Isolation, identification, antibiotic resistance profile and molecular analysis of Ornithobacterium rhinotracheal isolates from turkeys.

BACKGROUND: Ornithobacterium rhinotracheal (ORT) infects numerous birds, particularly chickens and turkeys. ORT is an emerging bacterial pathogen of global concern in the poultry industry. As ORT is rapidly spreading throughout commercial poultry, it requires intensive studies of its epidemiology, diagnostic procedures, molecular typing, virulence genes and antimicrobial resistance. OBJECTIVES: The present study was conducted in isolation and identification of ORT from slaughtered turkeys. METHODS: Cleft palate swabs of 200 were collected from slaughtered turkeys and cultured on blood agar. ORT was characterized using biochemical tests and PCR targeting the ORT 16S rRNA gene. Virulence genes of isolates were determined targeting adenylate kinase (adk), copA and virulence-associated protein D (vapD) genes. Additionally, diversity of ORT isolates was performed by enterobacterial repetitive intergenic consensus (ERIC) and RAPD PCR. Disk diffusion was used to determine the antibiotic sensitivity of the isolates. RESULTS: ORT was identified in 23 (11.5%) samples using both the biochemical tests and PCR. The result of detecting virulence genes showed that all the isolates (23: 100%) had the adk gene, whereas two (8.7%) isolates had the copA gene, and seven (30.43%) isolates had the vapD gene. Molecular typing of isolates revealed 21 different patterns by RAPD PCR assay using M13 primer and 20 distinct patterns by ERIC PCR test. Both ERIC and RAPD PCR were distinctive methods for investigating the genetic diversity of ORT isolates. The antibiotic resistance test showed that 18 (78.26%) isolates were resistant to gentamicin, amikacin, cefazolin, streptomycin and penicillin. All isolates (100%) were resistant to cloxacillin and fosfomycin. CONCLUSIONS: This study showed the prevalence of ORT in turkey and high resistance of this bacterium to many common veterinary antibiotics. Moreover, both ERIC and RAPD PCR are distinctive methods for investigating the genetic diversity of ORT isolates. These data may help monitor antibiotic resistance and typing of ORT in epidemiological studies and serve as the foundation for designing region-specific vaccines for future use.

Molecular characterization of trypanocide-resistant strains derived from a single field isolate of Trypanosoma evansi.

Four strains (SB-PR, SB-RS, SB-RD, and SB-RM) of Trypanosoma evansi (T. evansi) were used in this study. SB-PR is known to be trypanocide-sensitive, while the others are trypanocide-resistant to suramin, diminazene diaceturate, and melarsomine hydrochloride, respectively. SB-RS, SB-RD, and SB-RM are derivatives of a single field isolate of SB-PR. Trypanocide resistance will not only increase costs and decrease production efficiency but will also affect effective treatment strategies. Therefore, studies on this topic are important to avoid inefficient production and ineffective treatment. This paper aims to presents a comparative molecular characterization of the trypanocide-resistant strains compared to the parent population. Comparative molecular characterization of these strains based on a protein profile analysis performed with sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE), DNA fingerprinting of random amplified polymorphic DNA (RAPD), and the molecular characterization of expression-site-associated 6 (ESAG6), variant surface glycoprotein (VSG), and T. evansi adenosine transporter-1 (TevAT1) gene sequences. The results show three derived strains (SB-RS, SB-RD, and SB-RM) exhibit different banding patterns than SB-PR. According to the RAPD results, SB-RS and SB-RD are different strains with DNA fingerprint similarities of about 77.8 %, while the DNA fingerprint of SB-RM has a similarity of 44.4 % to SB-RS and SB-RD. No differences in VSG were found among the four strains; however, ESAG6 showed differences in both nucleotide and amino acid sequences, as well as in its secondary and 3D structure. In conclusion, all molecular analyses of the ESAG6 gene showed that SB-PR, SB-RS, SB-RD, and SB-RM are different strains. Furthermore, SB-PR, SB-RS, SB-RD, and SB-RM did not exhibit the TevAT1 gene, so the resistance mechanism was determined to be unrelated to that gene.

Identification of Sex-Associated Genetic Markers in Pistacia lentiscus var. chia for Early Male Detection.

Pistacia lentiscus var. chia is a valuable crop for its high-added-value mastic, a resin with proven pharmaceutical and cosmeceutical properties harvested from the male tree trunk. To achieve the maximum economic benefits from the cultivation of male mastic trees, it is important to develop early sex diagnosis molecular tools for distinguishing the sex type. Thus far, the work on sex identification has focused on Pistacia vera with promising results; however, the low transferability rates of these markers in P. lentiscus necessitates the development of species-specific sex-linked markers for P. lentiscus var. chia. To our knowledge, this is the first report regarding: (i) the development of species-specific novel transcriptome-based markers for P. lentiscus var. chia and their assessment on male, female and monoecious individuals using PCR-HRM analysis, thus, introducing a cost-effective method for sex identification with high accuracy that can be applied with minimum infrastructure, (ii) the effective sex identification in mastic tree using a combination of different sex-linked ISSR and SCAR markers with 100% accuracy, and (iii) the impact evaluation of sex type on the genetic diversity of different P. lentiscus var. chia cultivars. The results of this study are expected to provide species-specific markers for accurate sex identification that could contribute to the selection process of male mastic trees at an early stage for mass propagation systems and to facilitate future breeding efforts related to sex-linked productivity and quality of mastic resin.

Genetic relatedness and diversity of Capillaria species infecting bayad (Bagrus bajad) in upper Egypt.

BACKGROUND: This study investigates the genetic characteristics of Capillaria isolates from the infected fish, Bagrus bajad, and their relation to human Capillaria philippinensis using Random Amplified Polymorphic DNA (RAPD-PCR) analysis. Fifteen fish Capillaria were isolated and compared to identified human C. philippinensis using six primers: M-are, M-1, G-7, G-11, G-15, and G-18. RESULTS: All six primers successfully amplified DNA, highlighting their efficacy in distinguishing between human and fish Capillaria isolates. The analysis revealed distinctive banding patterns between fish and human isolates, with variations in size and number of DNA fragments. Additionally, genetic similarity analysis showed intriguing patterns of relatedness, with certain pairs exhibiting high similarity percentages. Comparative assessment of RAPD polymorphism demonstrated consistent findings of 100% polymorphism across all primers. The Unweighted Pair Group Method with Arithmetic Mean Algorithm (UPGMA) evaluated the closest relationship between human and fish isolates. These results underscore the utility of RAPD analysis in delineating the genetic diversity among Capillaria isolates from different hosts. CONCLUSION: Overall, this study contributes to our understanding of the genetic variability and relatedness among Capillaria isolates, shedding light on their evolutionary dynamics and zoonotic potential.

Development of SCAR Markers for Genetic Authentication of Metarhizium acridum.

In this study, molecular typing using Randomly Amplified Polymorphic DNA (RAPD-PCR) was conducted on 16 original isolates of Metarhizium acridum obtained from locusts (Schistocerca piceifrons ssp. piceifrons.) in Mexico (MX). The analysis included reference strains of the genus Metarhizium sourced from various geographical regions. The isolates were identified by phenotypic (macro and micromorphology) and genotypic methods (RAPD-PCR and Amplified Fragment Length Polymorphisms (AFLP), through a multidimensional analysis of principal coordinates (PCoA) and a minimum spanning network (MST). Subsequently, Sequences-Characterized Amplified Region (SCAR) markers were developed for the molecular detection of M. acridum, these markers were chosen from polymorphic patterns obtained with 14 primers via RAPD-PCR. Phenotypic and genotypic characterization identified the MX isolates as M. acridum. Of all the polymorphic patterns obtained, only OPA04 and OPA05 were chosen, which presented species-specific bands for M. acridum, and further utilized to create SCAR markers through cloning and sequencing of the specific bands. The specificity of these two markers was confirmed via Southern hybridization. The SCAR markers (Ma-160OPA-05 and Ma-151OPA-04) exhibit remarkable sensitivity, detecting down to less than 0.1 ng, as well as high specificity, as evidenced by their inability to cross-amplify or generate amplification with DNAs from other strains of Metarhizium (as Metarhizium anisopliae) or different genera of entomopathogenic fungi (Cordyceps fumosorosea and Akanthomyces lecanii). These SCAR markers yield readily detectable results, showcasing high reproducibility. They serve as a valuable tool, especially in field applications.

Characterizations of Ganoderma species causing basal stem rot disease in coconut tree.

The basal stem rot disease incidence ranged from 0 to 5% in Karnataka India during the year 2019-20. Twenty pathogenic isolates of Ganoderma sp varied with cultural characteristics and virulence on coconut seedlings of the variety Tipatur Tall. The identity of each isolate was confirmed through morphological characters and through ITS sequencing. Two isolates viz., G4 and G5 were identified as Ganoderma applanatum and remaining all isolates were identified as G. lucidum. The genetic diversity analysis of Ganoderma isolates was done using ten Random Amplified Polymorphic DNA (RAPD) and fifteen Inter Simple Sequence Repeat (ISSR) primers. Among the ten RAPD primers, only eight primers recorded polymorphism (33.30-66.70%). The primer SBS-Q3 exhibited the highest polymorphism of 66.70%. In case of ISSR primers, all primers recorded polymorphism (33.30-60.00%). The primer UBC866 was the most polymorphic primer with 60.0% polymorphism. RAPD and ISSR markers were compared for their efficacy in assessing the genetic diversity by taking the band frequency, Shannon's index, polymorphic information content, resolving power, and mean resolving power into consideration, and it was concluded that ISSR was marker of choice over RAPD. Supplementary Information: The online version contains supplementary material available at 10.1007/s13205-023-03872-w.

Limited genetic variability and spatial population structure in grasshoppers between natural and metal-contaminated areas in Egypt.

Pollutants in an environment can have long-term implications for the species living there, resulting in local adaptations with implications for their genetic structure. Heavy metal pollutants infiltrate soils and groundwater, bioaccumulate in food webs, and negatively impact biota. In this study, we investigated the degree to which the genetic structure and variability of the slender green-winged grasshopper (Aiolopus thalassinus (Fabricius) (Orthoptera: Acrididae)) were impacted by heavy metal pollution and distance. We used the random amplified polymorphic DNA-polymerase chain reaction (RAPD-PCR) method to examine the genetic variability of populations in 3 heavy metal-polluted and 3 unpolluted locations across varying geographical distances in Egypt. The heavy metal concentrations of cadmium, copper, lead, and zinc were measured from the grasshopper tissue and soils. Sixty-nine unique and polymorphic bands were produced by 4 primers. Cluster and principal component analyses separated the populations inside and outside Cairo into 2 main branches, which were further divided into smaller branches corresponding to their geographical regions. We found no differences in the Shannon genetic diversity index between populations or with increasing heavy metal concentrations in either the soil or the grasshopper tissue. Our results showed a greater genetic variation among populations than between populations within the same location, indicating populations within locations were less differentiated than those between locations. The moderate correlation between genetic similarity and spatial distance suggests geographical isolation influenced grasshopper population differentiation. Based on the RAPD analysis, environmental pollutants and geographical distances impact the A. thalassinus population structure, potentially restricting gene flow between sites even at small spatial scales.

Determination of antibiotic resistance patterns and genotypes of Escherichia coli isolated from wild birds.

BACKGROUND: Curbing the potential negative impact of antibiotic resistance, one of our era's growing global public health crises, requires regular monitoring of the resistance situations, including the reservoir of resistance genes. Wild birds, a possible bioindicator of antibiotic resistance, have been suggested to play a role in the dissemination of antibiotic-resistant bacteria. Therefore, this study was conducted with the objective of determining the phenotypic and genotypic antibiotic resistance profiles of 100 Escherichia coli isolates of gull and pigeon origin by using the Kirby-Bauer disk diffusion method and PCR. Furthermore, the genetic relationships of the isolates were determined by RAPD-PCR. RESULTS: Phenotypic antibiotic susceptibility testing revealed that 63% (63/100) and 29% (29/100) of E. coli isolates were resistant to at least one antibiotic and multidrug-resistant (MDR), respectively. With the exception of cephalothin, to which the E. coli isolates were 100% susceptible, tetracycline (52%), kanamycin (38%), streptomycin (37%), ampicillin (28%), chloramphenicol (21%), trimethoprim/sulfamethoxazole (19%), gentamicin (13%), enrofloxacin (12%) and ciprofloxacin (12%) resistances were detected at varying degrees. Among the investigated resistance genes, tet(B) (66%), tet(A) (63%), aphA1 (48%), sul3 (34%), sul2 (26%), strA/strB (24%) and sul1 (16%) were detected. Regarding the genetic diversity of the isolates, the RAPD-PCR-based dendrograms divided both pigeon and gull isolates into five different clusters based on a 70% similarity threshold. Dendrogram analysis revealed 47-100% similarities among pigeon-origin strains and 40-100% similarities among gull-origin E.coli strains. CONCLUSIONS: This study revealed that gulls and pigeons carry MDR E. coli isolates, which may pose a risk to animal and human health by contaminating the environment with their feces. However, a large-scale epidemiological study investigating the genetic relationship of the strains from a "one health" point of view is warranted to determine the possible transmission patterns of antibiotic-resistant bacteria between wild birds, the environment, humans, and other hosts. Video Abstract.

Phytotoxicity and genotoxicity study of reactive red 141 dye on mung bean (Vigna radiata (L.) Wilczek) seedlings.

BACKGROUND: Reactive Red (RR) 141 dye is widely used in various industrial applications, but its environmental impact remains a growing concern. In this study, the phytotoxic and genotoxic effects of RR 141 dye on mung bean seedlings (Vigna radiata (L.) Wilczek) were investigated, serving as a model for potential harm to plant systems. METHODS AND RESULTS: Short-term (14 days) and long-term (60 days) experiments in paddy soil pot culture exposed mung bean seedlings to RR 141 dye. The dye delayed germination and hindered growth, significantly reducing germination percentage and seedling vigor index (SVI) at concentrations of 50 and 100 ml/L. In short-term exposure, plumule and radical lengths dose-dependently decreased, while long-term exposure affected plant length and grain weight, leaving pod-related parameters unaffected. To evaluate genotoxicity, high annealing temperature-random amplified polymorphic DNA (HAT-RAPD) analysis was employed with five RAPD primers having 58-75% GC content. It detected polymorphic band patterns, generating 116 bands (433 to 2857 bp) in plant leaves exposed to the dye. Polymorphisms indicated the appearance/disappearance of DNA bands in both concentrations, with decreased genomic template stability (GTS) values suggesting DNA damage and mutation. CONCLUSION: These findings demonstrate that RR 141 dye has a significant impact on genomic template stability (GTS) and exhibits phytotoxic and genotoxic responses in mung bean seedlings. This research underscores the potential of RR 141 dye to act as a harmful agent within plant model systems, highlighting the need for further assessment of its environmental implications.

Capsular gene distribution and RAPD typing of Streptococcus agalactiae isolated from pregnant women.

Streptococcus agalactiae has different virulence factors, from which the capsule has the most significant role in the pathogenesis of this organism. We aimed to investigate the distribution of more prevalent capsular genes among different Random Amplified Polymorphic DNA (RAPD) types of S. agalactiae isolated from pregnant women. A total of 106 isolates were collected from 420 vaginal and rectal swabs obtained from pregnant women. The specimens were transferred using Todd Hewitt Broth and were cultured on a blood agar containing antibiotics. The S. agalactiae isolates were identified by the standard microbiological and biochemical tests. The genomic DNAs of S. agalactiae isolates were extracted using an extraction kit. Then, the PCR method was used to detection of the capsular genes. Moreover, The RAPD PCR was used to genotyping of the isolates. The colonization rate of the pregnant women was 25.23%, and there was a statistically significant correlation between the weeks of gestation and the probability of colonization (p-value < 0.05). Also, 31 (29.24%) and 18 (16.98%) pregnant women had a history of abortion and membrane rupture, respectively. In addition, 20 (18.86%), 32 (30.18%), 4 (3.77%), and 6 (5.66%) isolates carried genes encoding capsular types Ia, Ib, III, and V, respectively. None isolates had the type II capsular gene, and other 44 isolates were non-typeable. Nine clones (clusters) of S. agalactiae were observed in the present study with 70% similarity, and 53 different types were identified among the isolates. Except for capsular types III and V that belonged to clones 3, 5, 7, and 9, other capsular types were detected in different RAPD types. We found that the capsular types Ib and Ia were predominant among pregnant women in this area, indicating their significance for vaccine designation. Also, our isolates showed a lower genotypic diversity in RAPD typing. This may be due to the same sources of most isolates.

Characterization of Coniella granati Isolates Causing Pomegranate Decline in Italy.

Coniella granati, the causal agent of pomegranate crown rot, twig blight, and fruit decay, is an emerging worldwide pathogen with a heavy impact on pomegranate cultivation. In this study, we report the rapid spread of the fungus in Italian pomegranate orchards associated with crown rot symptoms and provide new results on fungal development, baseline sensitivity to different fungicides, and intraspecific variability by analyzing 11 isolates, representative of populations of the pathogen from comparable pomegranate orchards in different regions of Italy. In vitro assays showed that 25 to 30°C was the optimal range for both colony growth and conidial germination, corroborating the results previously obtained for Californian and Greek isolates. According to the baseline sensitivity assay on the response of colony growth and conidial germination to 10 fungicides, fludioxonil, thiophanate-methyl, tebuconazole, and cyprodinil were the most effective. Random amplified polymorphic DNA (RAPD) analysis, carried out using fourteen 10-mer primers, showed very low intraspecific variability (similarity coefficient >0.95), probably as a result of the recent spread of the pathogen in Italy and the uncommon occurrence of the sexual process as a source of genetic variability. In summary, this study provides new knowledge on C. granati that will be helpful for improving pomegranate crown rot management.

In vitro Genotoxicity and In silico Docking Analyses of the Essential Oils of Thuja orientalis.

Two main objectives were pursued to assess the reliability of Thuja orientalis essential oils (TOEO). The first objective was to extract TOEO, analyze them by GC-MS, and determine their in vitro genotoxicity against selected plants using the RAPD-PCR method. The second objective was to evaluate the in-silico toxicity of TOEO. The binding sites and energies of each content was calculated against B-DNA. In-silico analyses were performed using a simulation program, AutoDock Vina, and Toxicity Estimation Software Tools. 3-carene, cedrol, and 2-pinene were identified as the predominant components. In vitro studies showed that the TOEO had a more significant impact on reducing genomic stability in wheat compared to the amaranth. The lowest stability was determined as 39.78 % in wheat and 53.58 % in amaranth. Cedrol (-5,7 kcal/mol) and selinene (-5,6 kcal/mol) exhibited the highest binding affinity. The toxicity test indicated that components other than cyclohexene may have toxic effects, none of them were predicted to be mutagenic, and LD50 (mol/kg) values could vary between 1.33 and 1.55.

Metal accumulation and genetic adaptation of Oryza sativa to Cadmiun and Chromium heavy metal stress: A hydroponic and RAPD analyses.

This research was performed to assess the influence of Cd and Cr metals on growth, pigments, antioxidant, and genomic stability of Oryza sativa indica and Oryza sativa japonica were investigated under hydroponic conditions. The results revealed that significant metal influence on test crop growth, pigment content, metal stress balancing antioxidant activity in a dose dependent manner. Since, while at elevated (500 ppm) concentration of Cd as well as Cr metals the pigment (total chlorophyll, chlorophyll a, b and carotenoids) level was reduced than control; however antioxidant activity (total antioxidant, H2O2, and NO) was considerably improved as protective mechanisms to combat the metal toxicity and support the plant growth. Furthermore, the test crops under typical hydroponic medium (loaded with Cd and Cr as 200, 300, 400, and 500 ppm) growth conditions, effectively absorb the metals from medium and accumulated in the root and least quantity was translocated to the shoot of this test crops. Furthermore, typical RAPD analysis with 10 universal primers demonstrated that the genomic DNA of the test crops was adaptable to develop metal resistance and ensure crop growth under increased concentrations (500 ppm) of tested heavy metals. These findings suggest that these edible crops have the ability to accumulate Cd along with Cr metals, and additionally that their genetic systems have the ability to adapt to metal-stressed environments.

Genetic variations among the isolates of Bipolaris Maydis based on phenotypic and molecular markers

Abstract Maydis leaf blight, caused by Bipolaris maydis, is an important disease of maize crop in Khyber Pakhtunkhwa (KP) Pakistan. Fifteen isolates of the pathogen, collected across KP, were studied for variability based on phenotypic and molecular markers. Significant variability among the isolates was observed when assessed using phenotypic traits such as radial growth, spore concentration, fungicide sensitivity and virulence. The isolates were classified into six culture groups based on colour, texture and margins of the colony. Conidial morphology was also variable. These were either straight or slightly curved and light to dark brown in colour. Fungicide test showed significant variation in the degree of sensitivity against Carbendazim. Isolate Bm8 exhibited maximum radial growth on carbendazim spiked plates. Conversely, isolate Bm15 showed the lowest radial growth. Variations in virulence pattern of the isolates were evident when a susceptible maize variety Azam was inoculated with spores of B. maydis. Genetic variability amongst the isolates was also estimated by RAPD as well as sequencing of ITS region. The RAPD dendrogram grouped all the isolates into two major clusters. Average genetic distance ranged from 0.6% to 100%, indicating a diverse genetic gap among the isolates. Maximum genetic distance was found between isolates Bm9 and Bm10 as well as Bm2 and Bm8. Conversely, isolates Bm13 and Bm15 were at minimum genetic distance. Phylogenetic dendrogram based on sequencing of ITS region grouped all the isolates into a single major cluster. The clusters in both the dendrogram neither correlate to the geographical distribution nor to the morphological characteristics.

Resumo A ferrugem das folhas de maydis, causada por Bipolaris maydis, é uma doença importante da cultura do milho em Khyber Pakhtunkhwa (KP), Paquistão. Quinze isolados do patógeno, coletados em KP, foram estudados quanto à variabilidade com base em marcadores fenotípicos e moleculares. Variabilidade significativa entre os isolados foi observada quando avaliada por meio de características fenotípicas, como crescimento radial, concentração de esporos, sensibilidade a fungicida e virulência. Os isolados foram classificados em seis grupos de cultura com base na cor, textura e margens da colônia. A morfologia dos conídios também foi variável. Estes eram retos ou ligeiramente curvos e de cor marrom-claro a escuro. O teste de fungicida mostrou variação significativa no grau de sensibilidade ao carbendazim. O isolado Bm8 exibiu crescimento radial máximo em placas com adição de carbendazim. Por outro lado, o isolado Bm15 apresentou o menor crescimento radial. As variações no padrão de virulência dos isolados foram evidentes quando uma variedade de milho suscetível Azam foi inoculada com esporos de B. maydis. A variabilidade genética entre os isolados também foi estimada por RAPD, bem como sequenciamento da região ITS. O dendrograma RAPD agrupou todos os isolados em dois grupos principais. A distância genética média variou de 0,6% a 100%, indicando uma lacuna genética diversa entre os isolados. A distância genética máxima foi encontrada entre os isolados Bm9 e Bm10 e também entre Bm2 e Bm8. Por outro lado, os isolados Bm13 e Bm15 estavam a uma distância genética mínima. O dendrograma filogenético baseado no sequenciamento da região ITS agrupou todos os isolados em um único aglomerado principal. Os agrupamentos em ambos os dendrogramas não se correlacionam com a distribuição geográfica nem com as características morfológicas.

Tinea corporis intrafamilial infection in pets due to Microsporum canis

ABSTRACT Microsporum canis, one of the most widespread dermatophytes worldwide, is a zoonotic microorganism that transmits infection from reservoirs such as cats and dogs to humans. This microorganism is associated with Tinea corporis and other clinical manifestations; however, few studies have used genetic surveillance to determine and characterize the process of zoonotic transmission. In this study, we show a clear example of zoonotic transmission from a cat to an intrafamilial environment, where it caused Tinea corporis by infection with M. canis. Molecular characterization using the b-tubulin gene and Random Amplified Polymorphic DNA analysis made it possible to determine that the six isolates of M. canis obtained in this study belonged to the same genetic variant or clone responsible for reservoir-reservoir or reservoir-human transmission.

Genomic characterization of Puccinia triticina using molecular marker technology / Caracterização genômica de Puccinia triticina usando tecnologia de marcadores moleculares

Leaf rust, caused by Puccinia triticina, is the most common rust disease of wheat. The fungus is an obligate parasite capable of producing infectious urediniospores. To study the genetic structure of the leaf rust population 20 RAPD primers were evaluated on 15 isolates samples collected in Pakistan. A total of 105 RAPD fragments were amplified with an average of 7 fragments per primer. The number of amplified fragments varied from 1 to 12. GL Decamer L-07 and GL Decamer L-01 amplified the highest number of bands (twelve) and primer GL Decamer A-03 amplified the lowest number of bands i.e one. Results showed that almost all investigated isolates were genetically different that confirms high genetic diversity within the leaf rust population. Rust spores can follow the migration pattern in short and long distances to neighbor areas. Results indicated that the greatest variability was revealed by 74.9% of genetic differentiation within leaf rust populations. These results suggested that each population was not completely identical and high gene flow has occurred among the leaf rust population of different areas. The highest differentiation and genetic distance among the Pakistani leaf rust populations were detected between the leaf rust population in NARC isolate (NARC-4) and AARI-11and the highest similarity was observed between NARC isolates (NARC-4) and (NARC-5). The present study showed the leaf rust population in Pakistan is highly dynamic and variable.

A ferrugem da folha, causada por Puccinia triticina, é a ferrugem mais comum do trigo. O fungo é um parasita obrigatório, capaz de produzir urediniósporos infecciosos. Para estudar a estrutura genética da população de ferrugem da folha, 20 primers RAPD foram avaliados em 15 amostras de isolados coletadas no Paquistão. Um total de 105 fragmentos RAPD foram amplificados com uma média de 7 fragmentos por primer. O número de fragmentos amplificados variou de 1 a 12. GL Decamer L-07 e GL Decamer L-01 amplificaram o maior número de bandas (doze), e o primer GL Decamer A-03 amplificou o menor número de bandas, ou seja, um. Os resultados mostraram que quase todos os isolados investigados eram geneticamente diferentes, o que confirma a alta diversidade genética na população de ferrugem da folha. Os esporos de ferrugem podem seguir o padrão de migração em distâncias curtas e longas para áreas vizinhas. Os resultados indicaram que a maior variabilidade foi revelada por 74,9% da diferenciação genética nas populações de ferrugem. Esses resultados sugeriram que cada população não era completamente idêntica e um alto fluxo gênico ocorreu entre a população de ferrugem da folha de diferentes áreas. A maior diferenciação e distância genética entre as populações de ferrugem da folha do Paquistão foram detectadas entre a população de ferrugem da folha no isolado NARC (NARC-4) e AARI-11 e a maior similaridade foi observada entre os isolados NARC (NARC-4) e (NARC-5). O presente estudo mostrou que a população de ferrugem da folha no Paquistão é altamente dinâmica e variável.

Molecular genotyping of Salmonella spp. isolated from cheese samples of local stores in Al-Diwaniyah city, Iraq.

Background: Food safety is an important subject that the global cheese industry increases awareness of. This urges these economic sectors to elevate the level of research to minimize cheese contamination with pathogenic bacteria, such as Salmonella. Aim: Based on these merits, this study was conducted to genotype Salmonella spp. isolated from cheese samples of local stores in Al-Diwaniyah City, Iraq. Methods: The study used 41 samples of local fresh unsalted white cheese in a selective-growth-based isolation of Salmonella. These isolates were confirmed utilizing a slide-agglutination (SA) test and VITEK® 2 system (V2S). Then, the isolates were subjected to conventional PCR and sequencing techniques that both targeted the 16S rRNA gene. For subtyping, the Salmonella isolates were subjected to a random amplified polymorphic DNA (RAPD)-PCR method. Results: The results of both SA and V2S revealed the presence of 14 (34.2%) isolates of Salmonella spp. in the cheese samples. The PCR confirmed 6 (42.9%) of these isolates, which further were defined with close nucleotide similarity (98.03%) and (97.88%) to different world isolates, such as Salmonella enterica subsp. Arizonae and Salmonella enterica subsp. enterica serovar Typhi, respectively. The RAPD-PCR findings showed different fragments for all the tested isolates. Conclusion: The present study indicates that the samples of the local fresh unsalted white cheese contain different Salmonella genotypes, which could be originated from different contamination sources.

Cloning, sequencing, and characterizing of soil antibiotic active-producing Streptomyces species-specific DNA markers.

Association of the antibiotic activity of the soil Streptomyces isolates to their genetic profiles analyzed through RAPD-PCR fingerprints prompted us here in this study to use the most common bands as specific markers to identify homologous proteins within these isolates by cloning, sequencing, and characterizing these markers. Six out of twelve DNA bands ranged between 600 and 1350 bp previously obtained by RAPD-PCR analysis were purified out of the RAPD gels, and then cloned into pGEM-T Easy vector system. Success of the cloning process was confirmed by digesting purified plasmids with EcoRI. The clones namely No. 54, 55, 20, 56, 57, and 58 were sequenced using the DNA BigDye Terminator Sequencing System utilizing the M13 primer. Results indicated that the size of the inserted sequences is 599, 566, 522, 870, 857, and 254 bp, in clones No. 54. 55, 20, 56, 57, and 58, respectively. Homologous proteins of the six cloned sequences generated by DNA blast software indicated that the highest score of protein homology was scored for clone No. 54 with 87 % homology to putative secreted pectate lyase [Streptomyces coelicolor A3(2)]. The other clones showed less homology with 77 % homology for the clones No. 55 and 56, 73 % homology for the clone No. 20, and 55 % homology for the clones No. 57 and 58. The association of homologous proteins to the reported RAPD pattern is confirmed here for the first time, and the resulting DNA cloned fragments deserve further molecular analysis.

Comparative Assessment of SSR and RAPD markers for genetic diversity in some Mango cultivars.

Genetic improvement mainly depends on the level of genetic variability present in the population, and the degree of genetic diversity in a population largely determines the rate of genetic advancement. For analyzing genetic diversity and determining cultivar identities, a molecular marker is a useful tool. Using 30 SSR (simple sequence repeat) and 30 RAPD (randomly amplified polymorphic DNA) markers, this study evaluated the genetic divergence of 17 mango cultivars. The effectiveness of the two marker systems was evaluated using their genetic diversity characteristics. Additionally, the effects of SM (simple matching) and Dice similarity coefficients and their effects on mango clustering were evaluated. The findings showed that SSR markers generated 192 alleles, all of which were polymorphic (100%). With RAPD markers, 434 bands were obtained, 361 of which were polymorphic (83%). The average polymorphic information content (PIC) for RAPD and SSR was 0.378 and 0.735, respectively. Using SSR markers resulted in much higher values for other genetic diversity parameters compared to RAPD markers. Furthermore, grouping the genotypes according to the two similarity coefficients without detailed consideration of these coefficients could not influence the study results. The RAPD markers OPA_01, OPM_12 followed by OPO_12 and SSR markers MIAC_4, MIAC_5 followed by mMiCIR_21 were the most informative in terms of describing genetic variability among the cultivars under study; they can be used in further investigations such as genetic mapping or marker-assisted selection. Overall, 'Zebda' cultivar was the most diverse of the studied cultivars.

The potential of gamma irradiation on antioxidant capacity and genomic alterations in Calendula officinalis.

There are lines of evidence that ionizing radiations such as gamma rays can cause different biological effects on plants. Marigold (Calendula officinalis L.) is a member of the family Asteraceae. It possesses profound amounts of active ingredients. The aim of this study was to evaluate the changes imposed upon different dose levels of gamma radiation on some features of Calendula officinalis such as antioxidant activity, total phenolic compounds and flavonoid contents, antibacterial activity and genomic alterations. Calendula officinalis seeds were exposed to different doses of Gamma radiation (0, 10, 15, 20 and 25 GY). Total phenolics, flavonoids, antioxidant activity (measured by DPPH assay) using methanolic extracts of plants and antibacterial activity measured by the disc diffusion assay showed significant differences to the control samples. The samples treated with 10 GY gamma rays showed the highest total phenol and flavonoid contents. Antioxidant activity significantly differed between Gamma rays dose levels and it was the highest at 25 GY. Four bacterial strains including E. coli, Bacillus subtilis and Pseudomonas aeroginosa were used for the antibacterial assay. Extracts from plants treated with 25 GY gamma rays showed the highest antibacterial activity against the 4 bacterial strains. Random amplified polymorphic DNA (RAPD) and inter-simple sequence repeat (ISSR) markers were used to study the genetic variation. The polymorphism information content (PIC) for RAPD primers ranged from 3% to 13% and ranged from 6 to 13% for ISSR primers. Results indicated that ISSR markers were more efficient than RAPD markers, as they detected 25.57% polymorphic DNA bands compared to 21.31% polymorphism for RAPD markers.