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In March-April of 2020-2022, round and water-soaked spots surrounded by a brown border were observed on the green bean pods delivered to the markets of Isfahan from the southern provinces of the country. A bacterium with a fluorescent appearance and a spindle-shaped structure with irregular edges was routinely isolated from the 81 specimens collected. The genetic fingerprinting employing rep-PCR revealed a resemblance between the PCR product profiles of the isolates, which is consistent with their pathogenic and biochemical characteristics. The PCR reaction of representative isolates produced the expected-size fragments of 16Sr DNA through genus-specific primer sets. Subsequent analysis of the sequences of the fragments confirmed the affiliation of the isolates with Pseudomonas spp. The expected-sized amplicons were amplified and sequenced in nested-PCR tests using phaseolotoxin gene based primer pairs. The results showed that the nucleotide sequences were 99% homologous to Pseudomonas savastanoi pv. phaseolicola (Psp). By conducting a phylogenetic analysis with sequences of housekeeping genes gyrB and rpoD, the isolates were clustered with reference strains of Psp, confirming their identity. Considering the isolates’ morphological, biochemical and molecular characteristics, Psp was identified as the cause of halo blight disease in beans imported to Isfahan from the southern regions. In laboratory conditions, the persistence of the pathogen on seed was evaluated for more than one year. The long-term viability of Psp on seeds may lead to the ongoing prevalence of bean halo blight disease in the country in the forthcoming years, due to the cool and moist conditions resulting from spring rains.

Genetically modified (GM) plants are one of the great achievements of modern biotechnology in agriculture, which their cultivation has been increased in recent years. According to the international biosafety law, the import of genetically modified plants without the permission of Biosafety Committee into the country is prohibited. Therefore, we need an accurate method for assessing whether they are transgenic or not. In this study, samples of tomatoes were collected from various sources such as shops and greenhouses in Isfahan province to evaluate if they are transgenic. The initial screening of all samples was performed using primer pairs P35S F/R for CaMV35S promoter and NOS-1/NOS-3 for NOS terminator, amplified 195 bp and 180 bp of 35S promoter and NOS terminator sequences, respectively. The sequence of fragments was verified by comparison with known sequences in GeneBank and indicated a 100% homology, showing the studied samples are genetically engineered. The tomato samples were also tested for the presence of antisense polygalactoronase (PG) gene using the primer pair PG F/R, which amplified 384bp in all samples. Sequence analysis of this fragment resembled 100% similarity with the sequence of PG antisense gene in Gene Bank database. Results of this study showed that there is a transgenic tomato in the Iranian market. But, the consumers are not aware of its changed nature.

Potato soft rot is a common and important disease on potatoes. Fifteen pectolytic bacteria with green metallic colonies on EMB medium were isolated from the tubers showing surface cracking and soft rot symptoms, simultaneously. All the isolates grew at 37 ˚C, no starch digestion, they were not sensitive to erythromycin and negative for phosphatase. They could not use sugars of Xylose, Trehalose, α-methyl D-glycoside and Malonate, while they were able to use Inositol, Rhamnose and Arabitol sugars. Based on These characteristics the isolates recognized as P. carotovorum subsp. carotovorum. The isolates were further identified by PCR amplification with primer G1/L1 which produces two bands similar to those produced in P. carotovorum subsp. carotovorum, but not from P. carotovorum subsp. atrosepticum and Dickeya chrysanthemi. Digestion of the amplified PCR product with restriction enzyme Rsa I resulted in identical 385 bp, 280bp and 180 bp bands formation as described for P. carotovorum subsp. carotovorum. The results of physiological, pathogenicity, biochemical and molecular tests, confirmed that all bacterial isolates induced soft rot symptoms in potato tubers are P. carotovorum subsp. carotovorum.

The health aspects of vegetables containing high anthocyanin has increased the consumption of colored potatoes rich in anthocyanin in recent years. Anthocyanin’s in blue and purple potatoes are derived from delphinidin, while in the red ones they are resulted from pelargonidin. Three genes, D, P and R interact for production of delphinidin and pelargonidin within the anthocyanin biosynthesis pathway that eventually results in colored potato flesh. Studying genetic and allelic relations among these three major genes provides a better understanding of anthocyanin biosynthesis pathway in potato. In this research, 17 potato genotypes of varying tuber color were used to detect the three key genes of D, P and R using specific primers and to compare their restriction fragment patterns. Results showed that purple genotypes contained the dominant allele for the P locus and the white and the red genotypes had the recessive allele. The white colored potatoes contained only the r allele in a homozygous state while the red and purple colored potatoes had both R and r alleles in a heterozygous condition masking the phenotypical presence of r. Restriction fragment patterns of the PCR products clearly revealed that none of the white potato genotypes possessed the D allele but the colored genotypes contained both D and d alleles in a heterozygous condition with an unknown allelic arrangements. D is an important regulatory gene for both P and R genes and the dominant allele D is required for their expression and anthocyanin production in the potato tuber. In other words, having dominant alleles of P and R in a potato plant does not necessarily give colored potato if the dominant allele D is not present. Albeit, restriction fragment pattern procedure discriminated the colored genotypes from the white tuber ones in P and R genes but primarily determination of D locus dominant status as a key regulatory factor in anthocyanin biosynthesis pathway is more important.

The pistachio (Pistacia vera), a member of the cashew family, is a small tree originating from Central Asia and the Middle East. The tree produces seeds that are widely consumed as food. Pistacia vera often is confused with other species in the genus Pistacia that are also known as pistachio. These other species can be distinguished by their geographic distributions and their seeds which are much smaller and have a soft shell. Continual advances in crop improvement through plant breeding are driven by the available genetic diversity. Therefore, the recognition and measurement of such diversity is crucial to breeding programs. In the past 20 years, the major effort in plant breeding has changed from quantitative to molecular genetics with emphasis on quantitative trait loci (QTL) identification and marker assisted selection (MAS). The germplasm-regressioncombined association studies not only allow mapping of genes/QTLs with higher level of confidence, but also allow detection of genes/QTLs, which will otherwise escape detection in linkage-based QTL studies based on the planned populations. The development of the marker-based technology offers a fast, reliable, and easy way to perform multiple regression analysis and comprise an alternative approach to breeding in diverse species of plants. The availability of many makers and morphological traits can help to regression analysis between these markers and morphological traits.
In this study, 20 genotypes of Pistachio were studied and yield related traits were measured. Young well-expanded leaves were collected for DNA extraction and total genomic DNA was extracted. Genotyping was performed using 15 RAPD primers and PCR amplification products were visualized by gel electrophoresis. The reproducible RAPD fragments were scored on the basis of present (1) or absent (0) bands and a binary matrix constructed using each molecular marker. Association analysis between molecular date (as independent variable) and morphological data (as dependent variable) was performed using multiple regression analysis to identify informative markers associated with the yield related traits. Multiple regression analysis was conducted using stepwise method of linear regression analysis option of SPSS. Student t-test was performed to assess significance difference between mean trait estimates of genotypes where specific markers were present and absent. Markers shown significant regression values were considered to be associated with the trait under consideration.
Finally 11 primers were polymorphic and a total of 56 pieces (loci) were amplified that among these, 36 segments (64.29%) showed polymorphism with an average of 5.09% per primers and the rate of this polymorphism ranged from at least 25% for AJ05 primer up to 87.5% for OPAD02 primer. Polymorphic information content ranged from 0.095 (AJ05 and OPAD14) to 0.39 (OP 05), with an average of 0.23. Stepwise regression analysis between molecular data and traits was performed to identify informative markers associated with yield component traits. Nineteen RAPD fragments were found associated with six yield related traits. Some of RAPD markers were associated with more than one trait in multiple regression analysis that may be due to pleiotropic effect of the linked quantitative trait locus on different traits. However, to better understand these relationships, preparation of segregating population and linkage mapping is necessary. Also, these results could be useful in marker-assisted breeding programs when no other genetic information is available.
This investigation on molecular markers associated with yield traits in Pistachio has provided clues for identification of the genotypes with higher yield value. In breeding programs selection of quality material is often a time-consuming process, and thus marker-assisted selection could be of great useful in identification of promising genotypes with high value of yield traits. Some of RAPD markers can be used for elite selection of Pistachio, particularly when no other genetic information like linkage maps and quantitative trait loci are available for the species. The applications of the RAPD approach enable us to predict positive correlation between data generated by molecular markers and studied traits. Also, the marker–trait association identification will play an important role in plant MAS/QTL breeding programs, especially in plants where genetic information such as linkage map and QTL is not available.

Chickpea is commonly grown in large scales in the western part of Iran (Kermanshah، Hamedan، Lorestan and Chahar Mahal & Bakhtiari) and it forms a symbiotic relationship with rhizobia which promotes legumes growth through nitrogenfixation. Therefore، it is important to evaluate the population genetic structure of rhizobia nodulating chickpea in various areas to manage extension of chickpea cultivation. In this study، the characterization of chickpea nodulating rhizobia was carried out by PCR-RFLP analysis and nucleotide sequence determination of 16S rDNA region in addition to tracking and sequencing of nodC and nifH genes، which directly affected on Rhizobium- legume symbiosis. The results indicated that the diversity of rhizobia isolated from chickpeas in different sites is limited and they mainly belonged to Mesorhizobium cicer. Few strains of Agrobacrium tumefaciens were isolated from root nodules of chickpea growing in Lorestan soils indicating the absence of nodC and nifH genes in these isolates raising doubt about their nitrogen fixation capability. It seems that the long history of chickpea cultivation in western provinces and the specific relationship of chickpea with its symbionats are the main factors in predominance of M. cicer and absence of other Misorhizobium sp. in symbiosis with chickpea in Iran.

To characterize the distribution of melanin producing Medicago-nodulating rhizobia in western and north-western of Iran, 982 Sinorhizobium isolates were trapped from a combination of three alfalfa populations (Hamedani, Nikshahri, Kodi) and soil samples from eight sites across Kurdestan, Kermanshah, Eastern Azarbayjan and Lorestan provinces. All rhizobial isolates were examined for their ability to produce melanin (Mel+). According to the results, 24.1% of them were Mel+, as well as the reference strain S. meliloti GR4. Analysis of melanin production revealed more melanin producing isolates among different sites in comparison of different cultivars. Therefore, it can be concluded that the influence of field soil type in dominance of melanin producing strains is more than the host cultivars. The presence of 456 bp amplified fragment using primers designed from the mepA gene in 30 strains of rhizobial strains and its absence in 20 other strains was in accordance with the results of melanin production in the medium, showing that all strains producing melanin contained mepA gene. Multiple sequence alignment revealed that the sequence of mepA gene in S. meliloti is conserved, while this sequence in S. medicae had limited changes.

Armillaria root and crown rot is an important disease of woody plants including fruit, forest and ornamental trees in different regions. The disease is common in Isfahan and causes economical losses on a broad range of fruit trees. There only a few tolerant varieties to disease, and there is no efficient method in controlling the disease. Therefore, detection of the pathogen from soil is necessary to establish an IPM program for decreasing losses. In this study, specific primers were successfully used for rapid, sensitive and specific detection of Armillaria isolates from soil and wood samples. DNA was isolated directly from soil and wood samples and ITS region was amplified using universal primers ITS1 and ITS4 and specific primers AR1 and AR2 in nested PCR. The nested PCR method enables the detection of pathogen directly from soil and root samples without the need for isolation and cultivation of mycelium of Armillaria and in earlier steps of the disease progress.

Armillaria mellea the causal agent of root and crown rot of trees has a universal distribution and causes extensive economic disease on a broad host range of trees in gardens, forests and urban environments. The pathogen can survive under the bark as inoculum and there is no effective control method against the disease, so pathogen detection from soil and wood is important to predict disease severity and prevent disease dispersal from one tree to another. To achieve a rapid and sensitive detection method for A. mellea in soil and wood, sampling was performed from fruiting bodies, soil and wood and after isolation of pathogen on culture media, comparison was done among different detection methods using a semi-selective medium, baiting and molecular methods. Inoculum was prepared for two isolates of A. mellea and was inoculated to soil. Pathogen detection was done with soil direct culture method, baiting and nested PCR simultaneously. The culture medium was not effective to detect pathogen in long-term period. In baiting method, Pelargonium hortorum was used as a baiting plant that required long time to detect pathogen. The results revealed that nested PCR is an efficient method for detection of A. mellea.

To achieve a simple and sensitive Rosellinia necatrix detection method in soil, three detection methods including soil direct culture method, infected soil in culture media e. g. MEAR, MEAR + penconazole and MEAS, Nested PCR method and bait twig method were compared. Direct DNA Extraction from soil was done. Two specific primer pairs R2، R8 and R7، R10 in Nested PCR were used and the extracted DNA was purified using PVPP column. Among used methods, the direct culture method from soil was not suitable and wasn’t sensitive for detection. However, bait twig method was capable to detect, the pathogen in soil and root but it is time consuming and laborious method. In conventional PCR, using primer pairs R2-R8, 10 pg μl−1 of fungal DNA was detected; however in Nested PCR using R7-R10 primer pairs, 1fg μl−1 of fungal DNA was detected. Among used methods, the Nested PCR was better in detection of the fungus. Nested PCR method of detection in soil and root is more sensitive and reliable.

Considering the high level of morphological diversity in Iranian pomegranate cultivars, comparison of genetic variation among 24 pomegranate cultivars was evaluated using random amplified polymorphic DNA (RAPD) and inter simple sequence repeat (ISSR) markers. RAPD primers amplified 131 DNA fragments among which 29 were polymorphic (22.14%) and ISSR markers produced 173 amplification products, out of which 64 were polymorphic (37%). Mean PIC (polymorphic information content) was 0.128 for RAPD and 0.163 for ISSR. The results suggested that the ISSR markers produced much better reproducible bands and were more efficient in grouping cultivars. Pairwise similarity index values ranged from 0.353 to 1.0 (RAPD), 0.291 to 0.930 (ISSR) and mean similarity index values of 0.604 and 0.674 for RAPD and ISSR, respectively. The analysis of molecular variance (AMOVA) for RAPD and ISSR data showed no significant differences among the geographical regions and juice acidity of the used cultivars (P>0.05) indicated that genetic and geographic distances were not correlated.

Evaluation of genetic diversity is a considerable approach to screen within indigenous rhizobial population for compatible, highly effective strains, which can be further utilized as rhizobial inoculums. Genetic diversity of Rhizobium isolates from three clover species grown in soil samples of seven different geographical regions in Iran was investigated by Repetitive extra genomic palindromic fingerprinting (Rep-PCR) method. Out of 152 isolates tested in the study, 22 were identified as different strain types. Genome analysis of rhizobial isolates, as based on the fingerprinting profiles and the relating dendrogram, revealed considerable heterogeneity among isolates with different host specie's origin. The results demonstrate Rep-PCR fingerprinting method as a powerful technique to differentiate among closely related strains of Rhizobium leguminosarum bv. trifolii. The results also suggest that clover species tend to preferably absorb their rhizobial symbionts.

White root rot disease, caused by Rosellinia necatrix is one of the most important disease of fruit trees in Iran and worldwide. To detect the pathogen from soil and root, six infected soil samples were collected from cherry crown trees and two samples were collected as positive and negative control. DNA Extraction from eight soil samples including infected cherry roots by white cottony mycelium, roots infected by white mycelial fans and symptom less roots and two faba roots, with white cottony mycelium and symptom less root was performed. Detection of the Pathogen was done by two specific primer pairs R2، R8 and R7، R10 in Nested PCR reaction. The results showed the detection from six infected soil, positive control and in roots colonized by white cottony mycelium and roots by white mycelial fans and faba roots colonized by white cottony mycelium. No detection was achieved from symptom less roots. To verify the accuracy of the used detection method, ITS region was amplified and cloned in pTZ57R/T vector and sequenced. Comparison of the sequence showed %98. 99 similarities to the recognized isolate of R. necatrix. According to our findings, it seems that Nested PCR method could be useful to rapid detection of R. necatrix from soil and root of plants in orchards.

To understand the role of relative humidity rate, host genotype, inoculation method and growth stage in epidemiology of bean common blight, two greenhouse experiments were carried out monitoring epiphytic population size of Xanthomonas axonopodis pv. phaseoli (Xap) and disease severity. The result showed significant differences among genotypes, inoculation methods and growth stages for epiphytic population size and sam effects except genotypes for disease severity. The epiphytic population size was significantly higher on spray inoculated Khomein cultivar of bean during flowering (R6). However, the relative humidity rates did not significantly affect population dynamics of epiphytic Xap and the disease severity. Two field experiments were also carried out to determine the effects of irrigation systems (furrow irrigation and overhead sprinkler irrigation), inoculation method, growth stage and their interactions on epiphytic population size of Xap and disease severity. The result showed that the epiphytic population size and disease severity were higher on spray inoculated plants irrigated with overhead sprinkler system during pods filling (R8). In this study, a significant positive correlation was found between epiphytic population size of Xap and bean common bacterial blight severity.

To characterize the geographical distribution of medicago-nodulating rhizobia in western regions of Iran, 950 Sinorhizobium isolates were trapped from a combination of two local alfalfa populations (Hamedani, Nikshahri) together with a foreign cultivar ( Kodi) and soil samples from eight sites across Kurdestan, Kermanshah, Eastern Azarbayjan and Lorestan provinces. Also, a total of 45 isolates were obtained from nodules of naturally grown Melilotus officinalis (14 isolates) and Trigonella foenum-graecum (31 isolates) plants in Isfahan. On the basis of PCR partial amplification of the plasmid born nod box gene and chromosomal mucR gene of the isolates,16S ribosomal DNA PCR-restriction fragment length polymorphism analysis, and the nucleotide sequence, three isolates from alfalfa, seven isolates from M. officinalis and 13 isolates from T. foenum-graecum were proved to be Sinorhizobium medicae. The remaining isolates (943 from alfalfa, seven from M. officinalis and 18 from T. foenum-graecum) were identified as S. melilloti. Both species, S. meliloti and S. medicae, were recovered from nodules of all the hosts although S. meloti was clearly more dominant in nodulating different populations of alfalfa. Taken together, these results indicated that the abundance of S. meliloti is independent of the site of isolation and have a wide geographical distribution. In this study, the banding pattern resulting from PCR amplification of 16S rRNA gene, followed by digestion with Rsa I, clearly differentiated S. meliloti and S. medica strains, showing that PCR-RFLP is an appropriate method to discriminate medicago-nodulating rhizobian with relative rapidity.

Microsatellite DNA markers isolated from wild species khinjuk (Pistacia khinjuk Stocks.) were used to evaluate the genetic diversity available in Iranian pistachio cultivars. Out of the 27 SSR primers tested initially, 25 could amplify the DNA in different pistachio cultivars, of which 19 primer pairs produced clear bands. Based on the amplification profiles of the genotypes by the remaining primer pairs, eight primers produced a monomorphic product and other 11 microsatellites markers were found polymorphic among the genotypes. The number of putative alleles amplified by each polymorphic SSR locus ranged from two to eleven alleles with a total of 48 alleles. An average of alleles and observed heterozygosity per locus was 3.69 and 0.69 respectively, showing that these microsatellites are highly informative for pistachio fingerprinting. The UPGMA cluster plots based on nei index placed the 20 commercial pistachio cultivars into a major group containing three distinguished subgroups however, genotypes, namely, Ghazvini zudras and Sarakhs (wild P. vera), were clearly situated into two distinct clusters, distant from the domesticated genotypes studied here. Both Ghazvini zudras and Sarakhs are known as small-fruited genotypes which are grown in restricted regions. Therefore, the distinctness of these genotypes can be attributed to their geographical isolation and morphological characteristics. It seems that Ghazvini zudras probably originated from Sarakhs variety which posses an important role in development of pistachio cultivars. The present study revealed that the khinjuk pistachio microsatellites are well distributed in the genome of P.vera , and are informative for estimating the extent of genetic diversity and characterization of pistachio cultivars.

Aflatoxins are the most serious problem in production and export of pistachio of Iran. They are amongst the most toxic mycotoxins and are produced predominantly by Aspergillus flavus and A. parasiticus. Pistachio is susceptible to invasion by aflatoxigenic Aspergillus species and subsequent production of aflatoxins, during preharvesting, processing, transportation or storage. Various methods have been used to detect toxin in pistachio such as chromatography (TLC and HPLC), ELISA and PCR base methods. Some of these methods e.g. chromatography, are time consuming, labor-intensive and expensive. In this study, nuts sample of pistachio were collected from pistachio orchards in Kerman, Rafsanjan and Isfahan. Culture media AFPA and PDA, were used to isolate Aspergillus species. Two hundred and fifty isolates were obtained and based on microscopic and macroscopic studies, the isolates belonged to the genera, Aspergillus, Fusarium, Rhizopus, Alternaria and Cladosporium.

The identification of potato cultivars is a recurrent objective of potato research. The research is prompted by the increasing number of potato cultivars and the importance of seed purity. In developing a reliable method for identification of the imported potato cultivars and determining their genetic relationship, the capacity of 10 polymorphic simple sequence repeat markers (SSRs) was evaluated for the analysis of 28 commercial cultivars of potato. The number of alleles detected at different loci ranged from 3 to 10 alleles with a total of 57 for all loci and a mean of 5.7 alleles per locus. In the 28 potato cultivars analyzed, the number of heterozygous genotypes per locus varied between 6 to 28 with an average number of heterozygous genotypes per locus of 18, considering the 10 loci studied. Based on the resulting dendrogram of jacquard's similarity coefficient and UPGMA analysis, the potato cultivars were placed in two major groups. However, the results from similarity coefficient confirmed the close phylogenetic relationships among members in each cluster. The dendrogram derived from SSRs data clustered together Kenebek, Florida and Atlantic which are known as American potato cultivars, but Stanbuli, an old cultivar in Iran, was placed in concert with European cultivars. This finding might be an indication that this cultivar along with other unidentified cultivars, growing in local fields, has been introduced from European countries to Iran. The results obtained illustrate the appropriate utility of SSRs to assess genetic relationships of potato cultivars and develop a PCR- based tool for evaluation of potato seed purity.

To determine genetic diversity among some Iranian local varieties of alfalfa, six geographically diverse populations including: Bami, Rahnani, Nikshahri, Yazdi, Hamadani (from Isfahan), Hamadani (from Shiraz) along with Ranger, an American commercial variety, were evaluated using a set of 24 EST-SSR primers developed from cDNA library of Medicago truncatula and three microsatellite loci, identified from genomic library of M. sativa. Of the pairs of primers tested, four loci from EST-SSRs (AW9, BEE, TC6 and TC7) and genomic microsatellite (Afctt32), were found appropriate for assessing genetic diversity between these alfalfa genotypes. In total, 46 alleles were detected from the five loci in the samples of alfalfa examined. The number of alleles per locus in populations ranged from six to eleven and genetic diversity indices of loci were variable from 0.62 to 0.87 for the populations. Genetic relationship analysis of EST-SSR data revealed separation of Iranian populations from Ranger. It is likely that the parental origin of primary population from which Ranger has been derived is different from that of Iranian populations. Iranian local populations of alfalfa in this study were grouped in two main clusters. Alfalfa populations Hamadani and Rahnani, which are adapted to cold claimates, were grouped in one cluster and populations Bami, Yazdi and Nikshahri, belonging to the trpoical areas, were placed in the next cluster. The positioning of EST-SSR loci in coding regions of genome, possibly increases the usefulness of these markers to clarify inter specific genetic relationships among alfalfa populations.