فهرست مطالب h. rahimian

The beneficial characteristics of endophytic bacteria in general and environmental safety features of Bacillus spp. in particular, have made them potentially efficacious bacteria for plant growth promotion as well as plant defense elicitation against biotic and abiotic stresses. Given that the preservation of biodiversity and sustainability of the Hyrcanian forests in Iran is of utmost importance ecologically and environmentally, proper management of this ecosystem demands considerable attention and care. Studying the microbiome structure and activities, including determination of microorganisms living as endophytes or commensals can be regarded as the starting steps in this context. This study was aimed at the molecular identification and physiological characterization of endophytic Bacillus isolates recovered from the roots of hornbeam (Carpinus betulus L.) trees in some different locations of Hyrcanian forests.

Samples of hornbeam roots were randomly collected from Mazandaran and Golestan provinces, Iran, in the early spring of 2020. Root segments were surface-disinfected, washed in sterile distilled water (SDW) and crushed in drops of SDW using a sterile mortar and pestle. Isolation of bacteria was done by spreading drops of the suspension on plates of tryptic soy agar (TSA). Single colonies were selected after 3-4 days of incubation at 28˚C and restreaked on TSA to obtain pure cultures. Initial identification of the isolates was based on colony morphology, Gram-stain reaction, position of endospores, and production of catalase and oxidase. The isolates were characterized phenotypically and their diversity was assessed by comparing their DNA fragment patterns following their amplification by REP-, BOX- and IS50-PCR and electrophoresis on agarose gels. The data were converted into a binary matrix using TotalLab TL120 software, where the digits 1/0 represented the presence/absence of a DNA band. The similarity matrix and clustering were generated by the use of NTSYS 2.02e software based on the unweighted pair group method with arithmetic average (UPGMA) clustering algorithm. A representative isolate was selected for each of the DNA profile groups and a fragment of the 16S rRNA and HSP60 genes of each was amplified by PCR. PCR products were sequenced by Microsynth company (Switzerland) through the Sanger sequencing method. The nucleotide sequences were aligned and compared with those of a collection of Bacillus species retreived from GenBank using the BLASTn program. Phylogenetic trees were constructed using the Maximum-Likelihood and Neighbor-Joining methods with 1000 bootstraps in MEGA X software package. Antibacterial activity of Bacillus isolates against Brenneria roseae, the causal agent of hornbeam wetwood disease, was investigated using sterilized blank paper disk impregnated with extracts prepared in dichloromethane. The isolates were assessed for their capacity to produce Indole acetic acid (IAA), hydrogen cyanide, protease, and biofilm.

Thirty bacterial strains were isolated from hornbeam roots on TSA medium and subsequently characterized. The absence of bacterial colonies on the TSA plates inoculated with the final rinse water from the uncrushed bark fragments indicated the effectiveness of the surface disinfection procedure. The isolates were grouped into three clusters based on their phenotypic and genotypic characteristics. Comparison of the nucleotide sequences of the 16S rDNA and HSP60 with the sequences obtained from GenBank (NCBI) led to the identification of Bacillus thuringiensis, B. subtilis, and B. mycoides as the species constituting the three clusters, with B. thuringiensis being the predominant group. B. subtilis was the most populated and B. mycoides the least encountered species. Phylogenetic analysis based on the HSP60 gene sequence proved to be a reliable approach for determining the Bacillus species. IAA production level varied from eight to 75 mg/L among the species, with B. mycoides isolates producing the highest and B. subtilis the lowest levels of IAA. Most of the isolates had the capability to produce lipase, gelatinase, lecithinase, protease, and amylase. The isolates of B. thuringiensis produced higher levels of protease than the rest of the isolates. Biofilm production was particularly prominent in isolates of B. subtilis. The dichloromethane extract of B. subtilis isolate rAqa2 exhibited the highest growth inhibition (18.3 ± 0.25mm in diameter) of Brenneria roseae.

A high level of genotypic and phenotypic heterogeneity existed among the endophyte isolates recovered from the roots of C. betulus, despite their isolation from a common source species. This could be partly justified by viewing the Hyrcanian forests as a mixed-species ecosystem and a source of microbial consortia that could protect plants against stresses. This study is the first investigation on the molecular identification and characterization of endophytic Bacillus isolates from hornbeam roots in Iran.

The bacterial mosaic induced by Clavibacter tessellarius is one of the wheat seed-borne diseases that causes mosaic and chlorotic leaf spots on infected plants. The disease has previously been reported in a few regions within Iran. There is still no information on the disease's prevalence and distribution in the major wheat-growing areas of southern Iran. The disease may have gone unnoticed in part due to its mild symptoms and resemblance to those caused by viruses and certain non-infectious wheat diseases. This study aimed to determine the occurrence and distribution of wheat bacterial mosaic in wheat fields in southern Iran.

Between 2019 and 2020, surveys in wheat fields in four southern Iranian provinces, including Fars, Khuzestan, Kerman, and Bushehr were conducted, and 192 plant samples with symptoms resembling the bacterial mosaic were collected. Bacteria were isolated and purified using semi-selective media, and Gram-positive isolates were selected using Gram staining and KOH solubility tests. The isolates were identified using a polymerase chain reaction (PCR) with Clavibacter-specific primer pair CMR16F1/CMR16R1, as well as their phenotypic and pathogenicity characteristics. Additionally, a fragment of the gyrB gene from the representative isolates was amplified by PCR using primer pair 2F/4R. The PCR products were purified and directly sequenced. The obtained sequences were aligned, trimmed, and compared to those of related strains in the GenBank using the BLAST search method. A phylogenetic tree based on the gyrB sequences was constructed using the MEGAX software employing the neighbor-joining method.

In total, 400 bacterial isolates were obtained. Based on positive PCR results with specific primers for the genus Clavibacter along with the pathogenicity test on wheat, and the phenotypic and biochemical characteristics, 61 isolates were identified as C. tessellarius. In ten representative isolates, a ~530 bp fragment of the gyrB gene was amplified by PCR. The gyrB gene partial sequences were edited and deposited in GenBank under the accession numbers MZ014474–MZ01483. A BLAST search confirmed that all ten isolates shared the highest nucleotide identity (98.8% to 99.5%) with their corresponding sequences in C. tessellarius strains in GenBank. In the phylogenetic tree, these isolates with C. tessellarius strains from GenBank were clustered in a clade distinct from Clavibacter species and subspecies of Clavibacter michiganensis.

C. tessellarius was isolated from 53 out of 192 wheat fields (27.60%), exhibiting bacterial mosaic symptoms. The Fars, Khuzestan, Kerman, and Bushehr provinces had disease incidences of 35.7% (30 infected farms out of 84 sampled farms), 30.55%, 22.7%, and 7.1%, respectively, indicating the relatively widespread occurrence of the disease in these provinces. To our knowledge, the wheat bacterial mosaic is reported for the first time in the provinces of Khuzestan, Kerman, and Bushehr, and its occurrence in other parts of the country would not be unexpected.

Bacterial leaf streak of cereal was first reported from wheat and barley in the early twentieth century. This disease was observed in wheat and barley fields of Kerman province in 1987 and then isolated and identified from infected wheat and barley in different provinces of Iran, as Xanthomonas translucens pv. undulosa and X. t. pv. translucens respectively. In recent years, this disease became epidemic in Iran. For this reason, it was necessary to re-examine once again the different characteristics of the causal agents of this disease in different regions of Iran to determine if there is any possible genetic modification in these bacteria.

The phenotypic and genetic diversity of 12 bacterial strains isolated from wheat and barley (four isolates from wheat, six isolates from barley, one isolate from Phalaris sp. and one isolate from Lolium sp.) originated from Kerman, Kermanshah, and Fars provinces was studied. These isolates (selected from 67 isolates) were compared with three ancient Iranian standard strains (obtained from Unité de Phytopathologie, UCL, Louvain-la-Neuve, Belgium). The strains' pathogenicity test was performed on wheat and barley seedlings by spraying and injection methods. Then, the biochemical and physiological characteristics and SDS-PAGE profiles of the whole cell proteins of isolates were examined. Finally, using rep-PCR by BOX A1R, ERIC 1R, and ERIC 2 primers, the genetic diversity of isolates was performed.

All strains were Gram, oxidase, and urease negative, and catalase positive. All strains produced xanthomonadin pigment. The strains could produce hydrogen sulfide from cystine and peptone. Negative results were obtained for arginine dihydrolase, urease, starch hydrolysis, nitrate reduction, acetoin and indole production, and methyl red reaction. All isolates produced acid from cellobiose, fructose, galactose, di-mannose, arabinose, xylose, trehalose, arabitol, but not from salicin, mannitol, melibiose, rhamnose, dulcitol, sorbitol, and mesoinositol. They utilized acetate, citrate, and lactate but not d-tartrate and inulin. Only barley isolates could utilize lactose and hydrolyze lecithin and starch. There are no discriminating bands among PAGE profiles of wheat, barley, and standard strains. Genomic fingerprinting of isolates was performed, and the data were analyzed using NTSYS V 2.2 software and the UPGMA cluster analysis method. All isolates of wheat, Phalaris sp., Lolium sp. and one isolate from barley along with UPB922 standard strain were formed one group consists of three subgroups. Isolates of this group could infect wheat and barley plants. The rest of barley isolates, which were pathogenic only on barley, separated from the first group at a similarity coefficient of 84% and formed the second group, including the standard strain UPB922. Furthermore, this group had three subgroups. This grouping was completely related to the host range from the isolates. Based on biochemical, physiological, pathogenicity (host range) characteristics of strains and their genetic diversity compared to the standard strains, the first group, and the second group isolates were identified as X. t. pv. undulosa and X. t. pv. translucens, respectively.

It was found that the isolates are divided into two separate groups based on their host range. One group could contaminate wheat and barley plants, and another group infected only barley plants. Biochemical and physiological tests did not show much difference among the strains, but some tests were able to distinguish two pathovars, such as lactose utilization, the activity of lecithinase and lipase enzymes (hydrolysis of Tween 80), the tolerance of NaCl in the culture medium and the amount of slimes, produced on the nutrient agar medium containing glucose or sucrose. Rep-PCR tests could show the differences and variations within each pathovar. The grouping formed based on these tests showed a high correlation with the strain host range of pathovars. Using both BOX and ERIC primers showed that the genetic diversity of isolates was strongly related to the pathovars type and their host range. Since previous studies showed the same diversity in Iranian isolates, so it seems that there has not been a genetic change in the causal agent of this disease in Iran. The disease widespread occurred in recent years could be mainly due to environmental conditions and wheat and barley cultivars response to this disease.

Previous studies in Iran have explored the impact of using technology on improving students’ mathematical understanding. However, no study was conducted in relation to the impact of using technology on students’ mathematical misconceptions. This study explored the impact of using software in developing students’ misconceptions. In detail, the impact of using GeoGebra software on secondary school students’ misconceptions related to concepts such as angle scale, trigonometric angles, periodicity, minimum and maximum of trigonometric functions were explored using a two-tier diagnostic test. One of the novelties of this study is the use of a two-tier diagnostic test to explore misconceptions resulting from using the software.

The statistical population of this study comprises all grade 11 students of Golbahar and Golmakan in the academic year 2015-2016. Three classes were chosen from two different schools in these cities, one was considered as the control group (40 students) and the other two classes were considered as the experimental group (26 students). The instruments were a pre-test and a post-test (two-tier diagnostic test).‎ Four categories of misconceptions were identified based on the relevant literature and students’ responses to the pre-test. Finally, these misconceptions were analyzed by the chi-square test.

The findings showed that Geogebra software helped students in the experimental group enormously in understanding concepts such as periodicity‎, ‎identifying minimum and maximum of trigonometric functions‎, ‎and prevented developing misconceptions related to them. Analyzing students’ responses in the control group that received traditional teaching showed that several students did not able to calculate the periodicity of trigonometric functions. This difficulty observed both when students calculated the periodicity from the graphs and also when calculated the periodicity from the algebraic form of trigonometric functions. The strength of using the software includes observing many trigonometric graphs in the software environment, the ability to place trigonometric functions with different input on a coordinate axis and comparing them, and the manipulations performed by the students themselves on trigonometric graphs. These strengths prevented students from developing misconceptions about the concepts of frequency and minimum and maximum values. However, in relation to trigonometric angles‎, ‎using the software caused developing more misconceptions for the test group, and had no significant impact on preventing misconceptions in relation to the scale of angle‎. It seems due to the nature of the angle scale, in which the conversion from radians to degrees (or vice versa) is done by a series of mathematical operations, using Geogebra could not impact students’ misconceptions in this matter.

The results of this study indicate that teachers should be very cautious in selecting and using teaching aids in the classroom to prevent developing mathematical misconceptions associated with using the teaching aids. Therefore, we recommend mathematics education researchers and mathematics curriculum planners to conduct several studies on different softwares frequently used in mathematics classes, determine the pros and cons of these tools, and share their results with mathematics teachers. Sharing these results will help mathematics teachers to adapt their teaching accordingly based on the findings of these studies.

Wheat (Triticum aestivum) is the most important food crop in the world, but its yield is adversely affected due to plant pathogens particularly bacterial leaf blight (BLB) caused by Pseudomonas syringae pv. syringae (Pss). The management method presently in practice is insufficient to meet current safety and/or efficacy standards. Therefore, use of resistant genotypes is the best approach to manage BLB. The present study was undertaken to identify possible sources of resistance to Pss in cultivars and germplasms of wheat available. Two strains of Pss were isolated from symptomatic leaves of wheat and barley in Kerman province. The isolates were identified as Pss on the basis of physiological and biochemical characters, using specific primers and sequencing of 16S rRNA, ITS, and gyrB, rpoD, hrpL genes. Pss strains produced necrotic streaks on the susceptible wheat cv. Golestan. These strains were used for inoculation of 99 winter and spring wheat and durum (Triticum durum) genotypes to identify possible sources of resistance to BLB. The reaction of infiltrated seedlings fourth leaves infiltrated with bacterial suspension was scored seven to ten days after inoculation. Accessions were arranged in a randomized complete block design and three replications and five plants in each replicate were used. Among all the genotypes evaluated, only cv. Omid was found to be resistant to BLB. This wheat genotype can potentially be used in breeding wheat cultivars for resistance to BLB.

Citrus stubborn disease is caused by the helical mollicute Spiroplasma citri. The pathogen is naturally transmitted by sap feeding leafhopper vectors. In the present study attempts were made to detect S. citri in presumptive vectors. Leafhoppers were collected from various wild and cultivated plants in citrus growing areas in Jirfot. Fifteen leafhopper species were identified and 12 species were analyzed for the possibility of carrying S. citri with an antiserum prepared against a citrus strain of S. citri by indirect ELISA. S. citri was detected in leafhopper species by ELISA in Austroagallia sinuata, Psammotettix alienus, Circulifer haematoceps, Orosius albicinctus and Psamotettix striatus. However, S. citri was isolated only from the last three leafhopper species in LD10 medium. These leafhoppers were primarily captured from sesame fields. Therefore, these potential vectors and sesame farms may play a key role in the epidemiology of S. citri in the surveyed area.

Genetic male sterility is controlled by one pair of ressesive allele (aa) in sugar beet. This trait is used in most breeding programes. The exsistance of the character in a line or population facilitates transfer of important trait to the breeding material (for example resistance to plant disease). Also, it is possible to increase genetic diversity of monogerm populations by using genetic male sterility. The time and cost of transferring of this gene will be decreased, if the character is tagged with a molecular marker. Bulked segregant analysis using 302 RAPD primers in two F2 populations (231 and 261 population) was performed for the the identification of RAPD markers linked to the genetic male sterility gene. DNA preparation from 8 male fertile and male sterile plants were separately mixed. At first, the primers were tested on bulks. The primers with polymorphic bands were tested on individual plants of the bulks. Only if the polymorphism of the primers was confirmed, they were tested on the other individual plants. Finally, 10 and 6 markers were identified in 231 and 261 populations, respectively, which their distances to male sterility gene were lower than 50 cM. AB-8-18-600r marker was the nearest marker to male sterility gene. This marker showed only 3 and 1 recombination in 231 and 261 populations, respectively. The distance of this marker and genetic male sterility locus was estimated as 5.3 cM in combined F2 populations.

The effect of Rathayibacter tritici on the movement of Anguina tritici larva and nematode function as vector of ear rot bacterium was conducted in the laboratory (Agarose plates) and greenhouse conditions. The results showed that the contact of nematode larva with high concentration of bacterium or long duration of nematode-bacteria contact can decrease the movement and the efficiency of nematode function as the vector of the disease, and in some cases it resulted in the mortality of the nematode. No differences were detected in the mobility of larva in the concentrations less than 102 CFU and less than 0.5 hour of nematode-bacteria contact times and their controls (exposed to water alone). Movement of the nematodes appeared to be random under these conditions. It can be concluded that Rathayibacter tritici did not act as an attractant to Anguina tritici larva. These results suggest that the attachment of a large number of bacteria to nematode (as an essential vector of the bacterium) would induce nematode weakness and mortality. So it is possible that ear rot bacterium can parasite ear cockle nematode, or the nematode is a host for this bacterium .

The present experiment was aimed to evaluate the freezing tolerance of two cold tolerant (MCC426 and MCC252) and a cold susceptible (MCC505) chickpea genotypes. The study was carried out in a split-plot factorial design with three replications. Factorial arrangement of genotype and acclimation (acclimation and non acclimation) were imposed as main plot and temperatures (0, -4, -8, -12, 16, -20ºC) as subplot. The effect of freezing temperature (FT) on plant survival was significantly different among genotypes (p<0.05). According to the average effects of acclimation and FT, the plant survival in MCC426 and MCC252 was 40% and 31% respectively more than in MCC505. Lethal temperature for 50% response (LT50) and temperature resulting in 50% lower dry matter (DMT50) in MCC426 were –10.8ºC and –8.4ºC, respectively and were lower than the other genotypes. Acclimation increased the freezing tolerance such that MCC426 tolerated up to –12ºC without any mortality, however, at this temperature, plant mortality rates in MCC252 and MCC505 were 25.7% and 67.7%, respectively. Plant regrowth was affected by the intensity of FT, such that plant dry weight (PDW) and stem height (SH) in –12ºC decreased about 63% and 50%, respectively, compared with non - frozen control plants. The most freezing damage was observed in MCC505, -12ºC treatment caused 90% decreases in PDW and SH, but at this temperature, PDW and SH in MCC425 decreased 55% and 49% and in MCC252, the reduction was about 60%and 54%, respectively. It seems that the use of controlled experiments would contribute to the evaluation of freezing tolerance and screening programs in chickpea germplasm for the estimation of LT50 and DMT50 .

The fire blight with the bacterial causal agent, Erwinia amylovora (Burrill) winslow etal. is one of the most important diseases of the pome fruits that causes the economical losses to quince, pear and apple productions, respectively, in some parts of country. To determine the infection severity of the 43 raturally infected pear cultivars in collection of karaj horticulture research division and also for studing the reaction of these cultivars against disease, the evaluation was performed by the USDA standard system for these cultivars in collection. In this study, although the most of cultivars had been infected on the natural conditions, but disease severity was significantly different between them. By the USDA system, pear cultivars were divided in the 2 classes. Percentage of pear cultivars in very susceptible and moderately susceptible classes were 81.4 and 18.6 respectively. Also for dividing the pear cultivars, beside of USDA method, SPSS software and the cluster analysis were also by the UPGMA method and cultivars were divided into 3 clusters, but it seems that the classification of cluster analysis did not conform with USDA system. Correlation of I.V.S in the artificial tests and the disease severity by natural infection was very significant (r= -0.83).

Linkage maps are essential for genome studies in different crops. The objective of the study was to construct a linkage map using Khatoonie, a local melon accession in Iran because of its characteristics as a main accession in Iran. Hence, a cross between Khatoonie and the French line Charentais was made to obtain the F2 mapping population. To construct map, a total 42 RAPD primers was used. Linkage analysis indicated that 109RAPDs assigned to 13 linkage groups with a total length of 504.7 cM and distance between adjacent markers in average was 5.2 cM. Adding more markers to this basic map is on going because of being in lack of any linkage map for germplasm of Iranian melon. This map can be used in marker assisted breeding projects and for map-based cloning.