Search Results (218)

The cultivation of alfalfa is crucial for farmers as it is an excellent forage crop with a high nitrogen-fixing capacity, making it indispensable in crop rotations. Breeding programs face challenges in advancing more rapidly in genetic diversity to achieve a higher heterosis effect and, consequently, greater yield. In this study, we used 30 alfalfa varieties, which were used for molecular analyses by 5 ISSR primers and 13 RAPD primers. The results obtained highlighted the greater efficiency of ISSR primers in identifying genetic diversity. On the other hand, the simultaneous use of ISSR + RAPD allowed for clearer clustering of varieties that enabled more efficiently distinguishing the genetic diversity. The most efficient ISSR primer, A17, generated 31 polymorphic bands, while the most efficient RAPD primer, L-07, generated only 21 bands. Varieties such as “Pastoral” and “F1413-02” exhibited low similarity coefficients (0.39), suggesting their potential for enhancing genetic variability through crossbreeding, thereby increasing the potential of achieving a greater heterosis effect. Conversely, varieties with high similarity coefficients, such as ”Cristal” and “Viking” (0.81) are less suited for this purpose. The correlation between specific markers highlights that using both ISSR and RAPD markers together offers a clear understanding of genetic diversity in alfalfa, aiding in more effective selection for crossbreeding in breeding programs. Full article

The interaction of nano-fertilizers with commercially important crops can be a promising solution to increase both crop yield and quality. This study investigated the effect of iron oxide nanoparticles (Fe₃O₄ NPs) on four-week-old garden cress (Lepidium sativum L.) seedlings. Iron is an essential micronutrient for plants but is not always available in sufficient quantities, which can lead to chlorosis and even plant death. The seedlings were grown hydroponically, with three concentrations (1 mg/L, 5 mg/L, and 10 mg/L) of the NPs, alongside a control group with no additions. During the experiment, the following methods were employed: measurement of stem and root length, spectrophotometry to determine chlorophyll absorbance and concentration, and the RAPD technique to assess the genotoxicity of Fe₃O₄ NPs. The study demonstrated a significant increase in the shoot length of cress at all concentrations compared to the control group (p < 0.05; p < 0.01). The light absorption and chlorophyll concentration levels in the experimental groups significantly increased compared to the control group (p < 0.001). Genotoxicity analysis revealed that the genotoxic impact of the NPs on the garden cress genome was only 10%, a statistically insignificant level. The findings suggest that Fe₃O₄ NPs exhibit low genotoxicity and have the potential to enhance the growth and chlorophyll content of cress seedlings in hydroponic conditions. Full article

Fungi of the genus Aspergillus are widespread in the environment, where they produce large quantities of airborne conidia. Inhalation of Aspergillus spp. conidia in immunocompromised individuals can cause a wide spectrum of diseases, ranging from hypersensitivity responses to lethal invasive infections. Upon deposition in the lung epithelial surface, conidia encounter and interact with complex microbial communities that constitute the lung microbiota. The lung microbiota has been suggested to influence the establishment and growth of Aspergillus spp. in the human airways. However, the mechanisms underlying this interaction have not yet been sufficiently investigated. In this study, we aimed to enrich and isolate bacterial strains capable of inhibiting the germination and growth of A. fumigatus conidia from bronchoalveolar lavage fluid (BALF) samples of lung transplant recipients using a novel enrichment method. This method is based on a soft agar overlay plate assay in which bacteria are directly in contact with conidia, allowing inhibition to be readily observed during enrichment. We isolated a total of five clonal bacterial strains with identical genotypic fingerprints, as shown by random amplified polymorphic DNA PCR (RAPD–PCR). All strains were identified as Pseudomonas aeruginosa (strains b1–b5). The strains were able to inhibit the germination and growth of Aspergillus fumigatus in a soft agar confrontation assay, as well as in a germination multiplate assay. Moreover, when compared with ten P. aeruginosa strains isolated from expectoration through standard methods, no significant differences in inhibitory potential were observed. Additionally, we showed inhibition of A. fumigatus growth on Calu-3 cell culture monolayers. However, the isolated P. aeruginosa strains were shown to cause significant damage to the cell monolayers. Overall, although P. aeruginosa is a known opportunistic lung pathogen and antagonist of A. fumigatus, we validated this novel one-step enrichment approach for the isolation of bacterial strains antagonistic to A. fumigatus from BALF samples as a proof-of-concept. This opens up a new venue for the targeted enrichment of antagonistic bacterial strains against specific fungal pathogens. Full article

Despite quinoa (Chenopodium quinoa Willd.) gaining international popularity in the early 21st century for its nutritional benefits, there remains a critical need to optimize its cultivation practices in arid regions. Current research often overlooks the combined effects of supplemental irrigation and foliar treatments on quinoa’s yield and water efficiency, particularly under challenging environmental conditions like those in Borg El-Arab, Egypt. Field studies were conducted in Borg El-Arab, Alexandria, Egypt, during the winter seasons of 2021/2022 and 2022/2023 to determine the influence of supplemental irrigation (rainfed, 2000, and 4000 m³/hectare, respectively) and foliar spraying of sodium silicate (control, 200, and 400 ppm) on yield, yield components, seed quality, and water usage efficiency in quinoa cv. Chibaya grown in arid lands. Three replications were used in a split-plot design. The main plots were designated for irrigation, while the subplots were designated for foliar spraying. The results indicate that applying irrigation at a rate of 4000 m³/hectare significantly increased leaf dry weight per plant by 23.5%, stem dry weight per plant by 18.7%, total dry weight per 25 plants by 21.4%, leaf area per plant by 19.2%, and straw yield by 26.8% compared to the control treatment. There were no significant differences between irrigation with the rate of 4000 m³ or 2000 m³/hectare on biological yield kg/hectare, N (%), P (mg/100 g), and protein (%). The utilization of sodium silicate had no significance on all studied features except for straw yield kg ha⁻¹ at the rate of 200 or 400 ppm. The results regarding the RAPD1 primer revealed that the 2000+0 silicon treatment was the only treatment that resemble the control with no up- or downregulated fragment. Moreover, 20 upregulated fragments were observed in all treatments, while 19 DNA fragments were downregulated. Furthermore, the results obtained regarding the RAPD2 primer revealed that 53 fragments were upregulated and 19 downregulated. Additionally, the RAPD3 primer demonstrated that 40 DNA fragments were upregulated, whereas 18 downregulated DNA fragments were detected. It may be inferred that the application of irrigation at a rate of 4000 m3 ha⁻¹ might serve as a supplemental irrigation method. Spraying sodium silicate at a 400 mg L⁻¹ concentration could alleviate the dry climate on the Egyptian shore. Full article

During the last few decades, the main focus of numerous studies has been on the human breast milk microbiota and its influence on the infant intestinal microbiota and overall health. The presence of lactic acid bacteria in breast milk affects both the quantitative and qualitative composition of the infant gut microbiota. The aim of this study was to assess the most frequently detected cultivable rod-shaped lactobacilli, specific for breast milk of healthy Bulgarian women and fecal samples of their infants over the first month of life, in 14 mother–infant tandem pairs. Additionally, we evaluated the strain diversity among the most common isolated species. A total of 68 Gram-positive and catalase-negative strains were subjected to identification using the MALDI-TOF technique. Predominant cultivable populations belonging to the rod-shaped lactic acid bacteria have been identified as Lacticaseibacillus rhamnosus, Limosilactobacillus fermentum, Lacticaseibacillus paracasei, and Limosilactobacillus reuteri. Also, we confirmed the presence of Lactiplantibacillus plantarum and Lactobacillus gasseri. Up to 26 isolates were selected as representatives and analyzed by 16S rRNA sequencing for strain identity confirmation and a phylogenetic tree based on 16S rRNA gene sequence was constructed. Comparative analysis by four RAPD primers revealed genetic differences between newly isolated predominant L. rhamnosus strains. This pilot study provides data for the current first report concerning the investigation of the characteristic cultivable lactobacilli isolated from human breast milk and infant feces in Bulgaria. Full article

Jamun plant displays enormous diversity throughout Pakistan, which necessitates its screening, evaluation, and validation to document elite genotypes having better traits for the benefit of the fruit industry and farmers. Surveys were made in natural Jamun habitats across Punjab, Pakistan, and genotypes were marked based on visual diversity of trees and fruits. In total, 60 Jamun genotypes were selected for characterization based on phenotypic and genetic markers. Phenotypic characters related to trees, leaf, and flower along with fruit qualitative traits were assessed in situ. Results revealed significant diversity with high (>25%) coefficient of variance values and the first two components of correspondence analysis exhibited 41.71% variation among genotypes. A strong association was observed among traits like upright tree and round fruit shape (0.74), bluish-colored fruit and pinkish pulp (0.85), and elliptic-shaped fruit with low fruit waxiness (−0.72). Leaves of phenotypically characterized plants were brought to Wheat Biotechnology Lab., University of Agriculture, Faisalabad, Pakistan, where Jamun genotypes were investigated genetically using Random Amplified Polymorphic DNA (RAPD) and Inter Simple Sequence Repeat (ISSR) markers. A total of 132 bands were scored, of which 108 were polymorphic, corresponding to almost 81% polymorphism among collected genotypes. High polymorphism information content values were observed against RAPD (0.389) and ISSR (0.457) markers. Genotypes were compared in relation to genetic markers, which exhibited that almost 86% of genetic variability was attributed to differences among accessions, while 14% of variation was due to differences between collections of different areas. Findings of this study confirmed wide phenotypic and genetic distinctness of Jamun in Pakistan that can aid breeders for marker-assisted selection and germplasm enhancement for future crop improvement programs. Full article

The rapid spread of carbapenemase-producing strains has led to increased levels of resistance among Gram-negative bacteria, especially enterobacteria. The current study aimed to collect and genetically characterize the colistin- and carbapenem-resistant isolates, obtained in one of the biggest hospitals (Military Medical Academy) in Sofia, Bulgaria. Clonal relatedness was detected by RAPD and MLST. Carbapenemases, ESBLs, and mgrB were investigated by PCR amplification and sequencing, replicon typing, and 16S rRNA methyltransferases with PCRs. Fourteen colistin- and carbapenem-resistant K. pneumoniae isolates were detected over five months. Six carbapenem-resistant and colistin-susceptible isolates were also included. The current work revealed a complete change in the spectrum of carbapenemases in Bulgaria. bla_NDM-5 was the only NDM variant, and it was always combined with bla_OXA-232. The coexistence of bla_OXA-232 and bla_NDM-5 was observed in 10/14 (72%) of colistin- and carbapenem-resistant K. pneumoniae isolates and three colistin-susceptible isolates. All bla_NDM-5- and bla_OXA-232-positive isolates belonged to the ST6260 (ST101-like) MLST type. They showed great mgrB variability and had a higher mortality rate. In addition, we observed bla_OXA-232 ST14 isolates and KPC-2-producing ST101, ST16, and ST258 isolates. The colistin- and carbapenem-resistant isolates were susceptible only to cefiderocol for bla_NDM-5- and bla_OXA-232-positive isolates and to cefiderocol and ceftazidime/avibactam for bla_OXA-232- or bla_KPC-2-positive isolates. All bla_OXA-232-positive isolates carried rmtB methylase and the colE replicon type. The extremely limited choice of appropriate treatment for patients infected with such isolates and their faster distribution highlight the need for urgent measures to control this situation. Full article

Lacticaseibacillus spp. are genetically close lactic acid bacteria species widely used in fermented products for their technological properties as well as their proven beneficial effects on human and animal health. This study, the first to include such a large collection of heterogeneous isolates (121) obtained from international collections belonging to Lacticaseibacillus paracasei, aimed to characterize the safety traits and technological properties of this important probiotic species, also making comparisons with other genetically related species, such as Lacticaseibacillus casei and Lacticaseibacillus zeae. These strains were isolated from a variety of heterogeneous sources, including dairy products, sourdoughs, wine, must, and human body excreta. After a preliminary molecular characterization using repetitive element palindromic PCR (Rep-PCR), Random Amplification of Polymorphic DNA (RAPD), and Sau-PCR, particular attention was paid to safety traits, evaluating antibiotic resistance profiles, biogenic amine (BA) production, the presence of genes related to the production of ethyl carbamate and diaminobenzidine (DAB), and multicopper oxidase activity (MCO). The technological characteristics of the strains, such as the capability to grow at different NaCl and ethanol concentrations and different pH values, were also investigated, as well as the production of bacteriocins. From the obtained results, it was observed that strains isolated from the same type of matrix often shared similar genetic characteristics. However, phenotypic traits were strain-specific. This underscored the vast potential of the different strains to be used for various purposes, from probiotics to bioprotective and starter cultures for food and feed production, highlighting the importance of conducting comprehensive evaluations to identify the most suitable strain for each purpose with the final aim of promoting human health. Full article

We evaluated the specificity of the primers OPF-01, P54, and 1253 to identify A. fumigatus, A. flavus, and A. niger, respectively, with the RAPD-PCR method. Eighty-two isolates belonging to the sections Fumigati, Flavi, and Nigri were used. The isolates were identified by phenotypic (macro- and micromorphology) and genotypic (partial sequences of the BenA gene) methods. The RAPD-PCR method was used to obtain polymorphic patterns with the primers OPF-01, P54, and 1253. The specificity of the polymorphic patterns of the isolates of each species was evaluated through the UPGMA clustering method and logistic regression model. All isolates of the genus Aspergillus were identified at the section level by macro- and micromorphology showing the typical morphology of the sections Fumigati, Flavi, and Nigri, and the species were identified by the construction of the phylogeny of the partial sequence of the BenA gene. The patterns’ polymorphic strains obtained with the primers OPF-01, P54, and 1253 for the isolates of A. fumigatus, A. flavus, and A niger, respectively, showed the same polymorphic pattern as the reference strains for each species. To verify the specificity of the primers, they were tested with other species from the sections Fumigati, Flavi and Nigri. The results support that the primers OPF-01, P54, and 1253 generate polymorphic patterns by RAPD-PCR species specific to A. fumigatus, A. flavus, and A. niger, respectively. Full article

The spread of multidrug-resistant Acinetobacter baumannii in hospitals and nursing homes poses serious healthcare challenges. Therefore, we aimed to isolate and characterize lytic bacteriophages targeting carbapenem-resistant Acinetobacter baumannii (CRAB). Of the 21 isolated A. baumannii phages, 11 exhibited potent lytic activities against clinical isolates of CRAB. Based on host spectrum and RAPD-PCR results, 11 phages were categorized into four groups. Three phages (vB_AbaP_W8, vB_AbaSi_W9, and vB_AbaSt_W16) were further characterized owing to their antibacterial efficacy, morphology, and whole-genome sequence and were found to lyse 37.93%, 89.66%, and 37.93%, respectively, of the 29 tested CRAB isolates. The lytic spectrum of phages varied depending on the multilocus sequence type (MLST) of the CRAB isolates. The three phages contained linear double-stranded DNA genomes, with sizes of 41,326–166,741 bp and GC contents of 34.4–35.6%. Genome-wide phylogenetic analysis and single gene-based tree construction revealed no correlation among the three phages. Moreover, no genes were associated with lysogeny, antibiotic resistance, or bacterial toxins. Therefore, the three novel phages represent potential candidates for phage therapy against CRAB infections. Full article

Trueperella (T.) pyogenes is a mastitis-causing pathogen formerly known to cause severe clinical mastitis (CM), especially during the summer, leading to milk losses and low recovery rates. Unfortunately, its transmission behavior within herds is unclear. The diversity and occurrence of T. pyogenes were monitored to gain an initial insight into the infection transmission behavior of T. pyogenes in dairy herds and to lay a foundation for following targeted investigations. CM milk samples were collected from German herds, and one Swedish farm was sampled for isolates from subclinical mastitis. All in all, 151 T. pyogenes isolates from 16 herds were isolated, identified by MALDI TOF analysis and typed with RAPD PCR. Of these, 17 isolates originated from subclinical mastitis cases. We found that T. pyogenes mastitis occurred year-round, and clinical mastitis cases were caused by multiple strains (31 affected animals/28 strains). Instances of multiple cows being infected with the same T. pyogenes strain were rare and typically only involved a small number of animals at a time. However, if several quarters of a cow were affected, it was likely the same strain. Unlike clinical infections, subclinical T. pyogenes infections, in one investigated farm, harbored a dominant strain. Additionally, we found that T. pyogenes infections tended to persist and stay within a herd for a minimum of 7 months in the same or different cows. Full article

Non-Saccharomyces yeasts have recently garnered significant interest in oenology. When co-inoculated with Saccharomyces cerevisiae, they contribute to the improvement of wine quality from a sensory point of view. In the present study, a group of yeasts previously isolated from manna and honey by-products were subjected to a genotypic identification. The D1/D2 variable domains of the 26-sRNA gene and the ITS region of the 5.8S gene were sequenced. Additionally, a differentiation of strains was carried out by RAPD-PCR. All strains underwent in vitro screening. Subsequently, a micro-vinification experiment was conducted, focusing on strains with favourable technological characteristics: Lachancea thermotolerans, Starmerella lactis-condensi, and Candida oleophila. These strains were sequentially inoculated alongside a control strain of Saccharomyces cerevisiae. Technological screening revealed that some strains exhibited limited H₂S production, ethanol tolerance (up to 8% v/v), resistance to potassium metabisulphite (200 mg/L), osmotic stress tolerance (up to 320 g/L of glucose), and copper resistance (on average 5 mM). The findings from this study can guide the selection of new starters and co-starters for regional wine production. Full article

Beverages are an integral component of a person’s food package. Various types of microorganisms widely contaminate beverages. This review presents current research data aimed at identifying dominant microorganisms in beverages and molecular methods for their detection. Wine, beer, dairy drinks, and fruit juices were selected as the main objects of the study. The most contaminated beverage turned out to be fruit juice. As a result of a large number of independent studies, about 23 species of microorganisms were identified in it. At the same time, they are represented not only by bacterial and fungal organisms, but also by protozoa. Milk turned out to be the least contaminated in terms of detected bacteria. The most common pollutants of these beverages were Staphylococcus aureus, Bacillus cereus, and Vibrio parahaemolyticus. It has been established that among pathogenic genera, Salmonella sp., Campylobacter sp. and Shigella sp. are often present in beverages. One of the main tools for the quality control of beverages at all stages of their production is different types of polymerase chain reaction. The sequencing method is used to screen for microorganisms in beverages. The range of variations of this technology makes it possible to identify microorganisms in alcoholic and non-alcoholic beverages. The high specificity of methods such as PCR-RFLP, Rep-PCR, qPCR, End-point PCR, qLAMP, the molecular beacon method, and RAPD enables fast and reliable quality control in beverage production. Sequencing allows researchers to evaluate the microbiological diversity of all the studied beverages, while PCR varieties have demonstrated different fields of application. For example, PCR-RFLP, RAPD-PCR, and PCR allowed the identification of microorganisms in fruit juices, qPCR, LAMP, and the molecular beacon method in wine, LAMP and multiplex PCR in milk, and End-point PCR and Rep-PCR in beer. However, it is worth noting that many methods developed for the detection of microbial contaminants in beverages were developed 10–20 years ago; modern modifications of PCR and isothermal amplification are still poorly implemented in this area. Full article

This study aims at the identification and characterization of five actinobacterial strains with presumed belonging to the species Curtobacterium flaccumfaciens isolated from tomato and pepper plants, and establishing the potential role of both plants as natural reservoirs of this phytopathogen. Species identification was performed via MALDI-ToF MS, 16S rDNA sequencing and PCR. The strains were Gram-positive with a coryneform cell shape having yellow/orange-pigmented colonies; positive for catalase and esculin, and starch and casein hydrolysis; oxidase-, urease-, indole- and nitrate-reduction-negative and were strictly aerobic. All isolates produced antimicrobial substances against various phytopathogenic bacteria. Tomato and pepper plants were artificially infected with newly isolated strains in order to establish their role as natural reservoirs of the bacteria. Morphological alterations were observed only in the tomato plants, with defoliation of the first two to four leaves at the 28th day. Then, viable coryneform bacterial isolates (n = 73) were successfully re-isolated only from the stems of the infected plants. The similarity between the re-isolates and the respective initial isolates was confirmed phenotypically and genotypically by RAPD-PCR, confirming that solanaceous vegetables can act as reservoirs of C. flaccumfaciens. This is the first report of C. flaccumfaciens in Bulgaria. Full article

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. Full article