseases with alternative compounds such as essential oils and plant extracts. Antimicrobial peptides (AMPs) were also known as potential antibiotic and considered as a new antimicrobial agents. In the present study, the antibacterial and antifungal potential of essential oils, plant extracts and a recombinant peptide were investigated for their pathogenicity. For this purpose, the essential oils of Cuminum cyminum and Zataria multiflora and the plant extracts of Dorema ammoniacum and Ferulago angulata and a recombinant peptide were evaluated for their antimicrobial and antifungal effects on the P. tolaasii and T. harzianum pathogens.
Materials and Methods: In this study, essential oils of C. cyminum and Z. multiflora were extracted by hydrodistillation in a Clevenger-type apparatus. The plant extracts (D. ammoniacum and F. angulata) were prepared using maceration method. Recombinant peptides (Lactoferricin-Lactoferrampin) were produced from Adherent Human Embryonic Kidney 293 (HEK293). The antifungal properties of essential oils, plant extract and AMPs were studied on growth inhibition. Antibacterial activity of substances were tested against P. tolaasii via disk-diffusion method respectively. Minimum inhibitory concentration (MIC), Minimum bactericidal concentration (MBC) and Minimum fungicidal concentration (MFC) to the compounds were studied. All treatments were considered in a completely randomized block design with three replicates.
Results: The results of the antibacterial evaluations showed that the Chimera peptide have remarkable activity against pathogenic bacteria with a mean diameter of the zone inhibition region of 32.2mm, followed by C. cyminum and D. ammoniacum extracts with mean values of 7 and 6 mm respectively. Regarding to antifungal activity of substances isolated from above material, it was found that essential oils of C. cyminum and Z. multiflora completely prevent the growth of T. harzianum, followed by D. ammoniacum and F. angulate with maen values of 26 and 60.6 mm respectively. The lowest antifungal activity was observed for chimera peptide.
Discussion: The cultivated button mushroom is one of the most extensively cultivated mushroom in the world. The most serious diseases of cultivated mushrooms are caused by P. tolaasii and T. harzianum pathogens. The results of our study showed that the C. cyminum and D. ammoniacum have remarkable activity against pathogenic bacteria. Plant extracts, essential oils and their components have demonstrated strong fungistatic and antibacterial effects against pathogenic fungi and bacteria on cultivated mushrooms. Modes of action of essential oils in their interaction with bacteria have been quite revealed. It has been assumed that Thymol changes the permeability of cytoplasmic membrane of Gram-positive bacteria, acting as a proton exchanger, decreasing the pH gradient and causing a collapse of the proton motive force and eventually cell death. Regarding to antifungal activity, our results also showed that essential oils of C. cyminum and Z. multiflora completely prevent the growth of T. harzianum, followed by D. ammoniacum and F. angulate. One of the possible action mechanisms proposed that fungal growth may be reduced or totally inhibited due to monoterpenes present in essential oils which could increase the concentration of lipidic peroxides such as hydroxyl, alkoxyl and alkoperoxyl radicals and so bring about cell death. According to another mechanism, the essential oils would act on the hyphae of the mycelium, provoking exit of components from the cytoplasm, the loss of rigidity and integrity of the hypha cell wall, resulting in its collapse and death of the mycelium. In our study, a camel recombinant chimeric lactoferricin + lactoferrampin peptide was also tested for its antimicrobial activity. Our result showed that this recombinant peptide have remarkable activity against pathogenic bacteria. The details of the antibacterial mechanism for this recombinant peptide still are unknown. However, amino acid profile, sequence orientation and structural conformation of cationic peptides are the main features which make these peptides capable to inhibit bacterial growth by disrupting of the bacterial cell membrane. As a general results it can be concluded that natural plant-derived antimicrobial as well as recombinant peptides can be used as alternatives to synthetic pesticides, however, further studies on safety and toxicity of these substances should be carried out before use.
Conclusion: In summary, this study shows that essential oils and plant extracts isolated from above plant material have remarkable antifungal activities against T. harzianum, while the chimera peptide showed complete inhibition against P. tolaasii. Thus all of these substance could become alternative to synthetic antimicrobial compounds for control of mushroom pathogens. However, further studies on safety and toxicity of these substances should be carried out before use.
Marzieh Nourashrafeddin; Mohammad Farsi; Farajollah Shahriari; Javad Janpoor
Abstract
Introduction: Edible white button mushroom (Agaricusbisporus) is the most common edible mushroom in Iran and the world. The yield of this mushroom is less than the average of yield in the world because of strain degeneration and using strains with low yield. Most of the current hybrids are either identical ...
Read More
Introduction: Edible white button mushroom (Agaricusbisporus) is the most common edible mushroom in Iran and the world. The yield of this mushroom is less than the average of yield in the world because of strain degeneration and using strains with low yield. Most of the current hybrids are either identical or very similar to the first hybrids. Ongoing breeding programs are exploiting the variability in Agaricus germplasm to produce new varieties with better traits including higher yield and resistance to biotic and abiotic stresses. One of the breeding programs is F1 production from parental homokaryons crossing. These homokaryonsis were isolated among germinated basidiospores on the culture media. During the last decades, various molecular markers based on nucleic acid polymorphisms (such as Restriction Fragment Length Polymorphism, Random Amplification of Polymorphic DNA, Amplified fragment of Length Polymorphism, Inter Simple Sequence Repeat, Simple Sequence Repeat markers) have been used to differentiate homokaryons and heterokaryons. Microsatellites consist of short tandem repeat motifs distributed throughout the genome. Microsatellites are usually highly polymorphic due to a high degree of variation in the number of repeats among individuals. Microsatellite markers are multiallelic and co-dominant and thus tend to be more informative than other marker systems. Microsatellite markers have been widely developed in animals and plants and more recently in fungal species. The presence of microsatellites in the genome of A. bisporus was previously reported.
Materials and Methods: In this research, 160 germinated basidiospores were collected from commercially cultivated strain A15 and they were grown on compost extract agar (CEA). The mycelial growth rate of these160 isolates was evaluated at 25°C on CEA medium. 18 isolates with slow growing rate were selected from 160 isolates. In the next step, co-dominant SSR markers were used to homokaryons detection. Ten SSR primers showed polymorphism in parental control samples that were used to this experiment. The isolates were divided into two general homoallelic and heteroallelic groups and seven isolates from homoallellic group, which showed one-band pattern, characterized as putative homokaryon. Genetic similarity was calculated by NTSYSpc software version 2.02 e using UPGMA method. In the next step of experiment, the isolates (4 and 8) had minimum genetic similarity that was crossed to produce hybrid. In order to confirm the hybrid formation, PCR-SSR reaction with a primer (AbSSR 45) was performed.
Results and Discussions: Basidiospores were collected and allowed to germinate on CEA medium. Putative homokaryons were different in colony morphology and growth rate compared to the original heterokaryons. Mycelium samples showed different colony morphology including tomentose, apprised and strandy mycelium. Different growth rate can be affected by genetic factors in nucleus and mitoconderia. After four weeks, mycelium browning was appeared in liquid compost extract medium and created a disturbance in DNA extraction. To solve this problem, DNA was extracted from three-week old mycelium. Mycelium browning may cause by phenolic compounds produced by mycelium and enzymes that catalyze melanin biosynthesis reactions. Ten primers were used to homokaryon isolation. These primers were situated on the 9 linkage groups of 13 haploid chromosomes. Seven isolates were distinguished as putative homokaryon that showed one-band in all primers on the gel electrophoresis. The results of genetic similarity calculation showed that this index was variable between 0.17 to 0.67in 7 homokaryon isolates and the minimum genetic similarity (0.17) was observed between isolates 4 and 8. These two isolates were crossed and the result of this crossing was N1 hybrid. Also, other homokaryon isolates were crossed and mating incompatibility was observed in some of them. According to these observations, it is suggested that in future studies, in addition to genetic similarity, sexual incompatibility should also be considered. Hybrid N1 produced aerial mycelium and had higher growth rate in comparison to parental homokaryons and similar to heterokaryon control, had two-bands pattern. This two bands pattern indicates the presence of two non-sister nucleuse in each cells. Finally, the results showed that SSR marker can result to accurate detection of homokaryons.
Conclusions: The aim of the present study was screening homokaryon isolates of A.bisporus using SSR markers to obtain hybrid. Results showed that growth rate of homokaryon isolates were lower than the heterokaryons. Since, SSR markers were able to show high polymorphism in the isolates, thus it can be said that these markers are suitable to homokaryon screening. Final result of this study is N1 hybrid that can compare to commercially cultivated strains.
Hajar Shayesteh; Saeid Malekzadeh Shafaroudi; Kamal Ghous; Farajollah Shahriari
Abstract
Introduction Jujube (Zizyphus jujuba Mill.) as a valuable medicinal plant and adapted to different climatic conditions is widespread in many parts of Iran. Nowadays, beside the export of its fruit, jujube is also used as an herbal medicine to treat the diseases, so it has a high economic value. Study ...
Read More
Introduction Jujube (Zizyphus jujuba Mill.) as a valuable medicinal plant and adapted to different climatic conditions is widespread in many parts of Iran. Nowadays, beside the export of its fruit, jujube is also used as an herbal medicine to treat the diseases, so it has a high economic value. Study on genetic diversity is the first step to identify and preservation of germplasm. It is also considered as the basic principles of plant breeding. DNA markers seem to be the best way in determination of the genetic diversity. Inter simple sequence repeats (ISSR) markers are highly polymorphic and combine most benefits of Simple Sequence Repeats (SSRs) and amplified fragment length polymorphism (AFLP) to the generality of random amplified polymorphic DNA (RAPD).
Materials and Methods In order to study of the genetic diversity among 31 ecotypes collected from eight Jujube-rich provinces, including South Khorasan, Razavi Khorasan, Mazandaran, Golestan, Qom, Isfahan, Lorestan and Fars. Genomic DNA was extracted by CTAB method and polymerase chain reaction (PCR) was performed with 13 ISSR primers in which six most efficient primers were selected. Cluster analysis based on Dice similarity coefficient and Unweighed Pair Group Method with Arithmetic Mean (UPGMA) was carried out and POPGENe.3.2 software was used to determine the similarity of populations with each other.
Results and Discussion 84 loci were amplified and 70 of them (83%) revealed a proper polymorphism with the size between 200 and 2000 base pair. The average number of amplified and polymorphic bands per primer was 14 and 11.6 respectively. Primers with di-nucleotide repeats produced more polymorphic bands than ones with tri-nucleotide repeats. It seems that this is due to a higher frequency of sequences containing di-nucleotide repeats in intergenic regions and higher possibility of mutation revealed in more diversity in comparison to gene coding regions. Anchored primers with 1 or 2 nucleotides at the 5’ end make sure annealing only to the ends of SSRs in template DNA, so avoiding internal priming and smear formation. In addition, the anchor lets only a subset of the microsatellites to serve as priming sites. Primers (AC)8YT and (GA)8A with the higher percentage of polymorphism is recommended for further analysis. According to the cluster analysis, the ecotypes could be classified into seven main groups at the 0.85 level of genetic similarity. The most genetic similarity (0.95) was observed between ecotypes from Kalaleh and Doroh and also Noghab and Dustiran and the least genetic similarity (0.48) observed between Kangan and Borzaderan. POPGENe.3.2 software data indicated that populations of Isfahan and South Khorasan had the slightest difference while populations of Isfahan and Razavi Khorasan showed the most difference.
Conclusions In general results demonstrated that the total diversity of jujube ecotypes in Iran is summarized in the area of South Khorasan province. Given data showed that South Khorasan has been an original place of cultivation of this medicinal plant, this area could be considered as one of the important centers of jujube diversity. In addition, significant levels of diversity were observed among ecotypes belonging to Isfahan and Mazandaran provinces.
Mahdi Ghabouli; Ahmadreza Bahrami; Farajollah Shahriari; Jafar Zolala; Ali Mohammadi
Abstract
Plant tissue culture techniques are used as basic requirement of common plant transformation systems. In most cases of plant transformation, a reproducible regeneration protocol is the limiting step due to long time lasting, specialized facilities and well experienced persons. Furthermore, tissue culture ...
Read More
Plant tissue culture techniques are used as basic requirement of common plant transformation systems. In most cases of plant transformation, a reproducible regeneration protocol is the limiting step due to long time lasting, specialized facilities and well experienced persons. Furthermore, tissue culture procedures induce somaclonal variation among regenerated transgenic plants. Therefore, recently current studies in plant molecular biology prefer plant transformation procedures avoiding tissue culture phase. Various in plant a transformation procedures have been explored, among which the floral dip method is the most reliable in vivo transformation method. In this research, with the aim of evaluating the ability of floral dip method for genetic transformation of some Apiaceae plants, we studied Dill (Anethum graveolens), Fennel (Foeniculum vulgare), Coriander (Coriandrum sativum), Carrot (Daucus carrota), Parsley (Petrocelium sativum) and Celery (Apium graveolens). Arabidopsis thaliana was used as a model plant of experimental procedure. Flowers, in different stages of inflorescence development, were immersed in different suspension of Agrobacterium tumefaciens carrying the plant binary vector pBI121. This vector carries plant reporter gene uidA (gus) and the plant selectable marker gene npt II. Although, producing transgenic Arabidopsis plants with a high transformation rate of 4% verified the accuracy of experimental procedure, floral dip method was not successful for transformation of Apiaceae plants. Only one transformed celery plantlet, carrying nptII gene with no expression of GUS, was obtained bby screening more than 10000 seeds produced by treated plants from all the species. Transgenic Arabidopsis plants expressing gus reporter gene were confirmed through PCR and histochemical assays.
Bahman Hosseini; Haleh Hashemi; Farajollah Shahriari; Seyyed Hassan Marashi
Abstract
Abstract
Benzylisoquinoline alkaloids are a diverse group of nitrogenous compounds which is found in about 20% of plant species. Isolation of effective genes involved in morphine biosynthesis of opium poppy is very important in the production of specific which can be achieved using metabolic engineering ...
Read More
Abstract
Benzylisoquinoline alkaloids are a diverse group of nitrogenous compounds which is found in about 20% of plant species. Isolation of effective genes involved in morphine biosynthesis of opium poppy is very important in the production of specific which can be achieved using metabolic engineering techniques. In this biosynthesis pathway, the key enzyme SAT is Involved in the conversion of salutaridinol 7-O-acetyltransferase (EC 2.3.1.150) to salutaridinol-7-O-acetate, which is the immediate precursor of thebaine. In this project, the gene encoding this enzyme was isolated using primers which were designed on the basis of the gene sequence available on data banks (NCBI) for papaver somniferum. This gene IS then cloned in expression vectors under the Control of Camv 355 promoter. The result of this cloning was confirmed using different molecular methods such as enzyme digestion and PCR. Agro infiltration method was also used for transient expression of SAT gene. The result of evaluation showed that morphine and codeine were only Produced in the leaves of transgenic plants containing SAT transgen.
Keywords: Benzylisoquinoline alkaloids, Salutaridinol 7-O-acetyltransferase, opium poppy
