فهرست مطالب غلام خداکرمیان

Microbes that reside inside the plant tissues are known as endophytes. These microbes produce an array of compounds that have the potential to be used in modern medicine and agriculture. The aim of this study was to isolate, screen, and identify endophytic bacteria that have antimicrobial activity toward phytopathogenic bacteria. Aromatic plants such as Satureja khuzestanica are used in traditional medicine due to their secondary metabolites but information regarding their naturally occurring bacterial endophytes is limited.

In the present study, 27 strains of endophytes were isolated from S. khuzestanica. Based on the sequencing of the 16S rRNA gene, isolated bacterial strains with the highest activity against phytopathogenic bacteria belong to Bacillus, Streptomyces, and Pseudomonas genera. The bioactive secondary metabolites of these endophyte bacteria were extracted using ethyl acetate, followed by Gas Chromatography-Mass Spectrometry (GC-MS) analysis under standard conditions. The minimum inhibitory concentration (MIC) values were determined for five species of bacteria via the microbroth dilution technique. The crude extracts of the five selected bacterial endophytes indicated an antimicrobial activity against five phytopathogenic species.

Data analysis showed significant differences in antimicrobial activity that ranged from 0.312 mg/ml to 2. 5 mg/ml. The minimum bactericidal concentrations ranged from 0.625 mg/ml to 10 mg/ml.

The major compounds in GC-MS analysis of endophyte bacteria in this study were 2,4-di-tert-butylphenol, beta-d-glucopyranose, hexadecane, tetradecane,  eicosane, and dibutyl phthalate. This study reports for the first-time bacterial endophytes of S. khuzestanica, with antimicrobial activity against bacterial phypathogens. Our findings provide new insights into the antimicrobial activities of natural bacteria endophytes from S. khuzestanica.

In many parts of the world, medicinal plants have been used as an alternative medicine to promote human health and longevity since ancient times. Microbes residing within plant tissues are known as endophytes. The compounds produced by these microbes have the potential to be employed in modern medicine, agriculture, and other industries. Endophytic bacteria isolated from medicinal plants are valuable sources of novel bioactive compounds with diverse activities, including antimicrobial, anticancer, and antiviral properties. This study aimed to isolate, identify, and screen endophyte bacteria with antimicrobial activity against plant pathogenic bacteria. Aromatic plants such as Satureja khuzestanica are utilized in traditional medicine due to their secondary metabolites, but data regarding its naturally occurring bacterial endophytes is limited.

In the current study, 17 strains of bacterial endophytes were isolated in a Tryptic Soy Agar medium from the medicinal plant Satureja khuzestanica. Based on sequencing the 16S rRNA encoding gene, researchers isolated bacterial strains from the Bacillus, Streptomyces, and Pseudomonas genera with the highest activity against plant pathogenic bacteria. Under standard conditions, these endophytic bacteria’s bioactive secondary metabolites were extracted with ethyl acetate and analyzed using Gas Chromatography-Mass Spectrometry (GC-MS). The mass spectra of the compounds were compared to the National Institute of Standards and Technology (NIST) library’s database. Moreover, microbroth dilution techniques were used to determine the minimum inhibitory concentration (MIC) values for five different bacteria species.

The GC-MS analysis revealed the presence of a number of compounds, including dibutyl phthalate (DBP), eicosane, octadecanoic acid, hexadecanoic acid, and hexadecane 1,4- dicyclohexylbutane. Four of the selected bacterial endophytes exhibited antimicrobial activity against three plant pathogens: Ralstonia solanacearum, Pectobacterium carotovorum subsp., carotovorum, and Clavibacter insidiosus. Data analysis revealed significant differences in antimicrobial activity, with the minimum inhibitor concentration ranging from 0.312 mg/ml to 2. 5 mg/ml. Furthermore, we identified bioactive secondary metabolites with reported biological activities in antimicrobial, anti-inflammatory, and antioxidant properties with biotechnological applications in medicine, agriculture, and other industries based on an endophytic crude extract data analysis.

Our findings shed new light on the antimicrobial properties of naturally occurring bacterial endophytes in S. khuzestanica.

The causative agent of scab disease or potato scab, which are different species of the genus Streptomyces, has a relatively wide host range. This disease has caused a lot of economic damage in most of the cultivating areas. Researches on the virulence mechanisms of plant-pathogenic Streptomyces spp. have shown the importance of the thaxtomin phytotoxins as main pathogenicity determinants produced by several species. In addition, other phytotoxic specialized metabolites such as coronafacoil, Concanamycin and FD-891 along with the production of phytohormones and secretory proteins may have a role in the development or severity of the disease caused by species related to this genus. There are wide range of pathogenic factors in the species related to the Streptomyces genus. This phenomenon shows the potential of the species of each region to accept pathogenic genes and create new species, which is very important and decisive from the disease management point of view. The purpose of this review is to gain an understanding of the molecular mechanisms of plant pathogenicity to use improved management methods to control potato scab disease and other plant diseases caused by species related to this genus.

In this study,symbiotic bacteria of Sitotorga cerealella were identified. Containers containing barley were stored in a growth chamber at a temperature of 24 ° C and a relative humidity of 65% after contamination. To separate the bacteria of Sitotorga cerealella gastrointestinal tract,10 healthy larvae were selected from the reared population. After disinfection, the larvae gastrointestinal tract was removed with ethanol 70%, sodium hypochlorite and distilled water5%. Then, one milliliter of 0.1 M phosphate buffer with pH 7 was used to homogenize the gastrointestinal tract. The homogeneous and diluted contents were cultured in nutrient agar medium. To perform polymerase chain reactions from 5 μl of Master Mix ,2 μl of extracted DNA, 1.5 μl of RP2 primer and 1.5 μl of FD1 primer and 15 μl Distilled Water that the total volume of these materials should reach 25 microliters. The PCR process was performed using general primers FD1 and RP2 and 16srRNA gene sequence in a thermocycle.The PCR product sequencing was performed in collaboration with South Korea&amps Bioneer and the results were reviewed at the NCBI site. The phenotypic characteristics of the isolated bacteria were determined by standard bacteriological methods. After reviewing all the prepared colonies and comparing the obtained results, the highest frequency in terms of number of colonies was specific to Enterococcus mundtii and Enterobacter hormaechei was less abundant in the gastrointestinal tract of Sitotorga cerealella. Also, according to electrophoresis, the highest bands observed belong to Enterobacter hormaechei and the shortest band belongs to Enterococcus mundtii.

Garlic due to its antimicrobial activity, has a high potential in controlling of compatible bacteria in gut of pest insects. Developing the use of these compounds can be effective in plant pest management. In this study, the effect of Allicin from garlic extract on reducing the population of compatible bacteria of two moths larva Plodia interpunctella and Ephesti kuehniella gut was investigated. Initially, the two pests were bred in sufficient numbers. The Larva were used to isolation of guts bacteria. The bacteria were isolated and grouped based on phenotypic characteristics and comparison of protein electrophoresis pattern. Larvae were fed with feed containing tetracycline for three days to minimize guts bacteria, than a suspension of isolated bacteria was added to the larva diet. A series of dilutions of garlic extract were prepared with ratios of 1:1, 1:2, 1:4, 1:8, 1:16, 1:32, then 50 microliters of each dilution was added to each well in the plate and the bacterial suspension added to it. The growth inhibition zone diameter was measured and the results showed that garlic extract has antimicrobial activity against all bacteria. Antimicrobial activity of garlic was dependent on concentration and the highest activity was observed in7.816 mg/ml concentration. Bacillus safensis and Bacillus zhangzhouensis showed the highest and Serratia marsecesence showed the lowest susceptibility to allicin garlic extract. The highest and lowest MIC were related to Bacillus safensis and Serratia marcescence respectively

Hamedan province is one of the most important regions for cultivation of stone fruit trees, such as apricots (Prunus armeniaca) due to its suitable climatic conditions. Bacterial canker disease of stone fruit trees caused by Pseudomonas syringae pv. syringae, is one of the most serious diseases in this province, Iran and all over the world. The study was performed to identify the causative agent of the disease.

Samples of diseased apricots were collected in early spring and fall autumn of 2018. From the collected samples a total of 130 bacterial strains were isolated of which 65 representative’s strains were selected based on the color and type of colonies on the culture medium for further study. SDS-PAGE technique was used to group the representative’s strains. Pathogenicity of the representative’s strains were performed on apricot seedlings under greenhouse condition. The studied strains were identified based on their phenotypic features, using standard bacteriological method and followed by sequencing of the 16S rRNA encoding gene.

30 strains showed hypersensitivity reaction on geranium leaves.  According to the pattern of cellular soluble proteins on polyacrylamide gel (SDS-PAGE), the strains were divided into seven groups. BLAST of partial sequence of 16S rRNA encoding gene from this strain showed 98/7% similarity to P. syringae NCPPB 281. Another strain (FB46 strain) was identified as P. agglomerans due to 95.33% similarity with Pantoea agglomerans RSG19 by BLAST of 16S rRNA encoding gene sequence.

The results obtained are in agreement with of Vasebi et al. isolated P. syringae as a causative agent of apricot canker in East Azerbaijan province. P. agglomerans has also been reported as one of the factors associated with stone fruits, pome fruits and walnut trees in Alborz province. This is the first report for the presence of Pseudomonas syringae as a causative agent of apricot canker disease in Hamedan province. The bacterium Pantoea agglomerans is reported from the first time in the world as the causative agent which induce apricot canker symptoms.

Quorum sensing (QS) phenomenon is a cell density-dependent regulatory system that bacteria use to communicate with each other as a multicellular organism. Numerous Gram-negative and Gram-positive bacteria use this signaling system to synchronize genes expression and group behaviors including virulence factor secretion, antibiotic production, competence, biofilm formation, and bioluminescence. In this signaling system, bacterial cell-to-cell communication mediated by sending and sensing small signaling molecules known as autoinducers (AIs). For the prohibition of the quorum-sensing system in the bacterial cell, there is another regulatory system named quorum quenching (QQ). The function of this system is through the secretion of inhibitors (QSIs) and some enzymes (QQ enzymes) which interfere with communication in the quorum-sensing system. Due to the inhibitory potential of the QQ system to disrupt the communication network provided by the QS system, it introduced as a novel strategy to control plant bacterial diseases.

Plant rhizospheric microbes are diverse microorganisms which can be employed to assist plant health. Root exodates of potato as an edible plant can support many benefit bacteria, especially Bacillus spp. Awareness about diversity and phylogenetic of these bacteria has great potential to apply them as a plant growth promoting and antagonists against the root pathogens. For this purpose, healthy potato plants rhizospher samples were collected from Hamedan province and a total of 43 Gram and catalase positive bacterial strains were isolated. Antagonistic activity of the isolated strains towards Pectobacterium carotovorum subsp. carotovorum (Pcc) were evaluated on NA medium using dual culture in a complete randomized design, inhibition zone was recorded and analyzed. Results indicated that 11 strains were able to produce inhibitory zone against Pcc and showed significant differences at 1% level. Strain B57 with 3.16 centimeters and strain B4 with 1.16 centimeters inhibition zone, had the highest and lowest inhibitory respectively. Based on the phenotypic features antagonistic Bacillus strains were belongs to Bacillus subtilis, B. cereus and B. pumilus. Results of 16S rDNA gene sequencing from these three Bacillus species verified the results of phenotypic feature characterization. Phylogenetic position of the tested Bacillus strains showed that strain B34 clustered with 1000 bootstraps as B. cereus, strain B7 with 1000 bootstraps as B. subtilis and strain B57 with 1000 bootstraps as B. pumilus. Accession number for the 16S rDNA gene sequences of B7, B34 and B 57 Bacillus strains in NCBI are MG654658, MG654659 and MG654660 respectively. According to the litrature, one of the microbial factors that in the rhizosphere of potato plants prevents the establishment of pathogens is the diversity of bacilli in this area. Numerous studies have reported the role of rhizosphere bacilli in reducing root diseases of various plants.

In this study the possibility of inducing resistance was tested using five endophytic bacterial strains against wheat take–all disease caused by the Gaeumannomyces graminis var. tritici (Ggt) under greenhouse conditions. The results showed that disease severity in treated plants compared to control samples decreased 56.6% and wheat resistance against the pathogen has been substantially increased. Effect of five endophytic bacterial strains including Pseudomonas fluorescens, Serratia marcescens, Microbacterium phyllosphaerae, Bacillus pumilus and Streptomyces argenteolus were studied on some enzymes related to plant resistance at the 0, 24, 48 and 168 hours after inoculation with Ggt. Results showed the highest levels of the β–1, 4–glucanase and chitinase at 48 and 168 hours after inoculating of pathogen. The level of β–1, 4–glucanase in plant samples inoculated with Serratia marcescens showed 0.0291 U/mg protein compared to c+ (healthy control without endophytic bacteria and pathogen) with 0/0055 and cp (pathogen– infected control and untreated with endophytic bacteria ) with 0/0075. The level of chitinase for plant samples inoculated with Pseudomonas fluorescens and Serratia marcescens strains were 0.0056 and 0.0045 U/mg protein compared to c+ (healthy control without endophytic bacteria and pathogen) with 0/0030 and cp (pathogen– infected control and untreated with endophytic bacteria) with 0/0031, respectively.

Bacteria are an important compatible group of microorganisms which live within insect gut. Most of these microorganisms are useful to help the digestive system to work better. The aim of this study was to identify the symbionts bacteria associated with Plodia interpunctella and Ephestia kuehniella guts to use in future biotechnology studies. The larva from the two above-mentioned insects wereprepared and their guts organ were used to isolate symbiotic bacteria. The bacteria were isolated and purified on the enriched nutrient agar medium and their frequency was recorded. Based on the phenotypic characteristics the isolated bacteria were preliminary characterized. Then after comparing of their total soluble proteins by electrophoresis, they were kept for further studies. Identification of bacteria were done based on phenotypic characteristics with standard bacteriological methods as well as use of 16SrRNA encoding gene sequence and its comparison with NCBI data bank. Results indicated that the dominant symbiont bacteria in the Ephestia kuehniella gut were Bacillus toyonensis, Serratia marcescence and in Plodia interpunctella gut were Bacillus safensis, Bacillus zhangzhouensis and Bacillus wiedmannii. The accuracy of the results for phenotypic determination was verified by molecular analyzes using DNA sequences of the 16SrRNA gene comparission via NCBI sequences database. This is the first study on gut symbionts of Ephestia kuehniella and Plodia interpunctella in Iran.

Background and Objectives:

 Khuzestan is the only province with sugarcane cultivation. One of the limiting factors in the production of this crop is the occurrence of frost damages in some years. Since no comprehensive research has yet been conducted on detection and evaluation of bacteria with the potential to form nuclear ice in frostbite, this study was carried out to evaluate this subject.

 Materials and Methods:

 This study was carried out in the laboratory of Institute for Research and Training of Sugar and Auxiliary Industries. Endophytic and epiphytic strains of bacteria were isolated from sugarcane. To test the freezing potential of representative strains, four tests including, "determination of Ice nucleation activity by freezing in the tube", "freezing droplet test at -20 °C", "Ice nucleation test on sugarcane cut leaves in vitro" and "Ice nucleation test on sugarcane plant in greenhouse conditions" were performed. Also the genes of the nucleus were detected by 3076f / 3463r and 3308f / 3463r specific primers. 

Results:

 Results show that bacteria species B. gladioli, B. fungorum, B. cantaminans, M. huakuii, O. ciceri, M. proteolyticum, M. Foliorum, R. solanacearum , R. picketii and X. campestris had different degrees of symptoms. In addition, the genes of the nucleus were detected by 3076f / 3463r and 3308f / 3463r specific primers. For all of above mentioned bacterial strains, specifically strain X. campestris, no references have been reported of ice nucleation activity. To assess susceptibility or resistance of sugarcane, cultivar CP69-1062 were the most susceptible and highly damaged in the tests including "Ice nucleation test on sugarcane cut leaves in vitro" and "Ice nucleation test on sugarcane plant in greenhouse conditions". In addition, CP57-614 and CP48-103 cultivars showed minimum variation in frosting in compression to CP69-1062 cultivar. Two cultivars including CP73-21 and SP70-1143 exhibited a high degree of resistance to frostbite.

 Discussion:

 Due to the high variation among Ice-Nucleating bacteria and the sensitivity of commercial cultivars, our finding can be a suitable option for producers and encourage them to use resistant cultivars in the field.

The symptoms of bacterial leaf spot and blight on onion (Allium cepa L.) plants were observed in Fars (Eghlid) and Kermanshah (Bistoun) provinces with the rate of occurrence varying from 90 to 100% of the plants. In order to isolation and identification of the causal agent, suspicious samples were collected from infected fields. By culturing the samples on nutrient agar medium, 30 bacterial isolates were obtained. Pathogenicity test of the isolates on Allium spp. was performed and their pathogenicity was proved on onion, spring onion and leek. The isolates showed an identical total cell soluble protein pattern in SDS-PAGE. The isolates were similar to Pseudomonas syringae pv. porri based on phenotypic characteristics such as production of fluorescent pigment in KB medium, LOPAT tests, and utilization of different carbon sources. Genomic profiles generated with BOX-PCR placed the isolates into one groups. The results of the amplification and sequencing of the 16S rRNA, gyrB, and rpoD genes showed that the isolates belong to P. syringae pv. porri. This is the first report of the presence of the P. syringae pv. porri in Kermanshah province. Management of the disease seems to be necessary to prevent the disease epidemy.

During rice growing season, the symptomless rice seeds from different paddy fields in Guilan province, Iran, were collected. After isolation of epiphytic and endophytic bacteria, 39 isolates including 19 epiphytes and 20 endophytes were selected based on the predominant characteristics. Five Operational Taxonomic Units (OTUs) were obtained based on PCR-RFLP of 16S r-DNA in these isolates. According to biochemical tests and partial sequencing of 16S-rDNA, in both epiphyte and endophyte bacteria of rice seeds, the most populated OTUs (V and II) were identified as Pantoea ananatis and Pseudomonas oryzihabitans, respectively. Six representative isolates from these two OTUs were selected to be evaluated for their abilities for rice seed germination and growth enhancement. Among them, P. oryzihabitans was not beneficially effective. However, JpB1 isolate of P. ananatis was considered to be the most effective plant growth promoting isolate, since it showed stable beneficial effects on most surveyed characteristics in both rice seed germination and growth enhancement experiments.  Although, OpB3 isolate of P. ananatis produced IAA in higher amount and solubilized phosphate more than the other isolates followed by JpB1 and P. ananatis L3pB3, it was not beneficially effective on rice seed germination.

In recent years, there has been a considerable interest in the use of biological approaches, as an alternative to chemical fertilizers and pesticides to management of plant pathogens and improvement of crop productivity. Recently, endophytic bacteria have gained attention due to their efficient bio-control and plant growth promoting potentials. The objective of this study was to evaluate bio control and plant growth promoting ability of endophytic bacteria in challenging with Verticillium dahliae under in-vitro and greenhouse conditions. Endophytic bacteria were isolated from tomato plants and their bio-control activity was screened based on dual culture method. Antifungal and their plant growth promoting traits such as production of volatile compounds, antibiotics, proteases, chitinases, hydrogen cyanide, siderophore, indole acetic acid and phosphate solubilizing were evaluated. Their effects on seed germination and growth parameters of seedlings under in-vitro condition and on the control of disease and tomato growth were evaluated in greenhouse. In dual culture tests, FS67, FS167, FS300 and FS339 isolates showed significant antifungal activity and they were identified as Pseudomonas mosselli, P. fuorescence, Stenotrophomonas maltophilia and Acinetobacter calcoaceticus, respectively. All strains produced several kinds of antifungal and growth promoting agents under in-vitro conditions. They increased seed germination and growth parameters of seedlings. They also reduced the disease and improved the growth parameters of the plants in challenging with V. dahliae in greenhouse. Discussion and conclusion: The present study has shown that these endophytic bacteria have the bio-control and bio-fertilizer potentials, which make them suitable candidates as an alternative tool of chemicals in management of V. dahliae. Results indicated they might enhance tomato plant growth and health via various mechanisms and most of them probably employ more than one of these mechanisms.

Endophyte bacteria refers to the bacteria that live within plants. Fresh leaves of 23 sugar beet (Beta vulgaris L.) plants were collected from the west of Iran. After superficial disinfection, endophytic bacteria were isolated from the host tissue. Isolates were grouped based on their whole-cell protein electrophoresis patterns. One representative from each electrotype was selected and its morphological feature characterized according to the standard bacteriological criteria. The 16S rRNA encoding gene from these representatives was amplified using fD1 and rD1 universal primers, subjected to sequencing and aligned in the NCBI. The major occurring fingerprint types (electrotypes) were identified as Acinetobacter calcoaceticus, Pseudomonas aeruginosa, and Stenotrophomonas maltophilia (25, 23, and 22 strains respectively). The other minor occurring electrotypes were identified as Streptomyces spp, Acetobacter spp, and Agrobacterium spp. This is the first report of A. calcoaceticus, P. aeruginosa, and S. maltophilia as an endophyte in the leaves of sugar beet.

Phytopathogenic microorganisms affect plant health and burden a major threat to food production and ecosystem stability. Increasing the use of chemical pesticides for plant diseases control causes several negative effects on human and environment health. Furthermore, increasing public awareness about the side effects of them led to a research to find alternatives for these products. One of the alternative methods is bio-control utilizing plant associated antagonistic microorganisms.
In this study, 80 endophytic bacteria were isolated from tomato tissues. Their antagonistic activity screened based on agar diffusion test, against tomato bacterial wilt disease (Ralstonia solanacearum). They were identified based on the morphological, biochemical properties and 16s rRNA sequence analyses. These strains were evaluated in greenhouse and tested for their ability to induce the production of defense-related enzymes in plants e.g. Peroxidase (PO), polyphenoloxidase (PPO) and phenolics based on spectrophotometer method.
Results showed FS67, FS167 and FS184 strains had maximum inhibition zone forming. They identified as Pseudomonas mossellii, P. fuorescence and P. brassicacearum respectively. FS67 and FS167 strains significantly reduced disease in greenhouse. There was a significant increase in the activity of PO, PPO and phenolics in tomato plants treated with FS67, FS167 and pathogen.
Discussion and The present study has shown that P. mosselli and P. fuorescence might have the potential to control R. solanacearum. However, the good results obtained in vitro cannot be gained the same as those in greenhouse or field conditions. So, further experiments are needed to determine the effectiveness of these isolates under field conditions.This work support the view that increased defense enzymes activities could be involved, at least in part, in the beneficial effects of endophytic bacteria on plants growth in interaction of pathogens. This is the first report of antagonistic activity of Pseudomonas mossellii from Iran.

Growing evidence exists that agriculture affects antibiotic resistance in human pathogens. Beta-lactam antibiotics are the most commonly used antimicrobial agents in many countries. The abundance of beta-lactamase encoding genes can be used as an indicator of antibiotic resistance in the environment. So, to determine the beta-lactamase resistance genes, the abundance of culturable bacteria having bla-TEM genesin the soils under different land uses wasexamined.
44 Gram-positive and 34 Gram-negative bacteria plated on nutrient agar were isolated from agricultural, pasture and mining soils and selected to study the presence of TEM-class gene using PCR amplification. Antibiotic sensitivity test of bla-TEM諊╪힬솫 done adopting the Kirby-Bauer disk diffusion method and antibiotic discs used were: ampicillin, amoxicillin, vancomicin, streptomycin, tetracycline and gentamicin. Finally, five multi-drug resistant and bla-TEM isolates were identified using universal primers.
The highest level of beta-lactamase genes was observed in the Gram-positive and Gram-negative isolates from the pasture soils. In the agricultural and mining soils, a high abundance of bla-TEM isolateswasfound which also showed resistance to beta-lactam antibiotics. The identified multi-drug resistant and bla-TEM isolates were from these genera: Achromobacter, Bacillus, Brevibacillus, Aminobacter and Brevundimonas.
Discussion and The high number of bla-TEM bacteria in all the soils may be attributed to the other important feature of bla genes which is their capability to extrude toxic compounds like heavy metals in contaminated environments. Sensitivity of some bla-TEM bacteria to beta-lactam antibiotics was interesting. This result shows that bla-TEM genes confer resistance to beta-lactamase inhibitors in a different degree. Some of the identified isolates were pathogen. These pathogens in soils can transfer to plants and human which induce health problems. A high abundance of bla-TEM bacteria in the agricultural soil indicates the inefficiency of beta-lactam antibiotics.

Phylosphere is a site for living micro-organisms including bacteria. Plants species and plant leaf type have important role on diversity and density of bacteria live on them. There are several bacteria as epiphytic on aerial parts of plants and plant seeds which cause diseases in suitable conditions. Cotton bacterial blight has been appeared as epidemic in Iran particularly in Golestan province in recent years. Some suggestion propounds the presence of this bacterium on cotton seed surface. This study was conducted in Golestan province to identify the gram negative bacteria on seed surface. For this purpose, samplings were done randomly from cotton cultivar seeds Sahel, Varamin, Bakhtegan, Sepid and Khordad for planting. The seeds were incorporated into sterile distilled water and small amount of tween 20 as a detergent was added to it, and put to room temperature for one hour for better washing of bacteria on seed surface. Then the above suspension was streaked on bacterial culture media. The bacteria were isolated, purified and identified. The results revealed that Pseudomonas fluorescens, P. syringae, Pantoea annanas and Pectobacterium sp. were identified based on authentic references. The presence of Xanthomonas citri subsp. Malvacearum was not proved as causal agent of bacterial blight as epiphyte on cotton seeds.

Endophytic bacteria are important group of bacteria that produce phytohormones, antifungal and antibacterial agents, siderophore, nutrient competition and induced systemic resistance in the host, causing the biocontrol of plant pathogens. The aim of this study was to obtain endophytic isolates with antagonistic effects against the alfalfa wilt agent, Clavibacter michiganensis subsp. insidiosus in the laboratory and greenhouse. In order to conduct this research study, samples and isolation of bacteria from alfalfa fields in Hamedan province were performed. Then, in the laboratory conditions, their antagonistic effects against Cmi were investigated. The experiments were conducted in laboratory on NA (Nutrient Agar) culture medium in a completely randomized design with three replications by determining the pathogenic bacterium growth inhibition. The obtained data were analyzed by Statistical Analysis System (SAS) software and the means were compared by Duncan's multiple range test. Based on the laboratory studies results, eight antagonistic strains with inhibition diameter higher than 6 mm were selected for the greenhouse studies. In the greenhouse conditions, the strains were evaluated for their effects on increasing growth factors of alfalfa. The results showed that isolates 8 and 56 showed higher biocontrol efficacy. These isolates increased plant growth factors at 1% level statistical probability. The 16srRNA encoding genes of strains were amplified and sequenced. Result showed that they were belonged to the genera Bacillus subtilis, Pseudomonas sp., Escherichia coli, Sphingomonas paucimobilis and Paenibacillus glycanilyticus. These isolates have been effective in biocontrol of wilt bacterial agents in the laboratory and greenhouse conditions, and increasing plant growth factors. Endophytic bacteria including Bacillus subtilis and Sphingomonas paucimobilis showed effective performance against pathogenic bacteria. In addition they had great impact on plant growth characteristics such as fresh weight, dry weight and plant height. These effects may be due to the production of antibiotics and induced systemic resistance to biological control of bacterial wilt disease in the laboratory and greenhouse and increasing plant growth factors. These results are promising and may be used in the biocontrol and the management of soil-borne diseases.

Alfalfa witches’ broom (AWB) is a limiting factor for alfalfa growth and production in Iran, especially in the tropical regions of the country. AWB phytoplasma strains from two severely affected areas, Chahgeer (Abarkooh, Yazd province) and Juyom (Larestan, Fars province), were compared for main biologic, serologic and molecular characteristics. Based on disease symptoms in alfalfa farms, Chahgeer and Juyom AWB (CAWB and JAWB, respectively) strains were not differentiable. In dodder inoculated periwinkle and tomato plants JAWB phytoplasma induced stronger little leaf compared to the one induced by CAWB phytoplasma. In these experiments the two JAWB and CAWB phytoplasma strains are confirmed as vectored by different leafhopper species, Orosius albicinctus and Circulifer haematoceps respectively. Based on 16S rRNA gene and 16S-23S intergenic spacer region sequences, CAWB and JAWB were not differentiable, however no serologic relationship was observed between the two phytoplasmas in ELISA and DIBA tests using polyclonal antibodies prepared against each of them. Due to the lack of serological relationship, different insect vectors and induction of different symptoms in common host plants, CAWB and JAWB phytoplasmas should be considered as two different AWB strains both belonging to 16SrII-C subgroup.

Sugar beet seed plays an important role in the transmission of beneficial or detrimental bacteria. To identify and evaluate the effects of sugar beet seed-associated bacteria on seed germination, cleaned but not disinfected seeds were provided from Sugar Beet Research Department at Hamadan Agricultural Research Center. A total of 80 endophyte and epiphyte bacterial strains were isolated from the seeds. Based on the protein pattern and according to Laemmli method, three representative electrotypes were selected and their biochemical and molecular characteristics were evaluated. Based on the biochemical test results and sequencing of the 16S rRNA gene of the representative strains Stenotrophomonas rhizophila and Pseudomonas geniculata were identifiedas epiphytic and Stenotrophomonas maltophilia as endophytic species, respectively. To perform seed germination test, three different groups were generated based on the strain effect on seed germination. Seed germination test was conducted based on the inoculation of five representative strains from each group in completely randomized design with four replications. Each replication included a Petri dish containing 10 seeds. Results showed that seeds inoculated by S. maltophilia strain had the highest germination percentage (90), germination rate (0.0174 per hour), shoot fresh weight (100 mg), radicle fresh weight (126.2 mg), shoot dry weight (50 mg), radicle dry weight (43.7 mg), shoot length (6.5 cm), radicle length (8 cm), and the lowest seed uniformity compared with control. In order to improve germination indices and seedling growth, it is recommended to inoculate sugar beet seed with S. maltophilia bacterium.

Due to indiscriminate use of chemical pesticides and its impact on the environment, other methods including the use of epiphytic and endophytic bacteria to control pathogens is highly regarded. Since Medicago sativa is the most important forage crop in Iran and many parts of the world, this study was aimed to evaluate the inhibitory effect of alfalfa epiphytic bacteria on the causal agent of wilt disease in in-vitro.
Undiseased alfalfa leaf samples were collected from different parts of Hamedan province, before its flowering. After isolating epiphyte bacteria from alfalfa fields, the antagonistic effect of isolates was investigated by measuring the diameter of the inhibition zone against alfalfa bacterial wilt. Protein extraction was performed to assess the pattern of protein bands and the variety of the strains. Biochemical tests and PCR were used to identify the isolates.
A total of 30 epiphyte bacteria were isolated from alfalfa shoots. Bacterial isolates were identified as Bacillus, Pseudomonas, and Xanthomonas, based on biochemical, physiological and morphological tests. The highest bactericidal effect was related to isolating No. 16 and the lowest to isolates No. 13, and No. 6, respectively. Using molecular and dendrogram methods, isolate No. 16 was most closely related to Pseudomonas monteilii.
Epiphytic bacteria isolated in this study showed a good inhibitory effect against alfalfa wilt in vitro. This can be promising for the biocontrol of this disease. However, further studies are needed to prove the efficacy of the isolates in field conditions.

Molecular farming refers to the production of pharmaceutical proteins and industrial enzymes in plants by genetic engineering. T7RNA Polymerase is one of the most important enzymes which is used in various fields of biotechnology and molecular biology to produce RNA and study the structure and function of RNA in vitro. The in vitro synthesized RNA is employed in hybridization, microarray, anti-sense RNA and RNAi experiments. This enzyme has also applications in expression systems depending on the T7 promoter and terminator. T7RNA polymerase is a bacteriophage enzyme which acts mainly in the cytoplasm. This study aimed to clone and transform eukaryotic cells with the T7RNA Polymerase gene for production of T7 polymerase in tobacco plants. Specific primers with proper restriction enzyme sites were designed corresponding to the ends of the T7RNA polymerase gene and the gene was isolated and cloned into the plant expression vector, pCAMBIA1304. Successful of gene insertion was confirmed by PCR, restriction enzyme digestion and DNA sequencing. The recombinant construct was transformed into Agrobacterium tumefaciens LBA4404 strain for tobacco transformation. The transgenic tobacco plants were regenerated on selective media containing hygromycin and cefotaxime antibiotics. The presence and expression of the T7RNA polymerase gene in the transgenic plants was confirmed by PCR and RT-PCR techniques.

Alfalfa witches’ broom (AWB) is one of the most important alfalfa diseases in Iran. To characterize 16SrII group phytoplasmas associated with this disease, symptomatic and asymptomatic plants were collected during 2013-2015 and subjected to direct and nested polymerase chain reaction (PCR) using P1/P7, R16mF2/R16mR2 and R16F2n/R16R2. PCR amplicons of ~1.8, ~1.4 and ~1.25 kb respectively, were obtained only from all symptomatic plants. Restriction fragment length polymorphism (RFLP) analysis of R16F2n/R2 amplicons showed that the phytoplasma associated with AWB disease were members of 16SrII group subgroups 16SrII-D and -C. Blast analysis of these amplicon sequences and sequence homology of collected strains and strain sequences retrived from GenBank (AWB strains Chahgeer, Juyom and Bushehr) confirmed that AWB phytoplasmas collected from Bafg, Ardakan, Bahabad and Herat (Yazd province), Nikshahr (Sistan-Baluchestan), Bam, Zarand, Jiroft (Kerman province), Bushehr (Bushehr province), Tabas (South Khorasan province), Jowkar (Hamedan province) and Zardenjan (Esfahan province) cluster with phytoplasma strains enclosed in the 16SrII-D subgroup, while AWB strains from Chahgeer (Yazd province) and Juyom (Fars province) cluster with phytoplasma strains in the 16SrII-C subgroup. Based on these results the predominant strains of 16SrII phytoplasmas associated with AWB disease in Iran were classified in the 16SrII-D subgroup. In Ashkezar and Abarkouh in Yazd province entire alfalfa farm was infected with witches’ broom disease. In 3 year alfalfa stands in Ashkezar alfalfa farms were plowed due to high incidence of the disease.

Bacterial wilt disease caused by Ralstonia solanacearum is an important disease on potato in Hamedan province. During spring and summer 2011 infected plants were collected from potato fields to investigate the genetic diversity among R. solanacearum strains. A total of 52 strains were isolated on NA medium containing triphenyl tetrazolium chloride (TTC) from tubers and stems and their phenotypic characteristics revealed that they were belong to biovar 2A. PCR using ps96h and ps96i primers produced 148 bp bands in tested and standard strains. Genetic diversity of 26 selected strains were analyzed by PCR fingerprinting using, ERIC and BOX primers. The length of produced fragments ranged from 200bp to 1200bp in BOX primer and 200bp to 1500bp in rep primer. Tested bacterial strains were grouped based on the obtained molecular DATA from PCR using UPGMA method and Jaccard`s coefficient. Results showed five distinguished clusters. R. solanacearum strains were genetically divers although no direct connection was observed between geographic locations and PCR groups. The results of this study indicated that rep-PCR technique with two ERIC and BOX primers is capable to show genetic polymorphism among different populations of R. solanacearum.