RAPD Research Articles

Variabilidade genética de ameixa selvagem (Ximenia americana L.) baseada em marcadores rapd

ABSTRACT Several ethnobotanical studies highlighted the importance of yellow plum as a useful plant species in the Brazilian Northeast region. In order to domesticate and incorporate a native species in production systems it is necessary to obtain information on its development and genetic variation. This work aimed to analyze the genetic variability of individuals of X. americana, accessed by Random Amplified Polymorphic DNA (RAPD) molecular markers. 26 genotypes of X. americana, randomly collected at the private reserve Campo das Emas Farm located in the Caturité City, Paraíba State, Brazil, were used in the study. Through the RAPD technique it was possible to assess the genetic variations among native genotypes within a wild plum population. Based on Sokal and Rohlf dissimilarity coefficient it was possible to form four distinct groups using the UPGMA method. The Cophenetic Correlation Coefficient was 0.83, revealing variability in the consistency of the cluster pattern. The genotypes of X. americana have genetic diversity detected by RAPD markers, which have shown to be efficient in characterizing individuals. Genotypes 4, 8, 9, 11, 13, 14, 15, 16, 18, 21, 24, and 25 are the most divergent and should be used in conservation strategies and breeding programs.

Micropropagação e uniformidade genética de Kalanchoe daigremontiana (Crassulaceae)

ABSTRACT Kalanchoe daigremontiana is an ornamental species propagated exclusively asexually. However, no in vitro studies have been conducted to assess the relationship between changes in the culture environment and genetic variations of K. daigremontiana. Therefore, this study aimed to determine the optimal concentration of salts in the Murashige & Skoog (MS) medium and the optimal concentration of sucrose in the culture medium, in addition to the quality of light, in the development of in vitro plantlets from leaf explants of K. daigremontiana. Additionally, a genetic evaluation of the resulting plantlets was conducted to study phenotypic changes that could be attributed to somaclonal variation using Random Amplified Polymorphic DNA (RAPD) markers. For this purpose, leaf explants of K. daigremontiana were disinfected and inoculated in different salt concentrations (25, 50, and 100%) of MS medium without sucrose. The cultures were transferred to a growth chamber and subjected to three light conditions (white, blue, and red) with a 16-hour photoperiod, plus the absence of light, at a temperature of 27 ± 1 °C. Explants were also inoculated in 50% salt concentrations of MS medium supplemented with various concentrations of sucrose (control - without sucrose, 1.5, 3.0, 4.5, and 6.0%). K. daigremontiana leaf explants showed optimal development under white light and in any MS medium salt concentrations. A sucrose-free culture medium was viable, and no genetic variation was observed in the plantlets compared to the parent plants under the tested conditions.

Phylogenetic analysis of Salmonella enterica isolated from imported and local potato seed tubers

Human pathogens on plants are an emerging global health concern. Salmonella enterica exhibits a broad range of host associations, including plants, animals, and humans. Therefore, the rapid characterization of Salmonella on potatoes is essential for ensuring food safety, protecting crops, and preventing transmission to humans. The present study investigates the phylogenetic relationships of potato-borne S. enterica with clinical lineages. Among sixty-seven S. enterica isolates collected from local and imported potato seed varieties, 63 (94.02%) were confirmed to carry the invA gene, a specific marker for Salmonella. Random Amplified Polymorphic DNA (RAPD) analysis grouped these isolates into three distinct clusters, with Salmonella strains 7S-Moz-L and 1S-Kar-I showing genetic similarity to the clinical strain S. typhi 5S-CL2 (MZ708960). The invA protein is identified as a virulence factor relevant to human infections and may play a role in tuber colonization. Phylogenetic analysis of invA sequences revealed homology between Salmonella strain 9SF-Ast-L (MW319054), isolated from the Asterix potato variety, and the clinical strain S. typhi 5S-CL2. Moreover, Salmonella strain 1S-Kar-I [MW319050], isolated from the imported Karuda variety, showed an evolutionary relationship with Salmonella strains 7S-Moz-L (MW319052) and 8S-Qas-L (MW319053), both from local potato varieties. Overall, the study underscores the importance of identifying S. enterica in potato seeds prior to planting or transport to prevent the spread of quarantine pathogens and ensure public health safety.

Antimicrobial resistance, virulence gene profiling, and genetic diversity of multidrug-resistant Pseudomonas aeruginosa isolates in Mazandaran, Iran

BackgroundPseudomonas aeruginosa is a major cause of healthcare-associated infections (HAIs), particularly in immunocompromised patients, leading to high morbidity and mortality rates. This study aimed to investigate the antimicrobial resistance patterns, virulence gene profiles, and genetic diversity among P. aeruginosa isolates from hospitalized patients in Mazandaran, Iran.MethodsFrom September 2021 to April 2022, 82 non-duplicate P. aeruginosa isolates were collected from diverse clinical sources. Identification was confirmed using API 20 NE (bioMérieux, Marcy l’Etoile, France). Antimicrobial susceptibility testing was conducted using the Kirby-Bauer disk diffusion method according to CLSI guidelines to assess resistance to a range of antibiotics. The virulence profile (exoT, exoY, exoU, toxA, plcH, plcN, algD, aprA, lasB and exoS) of each P. aeruginosa isolate was determined by PCR. The genetic diversity among the strains was evaluated using the random amplification of polymorphic DNA (RAPD) technique. Clustering was based on a Dice similarity coefficient of ≥ 85%.ResultsOf the 82 total strains, P. aeruginosa exhibited the highest and lowest resistance toward ticarcillin-clavulanate (98.78%) and colistin (0%), respectively. Moreover, 100% of the P. aeruginosa isolates were MDR. The following prevalence of virulence factor genes was observed: aprA, lasB, algD, toxA, plcH, exoY, and exoT in 100% of isolates. The plcN, exoS, and exoU were identified 98.78%, 67.07%, and 45.12%, respectively. The RAPD patterns obtained with primers 272 and 208 had respectively 2–19 and 6–17 bands. According to the Dice similarity coefficient of higher than 85%, 56 and 39 clusters were recognized.ConclusionThe high rate of multidrug resistance combined with the widespread presence of virulence genes in P. aeruginosa isolates highlights the potential for increased infection severity, morbidity, and mortality in hospitalized patients. The substantial genetic diversity observed among isolates suggests that P. aeruginosa in this region may rapidly evolve, necessitating ongoing surveillance and more targeted antimicrobial strategies.Clinical trial numberNot applicable.

Impact of gamma-aminobutyric acid receptors modulators on renal and liver functional, molecular and histological characteristics in white male mice

The gamma-aminobutyric acid (GABA) receptors are considered the main receptors that inhibit neurotransmitters in the mammalian brain, and they have been proven to exist in non-neuronal cells. The study's purpose is to determine the impact of GABA modulation on renal and liver functions and molecular and histological characteristics using different doses of lorazepam. Lorazepam, one of the benzodiazepine drugs known for its modulatory effect on GABA receptors, has been used as a modulator to determine the impact of GABA modulation on renal and liver functions and molecular and histological characteristics in 30 albino male mice, out of which 21 were divided into 3 groups. Each group were treated with different dose of lorazepam (1 mg/kg, 2 mg/kg and 3 mg/kg body weight respectively). The rest 9 of animals were considered as a control group. Histological and functional parameters were studied in kidney and liver functional states to evaluate the impact of GAPA modulation using lorazepam. Total genomic DNA was extracted from liver, brain, kidney and blood and random amplification polymorphic DNA (RAPD) technique were used to detect the molecular impact of GABA modulation on the genomic DNA. The data were analyzed using SPSS Version 20.0 (SPSS Inc.), with means and standard deviations (SD) calculated for quantitative variables, and ANOVA applied for comparing group means. Functional parameters (blood urea, creatinine, GOT, GPT, GGT) and histopathological examination showed a significant change in treated groups compared to the control group. In both the liver and kidneys of mice, with increasing doses of lorazepam, there was an increase in the severity of congestive phenomena in blood vessels with the appearance of hemorrhages, signs of inflammation, and cell degeneration and necrosis. The molecular investigation indicated substantial changes in RAPD profiles of treated groups, with normal bands disappearing and novel bands appearing in contrast to the control group. The RAPD profiles of the treated and control samples revealed 432 bands, with 109 as control bands, 167 (loss of normal bands and emergence of novel bands) as polymorphic bands, and 156 as homomorphic bands. It is concluded that GABA modulation by lorazepam increases the functional and histopathological alterations, producing variations in the genomic DNA.

Conservation Genetics and Breeding using Molecular Genetic Markers in Japanese Quail (Coturnix japonica).

The Japanese quail (Coturnix japonica) is a small migratory bird whose main habitats are located in East Asia, Russia, China, Japan, Korea, and India. The Japanese quail was first introduced into the Iraqi research sector in the early 1980s. This investigation aimed to identify the genetic divergence between the available genetic lines of the Japanese quail in Iraq as a first step to conducting further conservation and breeding, benefiting from studying the genetic diversity related to productivity, adaptation, and immune susceptibility. In this study, we harnessed the random amplified polymorphic DNA (RAPD)-polymerase chain reaction (PCR) protocol to investigate the genetic structure, diversity, and differentiation of seven distinct genetic lines of these birds with white, brown, wild grey, and yellow plumage guided by 20 molecular genetic markers. Our findings showed a relatively high polymorphism level of these 20 markers, which was adequate for describing genetic variation within and between the quail lines under study. Overall, the pairs of the White male and Brown male, Brown female and Grey male, and Grey female and Grey male lines were the most genetically distant. Conversely, the White male and Grey male lines were the most similar. The genetic differences established between these lines can enable us to suggest recommendations for further conservation genetics and breeding of this species. In particular, we propose that choosing animals (lines) with the greatest genetic distances, i.e., the least genetic similarities, can help preserve the highest genetic variability within the population. This proposed approach mirrors many contemporary conservation strategies, and information derived directly from this study can potentially be used to improve breeder selection regimes for additional conservation initiatives ex situ.

Genetic Assessment and Positioning of Algerian Barley Landraces with Respect to Landraces from the Middle East and Europe Using RAPD and SSR Markers.

Landraces are a critical genetic resource for resilience breeding, offering solutions to prepare agriculture for the challenges posed by climate change. Their efficient utilisation depends on understanding their history and genetic relationships. The current study investigates the phylogenetic relationships of barley landraces from Algeria, varieties from the Near and Middle East, traditional landraces, and modern cultivars from Europe. Using a core set of 33 varieties, including the wild ancestor Hordeum spontaneum from Armenia, genetic diversity was analysed with Random Amplified Polymorphic DNA (RAPD) and Simple Sequence Repeat (SSR) markers spanning all barley chromosomes. Based on the SSR-based phylogeny, the Algerian varieties are well clustered with those from the Near East, while distinct from the European varieties. The findings from RAPD markers partially support these results. Using exclusively traditional landraces, where a region of origin can be defined, the SSR markers are analysed separately for each chromosome individually, and the resulting clades are represented by the respective region of origin. This strategy resolves qualitative differences in geographic resolution, depending on the chromosome. While marker HvB23D (chromosome 4) separated the wild H. spontaneum from all domesticated genotypes, markers Bmag19 and Hv13GIII (chromosome 3) reveal four distinct geographic clusters (Maghreb, Near and Middle East, West Europe, Central Europe). These biogeographic patterns suggest a model, where divergence of domesticated barley due to human activity interacted with introgression of individual chromosomes from wild barley, yielding adaptive diversity. These biogeographic patterns suggest a model in which the divergence of domesticated barley, driven by human activity, interacts with the introgression of chromosomes from wild barley, resulting in the creation of adaptive genetic diversity. Our research advances our knowledge of barley landraces' functional genomics and highlights their potential in molecular breeding, particularly for developing resilient varieties suited to diverse environmental conditions.

DNA markers for early detection of somaclonal variation in Berangan banana

Banana is Malaysia’s second most cultivated fruit, with 50% of the cultivation focusing on Berangan and Cavendish types. Bananas have been identified as one of the important fruits in the National Agrofood Policy (2011-2020) due to their demand in the domestic and export markets. Thus, FGV is committed to achieving a 50% market share of local banana production through tissue culture propagation techniques. This technique enables the mass production of banana planting material in a short period. However, one downside of this technique is the random occurrence of somaclonal variation, which has been reported for various crops. FGV’s clonal propagation aims to produce true-to-type banana planting materials, therefore, somaclonal variants are undesired. Hence, we aimed to establish a marker-based screening method for early detection and elimination of Berangan somaclonal variants prior to field planting. Berangan leaf samples with and without somaclonal variation were collected from different sites. The samples’ DNA was extracted using an in-house cetyltrimethylammonium bromide (CTAB) extraction method and screened using 145 Random Amplified Polymorphic DNA (RAPD) markers. RAPD markers that could distinguish somaclonal variation in the Berangan samples were developed into Sequence Characterized Amplified Region (SCAR) markers for routine screening. The SCAR markers were validated for their ability to reproduce the specific bands from the RAPD results. Of the 145 RAPD markers, three markers do not produce any bands, 47 RAPD markers produce monomorphic bands and 95 RAPD markers produce polymorphic bands. Three out of the 95 polymorphic RAPD markers were able to selectively distinguish the somaclonal variation in samples from similar sources. The three RAPD markers were developed into SCAR markers, and they successfully reproduced the specific bands from their respective RAPD results. Hence, the developed SCAR markers will be utilized for the early detection and elimination of Berangan somaclonal variants from selected sources.

Marker Assisted Selection: A Novel Approach for Crop Improvement

The process of marker-assisted selection, or marker-aided selection (MAS), selects a trait of interest indirectly by considering a marker linked to the trait (e.g., quality, productivity, disease resistance, and biotic stress tolerance) rather than the trait itself. This integration of marker data with traditional selection approaches has become a widely studied and recommended method for advancing breeding programs. This technique has been extensively researched and recommended for animal and plant breeding. Here, we combine marker data with conventional selection to choose the best candidate for a future breeding program. Nowadays, the majority of MAS investigations employ DNA-based markers such as microsatellites, single nucleotide polymorphisms (SNPs), random amplification of polymorphic DNA (RAPD), amplified fragment length polymorphism (AFLP), and restriction fragment length polymorphism (RFLP). In this case, we discussed several kinds, techniques, and other features of marker Assisted Selection.

Investigation of clonal relationship in hospital-associated Candida parapsilosis isolates.

We aimed to investigate the clonal relationship and antifungal susceptibility of C. parapsilosis isolated from hospitalized patients and to determine whether it is due to transmission or not and the spread status of resistant isolates. Between January 2017 and June 2019, totally 277 C parapsilosis isolated from blood, urine and catheter samples of adult or pediatric in-patient (intensive care and service) who applied to Mycology laboratory in our hospital were included in the study. All isolates were identified using conventional methods, API 20C AUX (Biomerieux, France) semi-automated system and confirmed by MALDI-TOF MS Biotyper Smart (Bruker Daltonik GmbH, Germany). Randomly amplified polymorphic DNA (RAPD) PCR method was used for molecular genotyping of isolates. MIC values for fluconazole, anidulafungin and amphotericin B were determined according to the M27-A3 CLSI broth microdilution reference method guideline. Seven different band patterns (A-G) were detected in 277 isolates by RAPD PCR method. According to the rank order of the isolates, 170 (61.37%) C, 65 (23.47%) A, 18 (6.50%) G, 11 (3.97%) B, six (2.17%) E, two (0.72%) F and one (0.36%) D patterns were determined. When the band patterns of the isolates were evaluated according to the years, it was detected that C pattern continued between 2017 and 2019 and that all isolates continued to spread only as C pattern in 2019. While 211 (76.17%) of the isolates were resistant to fluconazole (≥ 8µg/ml), two (0.72%) were resistant to amphotericin B (≥ 2µg/ml) and two (0.72%) were intermediate to anidulafungin. It is noteworthy that the spread of the C pattern in C. parapsilosis strains has increased over the years and is the main pattern isolated from the whole hospital. The detection of high fluconazole resistance in C. parapsilosis isolates in our hospital may also be related to the dominant pattern.

Efficient micropropagation protocol and assessment of genetic fidelity of micropropagated plantlets of Pueraria tuberosa

The aim of this study was to develop an efficient micropropagation protocol for Pueraria tuberosa, a medicinally valuable woody liana species. The nodal explants derived from in vitro-grown seedlings were cultured on Murashige and Skoog (MS) medium containing various combinations of 6-benzylaminopurine (BAP), thidiazuron (TDZ), indole-3-acetic acid (IAA), and indole-3-butyric acid (IBA). These combinations led to the substantial development of multiple shoots. The combination of TDZ (0.57 µM/L) and IBA (0.12 µM/L) resulted in the highest regeneration rate (85%), maximum number of shoots (5.52 ± 0.67), and best shoot length (4.96 ± 0.33 cm). The most effective in vitro rooting treatment consisted of half-strength MS medium accompanied by 2.46 µM/L IBA and a 10-minute pulse treatment with the same concentration of IBA. This treatment resulted in the highest root formation (100%) and maximum roots per shoot (16.72 ± 1.81), with the highest root length (3.58 ± 0.54 cm) among all treatments tested. After four weeks under field conditions, hardened in vitro plantlets showed an 88.2% survival rate. The genetic fidelity of regenerated plants was assessed using inter-simple sequence repeat (ISSR) and random amplified polymorphic DNA (RAPD) markers, which produced monomorphic bands, demonstrating the genetic homogeneity of regeneration and reliability of the in vitro propagation method.

Genetic diversity of several local gogo rice cultivars from Batang Lubu Sutam District using RAPD (Random Amplified Polymorphic DNA) molecular markers

Abstract It is known that many farmers have abandoned local gogo rice in Padang Lawas Regency, because they have many shortcomings such as long maturity, low total grain count, and high plant height. However, in addition to its shortcomings, there are advantages such as resistance to stress. Characterization in plant breeding can be done in terms of agronomy, morphology, nutritional content, and molecular markers. Utilization through RAPD molecular markers is expected to identify characteristics of gogo rice plants so that genetic improvement can be carried out. This study aims to determine the diversity of genetic information of gogo rice from Padang Lawas Regency based on RAPD molecular markers. This study was conducted from October 2023 to April 2024. The genetic material in this study was 14 local gogo rice cultivars introduced. Samples of gogo rice cultivars were isolated and amplified using 2 RAPD primers, namely OPB 7 and OPD 2. The UPGMA cluster analysis method and PCoA analysis used MVSP software version 3.2. The results of amplification with 5 RAPD primers produced a percentage of polymorphic bands of 100%. The diversity of 14 local gogo rice cultivars is included in the moderate category with a similarity coefficient of 40%.

Cryopreservation of Fegra Fig (Ficus palmata Forssk.) and Genetic Fidelity of the Recovered Micropropagated Plants

Cryopreservation allows for the long-term storage of biological materials, ensuring their viability for future use and preserving biodiversity. This study aimed to develop an efficient protocol for the long-term in vitro conservation of fegra fig. In vitro precultured shoot tips of fegra fig with 0.3 M sucrose showed the highest regrowth percentage (100%) before plunging into liquid nitrogen. After plunging into liquid nitrogen, the regrowth percentage was 43.33%. Following cryopreservation, the recovered shoots were rooted and acclimatized to ex vitro conditions. The genetic fidelity of the acclimatized 8-week-old fegra fig plantlets after cryopreservation with their mother plant was tested using randomly amplified polymorphic DNA (RAPD), inter simple sequence repeats (ISSR), and start codon targeted (SCoT) molecular markers. Genetic similarity between the stored plantlets with their mother plant was 100% ensuring uniformity and true-to-type regenerated plants following cryopreservation. This study presents, for the first time, an efficient protocol for the genetic conservation of fegra fig.

Analysis of molecular genetic diversity of Nephelium lappaceum L. plant in North Sumatra using RAPD markers

Abstract Achieving high fruit production requires development with quality superior seeds and good management professional. In fact, it is very difficult to identify the advantages of perennial plants such as rambutan which are only based on morphology because it has a long lifespan, so identification with molecular marker technology is needed. This study aimed to obtain achieve data about genetic diversity and molecular characterization of genetic banding pattern of Rambutan (Nephelium lappaceum L.) in North Sumatra using RAPD (Random Amplified Polymorphic DNA) markers. Research was conducted at Molecular Genetics Laboratory, Faculty of Agriculture, Universitas Sumatera Utara, Medan from January 2024 to March 2024. Based on nine plant samples from 3 districts in North Sumatra, namely: Binjai (BJ), Deli Serdang (DS), and Langkat (LG) where from each 3 different samples accessions were taken from each district. Determine of band size use a UVITEC Cambridge FireReader computer software and determine of genetic sameness relationship use Multivariate Statistical Package (MVSP ver 3.2) online software. Base on 9 samples isolated of rambutan and amplify use primers OPK-16, OPK-7, OPK-19 and OPD-8, obtained band sizes range from 100-4,600 bp and base on phylo-genetic UPGMA results were divided into two groups. Group I consist of LG 1 and Group II consisted of LG 2, DS 2, BJ 2, LG 3, BJ 1, BJ 3, DS 3, DS 1 with genetically similar points range from 0.48-1.

Genetic variation based on RAPD (Random Amplified Polymorphic DNA) markers in western tarsiers (Cephalopachus bancanus) from South Sumatra and Bangka Island

Genetic variation based on RAPD (Random Amplified Polymorphic DNA) markers in western tarsiers (Cephalopachus bancanus) from South Sumatra and Bangka Island

Assessment of Genomic Integrity of Vitex negundoL., An Important Indian Medicinal Plant, Using RAPD Markers

Vitex negundo is an Indian medicinal plant containing steroids, flavonoids, lignans and terpenoids that can be used as a precursor for commercial production. An efficient marker system such as Random Amplified Polymorphic DNA (RAPD) was used to assess the genetic integrity of V. negundo. The straightforward method RAPD can be used to evaluate genomic integrity because it uses a small amount of DNA for PCR amplification. Six out of thirteen RAPD primers generated 150 distinct bands, of which 31 were polymorphic, with an average of 5.16 polymorphic bands per primer. A maximum of up to 32 fragments were amplified, and an average of 25 per primer, and the amplicons varied in size between 100 and 2000bp. The percentage of polymorphism ranges from 12.9 to 22.5, with an average of 16.6. The PIC values ranged from 0.11 to 0.63 for RAPD primers. The study pointed out that RAPD markers evaluate the genetic fidelity in Vitex negundo. The UPGMA cluster analysis grouped all in vitro raised plantlets treated with different growth regulators such as BAP, DPU, TDZ, and mT. The principal component analysis also substantiates this clustering pattern. Thus, the phylogenetic relationship and a high genetic variation revealed in the present study could provide baseline data for the conservation and improvement of this plant in future. Also, the molecular marker identified in this study will be helpful in the authentication of this species to prevent adulteration in herbal medicine.

Prevalence and Diversity of Plant Parasitic Nematodes in Irish Peatlands

The prevalence of plant parasitic nematodes (PPN) in the Irish peatlands was investigated in five different peatland habitats—raised bog, cutover scrub/woodlands, fens and peat grasslands, which were further sub-categorised into fourteen different sub-habitats. Within the raised bog habitat were healthy bog hummock (HBH), healthy bog lawn (HBL), degraded bog hummock (DBH) and degraded bog lawn (DBL) and the fen habitats were fen peat (FP) and rich fen peat (R-FP). Cutover scrub or woodland habitat included cutover scrub rewetted (C-RW), cutover scrub non-rewetted (C-NRW), woodlands rewetted (W-RW) and woodlands non-rewetted (W-NRW). Grassland included wasted peat (WP), rough grazing (RG-I) and improved fen peat grassland (IFPG-RW and IFPG-NRW). Soil samples from peatlands were all collected between July and December 2023 when the temperature ranged from 12 to 20 °C. One half of each sample was used for molecular nematode analysis and the other half for morphological identification of nematodes. For the morphological identification, a specific nematode extraction protocol was optimised for peatland soils, and the extracted nematodes were fixed onto slides to be studied under a high-power light microscope. Subsequently, the other part of the soil was processed to isolate total DNA, from which the 18S rRNA gene was sequenced for the identification of nematode taxa. The extracted DNA was also used for randomly amplified polymorphic DNA (RAPD) fingerprinting analysis to determine banding patterns that could classify different bog habitats based on PPN random primers. Compared to that in the climax habitats (HBH, HBL, DBH, DBL, FP, R-FP), PPN prevalence was recorded as being higher in grasslands (WP, RG-I, IFPG-RW and IFPG-NRW) and scrub/woodland ecosystems (C-RW, C-NRW, W-RW, W-NRW). The results indicate that nematode populations are different across the various bog habitats. Emerging and current quarantine PPN belonging to the families Pratylenchidae, Meloidogynidae, Anguinidae and Heteroderidae were noted to be above the threshold limits mentioned under EPPO guidelines, in grassland and wooded peatland habitats. Future actions for PPN management may need to be considered, along with the likelihood that these PPN might impact future paludiculture and other crops and trees growing in nearby agricultural lands.

One-Step Soft Agar Enrichment and Isolation of Human Lung Bacteria Inhibiting the Germination of Aspergillus fumigatus Conidia

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.

Study of Genetic Diversity of Aromatic Rice Genotypes Using Random Amplified Polymorphic DNA Markers

The study of genetic variation in aromatic rice is necessary for enhancing and preserving its genetic makeup. In this study, we amplified four Random Amplified Polymorphic DNA (RAPD) markers from the genomes of 11 different aromatic rice genotypes in order to evaluate the genetic variation and relatedness in aromatic rice. The genotypes were collected from different sources- five of which were obtained from the Bangladesh Rice Research Institute, one from the Bangladesh Institute of Nuclear Agriculture, and the remaining five were local genotypes. In RAPD profiling, twenty-seven of the thirty-six bands that were generated by the four primers were polymorphic, resulting in an estimated average polymorphism of 47.22 percent. The average value of the polymorphic information content was 0.309 with a range from 0.165 to 0.456, indicating that the primers were effective to detect genetic diversity between the genotypes. The genetic diversity and Shannon information index across all of the genotypes and primers were 0.176 and 0.260, respectively, which indicated that there was a certain amount of genetic variation in the studied aromatic rice genotypes. Kalizira and Binadhan-9 genotypes had the smallest genetic distance (0.057), while it was the highest (0.406) between the BRRI dhan 34 and BRRI dhan 50. The genotype pairs BRRI dhan 38 vs BRRI dhan 34, BRRI dhan 38 and Kalizira, and Zirakatari and Kalizira had also a higher level (0.365) of genetic distances. The genotypes were grouped into two main clusters in the UPGMA dendrogram. Cluster I included five genotypes: BRRI dhan 34, Basmoti, Atashail, Kalizira, and Binadhan-9. Cluster II included remaining six genotypes: BR5, BRRI dhan 38, BRRI dhan 50, Uknimodhu, BRRI dhan 37, and Zirakatari. Documentation of the genetic variation and relatedness among the aromatic rice genotypes under this investigation could be useful for future research into the development of aromatic rice and related fields. J Bangladesh Agril Univ 22(3): 277-284, 2024

Analysis of antimicrobial resistance and genetic diversity of Acinetobacter baumannii in a tertiary care hospital in Haikou City.

This study addresses the distribution and antimicrobial resistance of Acinetobacter baumannii (A. baumannii) in a medical facility in Haikou City, aiming to provide essential insights for enhancing in-hospital treatment and prevention strategies. We conducted a retrospective analysis of 513 A. baumannii isolates collected from a tertiary care hospital in Haikou between January 2018 and December 2020, focusing on their antimicrobial resistance patterns. Random Amplified Polymorphic DNA (RAPD) analysis was performed on 48 randomly selected A. baumannii strains. Using Gel-pro4.0 and NTSYSspc2.10 software, we constructed dendrograms to assess the genetic diversity of these strains. Our results indicate that males between 60 and 70 years old are particularly vulnerable to A. baumannii infections, which are most frequently detected in sputum samples, with a detection rate exceeding 70%. Alarmingly, over 50% of the isolates were identified as multi-drug resistant. The RAPD-PCR fingerprinting cluster analysis demonstrated substantial genetic diversity among the strains. Using primer OPA-02 at a 45% similarity coefficient, the strains were categorized into four groups (A-D), with group A being predominant (39 strains). high-prevalence areas like the Neurosurgery and Intensive Care Medicine Wards require enhanced surveillance and targeted interventions to manage Group C infections effectively. Additionally, the varied presence of other groups necessitates customized strategies to address the specific risks in each ward. Similarly, primer 270 at a 52% similarity coefficient classified the strains into five groups (E-I), with group E being most common (36 strains). The study highlights a concerning prevalence of antimicrobial resistance, particularly multi-drug resistance, among A. baumannii strains in the Haikou hospital. The significant genetic diversity, especially within groups A and E, underscores the need for tailored hospital treatment protocols and prevention measures. These findings contribute to the growing body of research on antimicrobial resistance, emphasizing the urgent need for effective management strategies in healthcare settings.