Pure Isolates Research Articles

Health-Promoting Properties of Anthocyanins from Cornelian Cherry (Cornus mas L.) Fruits.

The cornelian cherry is a plant that annually provides fruits, drupe-type, ranging in color from yellow through pink, red, carmine, and almost black. Cornelian cherry bears abundant fruit in temperate climate conditions, which means that its dark-colored fruits can be treated as an excellent source of anthocyanins. After consuming, anthocyanins have a protective function in the human body. Raw fruit extracts and their pure isolates, rich in anthocyanins, have a wide spectrum of health-promoting properties. This review focuses on the health-promoting properties of anthocyanins from fruits of cornelian cherry, documented in research conducted in vitro, in vivo, and in humans. The results obtained so far confirm the beneficial effects of anthocyanins on the blood parameters, whose values are important in predicting and assessing the risk and progression of cardiovascular and metabolic diseases. A beneficial effect on molecular and histopathological changes in target organs such as the heart, brain, kidneys, and liver has also been demonstrated. Anthocyanins from cornelian cherry have a strong antioxidant effect, which explains their protective effects on organs and anticancer effects. Moreover, they have antiglycemic, antihyperlipidemic, anti-inflammatory, and antimicrobial properties. The work highlights the perspectives and directions of necessary research.

Biopreservative Efficacy of Some Common Spices Sold in Dakingari Market on Hot-Break Locally Processed Tomato Paste

The research investigated the biopreservative efficacy of some common spices sold in dakingari market as substitutes for chemical preservatives on hot-break locally processed tomato paste (Platinum 7 F1 variety). After concentration, the paste was divided into thirty one (31) portions labeled A–O2. The spices were added to tomato paste in the following proportion of ginger; garlic; onion; pepper; ginger and garlic; ginger and onion; ginger and pepper; garlic and onion; garlic and pepper; onion and pepper; ginger, garlic and onion; ginger, garlic and pepper; garlic, onion and pepper; onion, pepper and ginger; ginger, garlic, onion and pepper 3 and 5 percent. Each mixture (450g) was evenly homogenized, filled up into air-tight Mayonnaise jars and stored at room temperature over a period of 9 weeks. Pure cultures were obtained from distinct colonies by repeating streaking on fresh agar plates and subjected to microscopic examination and biochemical tests such as Gram staining. Microbial examination and yeast counts of the samples were enumerated on a weekly basis while the pure microbial isolates were identified. Results showed that those without fungi growth are; Sample G (ginger and onion), Sample O (onion, pepper and ginger), Sample D (onion), Sample E (pepper), Sample M (ginger, garlic and pepper) and Sample L (ginger, garlic and onion), Sample I (garlic and onion) (3 and 5%). The microorganisms were identified as organism: Rhizopus orazae, Penicillium citrinum, Aspergillus micronesiensis, Aspergillus flavus, Aspergillus niger , Phoma multristrata, Lactobacillus plantarum, Lactobacillus brevis, Leuconostoc mensenteroides, Lactobacillus acidophilus, Lactobacillus fermentum. The study concluded only Sample D (onion) and Sample N (garlic, onion and pepper) that has no bacteria growth (3 and 5%).The study concluded that combined samples with combination of garlic or ginger (3 and 5%) suitably preserved tomato paste for 9 weeks without deterioration. The use of natural products such as ginger, onion and garlic as natural preservatives could significantly improve tomato utilization in household production in Dakingari and Nigeria at large where refrigeration is greatly hampered by erratic power supply. Further investigation for other biopreservatives is recommended to ascertain the safety consumption of tomato paste of the people in the study area and the need to look into more new natural bio-preservatives which can promote human health besides acting as food preservative.

Prevalence of Multi Drug Resistance Escherichia coli Bacteriuria among Antenatal Women in a Tertiary Hospital in Enugu, Nigeria

Background: Urinary Tract Infection (UTI) due to Escherichia coli (E. coli) is a common health problem among women. Pregnant women are at increased risk due to the physiological changes associated with pregnancy. The present study was designed to determine the prevalence of E. coli UTI and its antibiotic susceptibility pattern among pregnant women at the antenatal clinic of the Enugu State University of Science and Technology Teaching Hospital, Enugu, Nigeria.
 Materials and Methods: A cross sectional study was carried out on a total of 200 pregnant women with and without symptoms of UTI. Mid-stream urine samples were collected and inoculated into Cysteine Lactose Electrolyte Deficient (CLED) and MacConkay. Colony counts > 102 cfu/ml of urine were regarded as significant bacteriuria. Pure isolates of bacterial pathogen were characterized by colony morphology, gram-stain and standard biochemical procedures. The method of agar disc diffusion susceptibility testing was used to determine the antibiotic resistance and sensitivity of the isolates.
 Results: The overall prevalence of UTI was 15.4% while 48.5% were symptomatic cases and 5.0% asymptomatic cases. E,Coli was the most frequently isolated organism with a prevalence of 53.4% (111 samples) while other pathogens accounted for 46.6% (89 samples). E. coli showed the highest resistance to the antibiotic Ampicillin 100%, 78% to Tetracycline and 70% to Rocephine but a low resistance of 16.7% against Nitrofurantoin and Ceftriazone respectively.
 Conclusion: UTI due to E. coli in pregnant women in Enugu is relatively high and shows high resistance to routine antibiotics. It is therefore, strongly recommended to undertake a routine culture as part of the antenatal care for pregnant women.

Food Safety Aspects of Breeding Maize to Multi-Resistance against the Major (Fusarium graminearum, F. verticillioides, Aspergillus flavus) and Minor Toxigenic Fungi (Fusarium spp.) as Well as to Toxin Accumulation, Trends, and Solutions-A Review.

Maize is the crop which is most commonly exposed to toxigenic fungi that produce many toxins that are harmful to humans and animals alike. Preharvest grain yield loss, preharvest toxin contamination (at harvest), and storage loss are estimated to be between 220 and 265 million metric tons. In the past ten years, the preharvest mycotoxin damage was stable or increased mainly in aflatoxin and fumonisins. The presence of multiple toxins is characteristic. The few breeding programs concentrate on one of the three main toxigenic fungi. About 90% of the experiments except AFB1 rarely test toxin contamination. As disease resistance and resistance to toxin contamination often differ in regard to F. graminearum, F. verticillioides, and A. flavus and their toxins, it is not possible to make a food safety evaluation according to symptom severity alone. The inheritance of the resistance is polygenic, often mixed with epistatic and additive effects, but only a minor part of their phenotypic variation can be explained. All tests are made by a single inoculum (pure isolate or mixture). Genotype ranking differs between isolates and according to aggressiveness level; therefore, the reliability of such resistance data is often problematic. Silk channel inoculation often causes lower ear rot severity than we find in kernel resistance tests. These explain the slow progress and raise skepticism towards resistance breeding. On the other hand, during genetic research, several effective putative resistance genes were identified, and some overlapped with known QTLs. QTLs were identified as securing specific or general resistance to different toxicogenic species. Hybrids were identified with good disease and toxin resistance to the three toxigenic species. Resistance and toxin differences were often tenfold or higher, allowing for the introduction of the resistance and resistance to toxin accumulation tests in the variety testing and the evaluation of the food safety risks of the hybrids within 2-3 years. Beyond this, resistance breeding programs and genetic investigations (QTL-analyses, GWAM tests, etc.) can be improved. All other research may use it with success, where artificial inoculation is necessary. The multi-toxin data reveal more toxins than we can treat now. Their control is not solved. As limits for nonregulated toxins can be introduced, or the existing regulations can be made to be stricter, the research should start. We should mention that a higher resistance to F. verticillioides and A. flavus can be very useful to balance the detrimental effect of hotter and dryer seasons on aflatoxin and fumonisin contamination. This is a new aspect to secure food and feed safety under otherwise damaging climatic conditions. The more resistant hybrids are to the three main agents, the more likely we are to reduce the toxin losses mentioned by about 50% or higher.

First report of Sphaerulina azaleae causing leaf spot on Rhododendron pulchrum Sweet in China.

Rhododendron pulchrum Sweet is a popular ornamental evergreen shrub, renowned for its exquisite flowers. In November 2020, leaf spot disease was observed at Qujing Normal University (25.527°N; 103.744°E), Qujing, Yunnan, China. Symptoms were observed on 25-35 % leaves. This led to early leaf drop, significantly reducing its ornamental value. The infected leaves exhibited irregular, dark brown to blackish spots, with random distribution. To investigate the causal agent, ten symptomatic leaves were collected for pathogen isolation. Leaf spots bearing ascomata were picked with a sterile needle, placed on water agar, and then incubated at 26 ℃ for 24 h. Individual germinated spores were transferred onto PDA for further purification and morphological study. Two pure isolates (chl01 and chl02) were obtained. The colonies on PDA were circular, raised, pink-white at the center, dark brown at the margin, and dark brown to black from reverse after four weeks at 26 °C. Moreover, colonies on PDA sporulated following a 5-hour UV light exposure in a laminar flow hood and a subsequent 14 h light: 10 h dark (14:10) cycle for 5 days at 26 °C. The conidiophores are reduced to conidiogenous cells which are 1.5-3 × 1.5-2.5 µm (n = 10), integrated, hyaline, proliferating sympodially near the apex, cylindrical, and widest at the base. Conidia are hyaline, slightly verruculose, guttulate, cylindrical, straight or irregularly curved, subobtuse at the apex, truncate at the base, and 19-28 × 3-4 µm (n= 30), with 1-3 septa. The morphological features were similar to the genus Sphaerulina (Quaedvlieg et al. 2013). To confirm the candidate pathogen, we performed PCR and gene sequencing using specific primers ACT-512F/ACT2Rd, EF1-728F/EF2, ITS4/ITS5, LSU1Fd/LR5 and fRPB2-5F/fRPB2-414R for actin (ACT), the partial translation elongation factor 1-alpha (TEF1), internal transcribed spacer region (ITS), large subunit ribosomal RNA gene (LSU), and RNA polymerase II subunit (RPB2) genes, respectively (Choi et al. 2020). Sequences were deposited in GenBank with accession numbers OR359635 (ACT), OR359637 (TEF1), OR335339 (ITS), OR335299 (LSU) and OR359636 (RPB2). BLAST searches showed 97.34% (ACT), 99.47% (TEF1), 100% (ITS), 100% (LSU) and 100% (RPB2) identity with those of Sphaerulina azaleae CBS 128605 from GenBank, respectively. A multilocus phylogenetic tree was generated using maximum likelihood (ML) based on the concatenated ACT-TEF-ITS-LSU-RPB2 sequence, placing our isolates within the S. azaleae group with high confidence. To fulfill Koch's postulates, three R. pulchrum plants in the landscape (three leaves per plant) were inoculated by pipetting 0.2 ml spore suspension (1 × 106 conidia/ml) onto the surface of each leaf (Khoo et al. 2022). Three additional plants in the landscape were inoculated with sterile water on similar aged leaves. The leaves were enclosed in plastic bags for 72 hours. At 7 days postinoculation, infected leaves exhibited the symptoms as described above, whereas the controls showed no symptoms. This experiment was repeated twice. The same fungus was reisolated from the infected leaves and identified based on morphology and Sanger sequencing. It is worth noting that S. azaleae can cause leaf spot on Rhododendron yedoense f. poukhanense (Choi et al. 2020). To our knowledge, this is the first report of S. azaleae causing leaf spot on R. pulchrum Sweet in China, indicating that appropriate management strategies are needed.

Microbes Isolated from Landfill Soil Utilize Polyethylene Terephthalate (PET) as Their Sole Source of Carbon: An Unexplored Possibility of Bioremediation in Bangladesh

Plastic products are so extensively used that they continue to strain the already overburdened waste management system and, inevitably, the global climate. Biodegradation is a sustainable remedy. Here, we report a few microorganisms isolated from landfill soil near Dhaka that thrive especially on polyethylene terephthalate (PET) polymers. Soil samples were subjected to three enrichment cycles that contained no carbon except PET. Pure isolates were recovered and incubated on minimal agar containing PET as the sole carbon. A morphological examination was carried out. Potential PET-degrading enzyme sequences from the isolates and other microalgae were analyzed for homology using BLASTP and TBLASTN, and multiple sequence alignment (MSA) was performed to assess conserved domains. Six isolates were obtained. Two isolates grew around the PET film but did not grow sufficiently in other areas of the minimal agar. Two other isolates with greenish pigmentation flourished around the PET film as well as on other areas of the agar. One of the green cells resembled Aphanocapsa, with irregular shapes and occasionally brown dense bodies, while the others looked round like Microcystis. Homology analysis revealed the hypothetical PETases in green cells contained the highly conserved catalytic triad (Ser-His-Asp) at the active site, as always found in alpha-beta hydrolase fold containing enzymes. Microbes isolated from two landfill sites in the vicinity of Dhaka have been adapted to utilize PET as a carbon source. In the future, sequencing and further characterization would be necessary to validate the findings. Microalgal systems demand increased focus, given their potential to offer valuable resources for bioremediation.

First Report of Verticillium Wilt caused by Verticillium dahliae on Chilli in South Korea.

Chili pepper (Capsicum spp.) is an important crop in South Korea and is widely used in Korean cuisine, cultivated across a land area of roughly 29.8 thousand hectares, with a total production of 69 thousand tons (Lee et al., 2005; Statista, 2022). In September 2020, two farmer fields in Samcheok (37.444039°N, 129.135875°E; 37.633738°N, 128.911731°E), Gangwon province, South Korea, it is observed that chili pepper leaves showing yellowing and wilting symptoms, with an estimated disease incidence of approximately 10-15%. To identify the causal agents six affected plants were brought to lab. All the plants exhibited vascular discoloration in stem and root. After surface sterilizing small pieces of discolored tissue in 1% NaOCl for 30 s and rinsing twice in sterile distilled water, the tissue pieces were placed on water agar and incubated at 25°C for 10 days. Six pure isolates with consistent morphological characteristics were obtained by single spore isolation. Two representative isolates, NC17601 and NC20845 were selected for identification based morphological and molecular characters. Colonies on potato dextrose agar (PDA) during 10 days of incubation at 25°C in the dark were cottony white initially but progressively became dark as the formation of melanized microsclerotia. Conidiophores were hyaline, vertically branched or not, and had 2 - 4 phialides per node. Phialides were subulate and tapering from base to tip. The colonies produced abundant conidia, which were hyaline, single celled, smooth walled, cylindrical to oval, clustered on phialides, and 3.8 - 7.2 ×2.1 - 3.9 ㎛(mean ± SD: 5.2 ± 0.7 × 2.8 ± 0.5). Microsclerotia were aggregated form, various size and shape, and brown. These are the typical morphology of Verticillium dahliae (Inderbitiz et al. 2011; Yu et al. 2016). The molecular identification was later confirmed through PCR amplification, and sequencing targeting the translation elongation factor 1 alpha (TEF), actin (ACT), tryptophan synthase (TS), and glyceraldehyde-3-phosphate dehydrogenase (GPD) genes using the primer sets described by Inderbitiz et al. (2011). The resulting sequences were deposited in GenBank with accession numbers LC761935 to LC761942. The maximum likelihood tree based on combined sequences of ACT, GPD, TEF and TS was inferred using RAxML- HPC2 on XSEDE as implemented on the CIPRES web server. The phylogenetic tree showed that the isolates were sit together with V. dahliae isolates (Ex-type PD322, PD227 and PD502). Pathogenicity tests using two isolates (NC17601 and NC20845) were conducted in the greenhouse, where 10 two weeks old seedlings per isolates (cv. Bigstar) were root-tip cut and then soaked in a fungal spore suspension of 107 conidia ml-1 for 1 h, while 10 seedlings were treated with sterile distilled water as a control. All the treated plants were maintained at 25°C (night)/ 25°C (Day) under natural light. After three weeks, all inoculated plants exhibit growth stunting with vascular discoloration in the stem and roots as compared to asymptomatic control plants. The isolates of V. dahliae were consistently re-isolated from discolored root tissues and identified based on morphological characteristics, thus fulfilling Koch's postulates. In South Korea, V. dahliae has been reported to cause wilt disease in various crops, including Kimchi cabbage and radish (Dumin et al. 2020; Choi et al. 2023). To the best of our knowledge, this is the first report that V. dahliae causing Verticillium wilt of chili pepper in South Korea. Overall, Verticillium dahliae is considered to be a significant threat to agriculture in South Korea, and efforts are being made to develop effective control strategies to mitigate its impact on crops.

Actinomycetes isolated from rhizosphere of wild Coffea arabica L. showed strong biocontrol activities against coffee wilt disease.

Coffee, the second most traded commodity globally after petroleum and is the most exported cash crop of Ethiopia. However, coffee cultivation faces challenges due to fungal diseases, resulting in significant yield losses. The primary fungal diseases affecting coffee production include coffee berry disease, wilt disease (caused by Gibberella xylarioides), and coffee leaf rust. In this study, we aimed to isolate potentially antagonistic actinomycetes from the root rhizosphere of wild Coffea arabica plants in the Yayo coffee forest biosphere in southwestern Ethiopia. Soil samples were collected from the rhizosphere, and actinomycetes were selectively isolated and identified to the genus level by morphological, physiological, and biochemical characterization. These pure isolates were screened for their antagonistic activity against Gibberella xylarioides in vitro using a dual culturing method. Promising isolates demonstrating strong inhibition of fungal mycelial growth were further investigated through in vivo experiments using coffee seedlings. A total of 82 rhizobacteria were isolated. These isolates' inhibition of fungal mycelial growth varied from 0% to 83.3%. Among them, four isolates MUA26, MUA13, MUA52, and MUA14 demonstrated the highest percentage inhibition of fungal mycelial growth: 83.3%, 80%, 76.67%, and 73.3%, respectively. Seedlings inoculated with MUA13, MUA14, and MUA26 during the challenge inoculations (Rhizobacteria + Gibberella xylarioides) exhibited the lowest disease incidence compared to the infected fungi (P < 0.05). Notably, the seedlings inoculated with MUA26 demonstrated the highest disease control efficiency, reaching 83% (P < 0.05). MUA26 was found to produce extracellular enzymes, including chitinase, protease, and lipase, which acted as inhibitors. In summary, this study highlights that MUA26, among the actinomycete isolates, exhibited significant antagonistic activity against Gibberella xylarioides f.sp. coffea. Its efficacy in controlling coffee wilt disease, both in vitro and in vivo, positions it as a potential bioinoculant for managing coffee wilt disease.

Biochemical and Fe-resistant characteristics of indigene bacteria from a high iron concentration landfill in Indonesia

Bacteria isolated from leachate with high heavy metal concentration mostly have potential for bioremediation. The aim of this study was to isolate bacteria from leachate of a landfill containing high concentration of Fe. The leachate sample was collected from Gampong Jawa Landfill, Aceh, Indonesia and used to isolate the bacteria by spread method. The purification of the isolates was carried out through four quadrant plate method. Pure isolates were characterized based on the microscopic or macroscopic appearance and biochemical tests. Fe-resistance was tested by disc diffusion method and constructing the growth curve based on the optical density of broth culture media. A total of 27 pure isolates were obtained and identified, representing eight different genera: Pseudomonas, Bacillus, Neisseria, Micrococcus, Staphylococcus, Proteus, Acinetobacter, and Escherichia. Of eight isolates (each genus was represented by a single isolate), Pseudomonas sp. and Bacillus sp. were considered resistant to Fe exposure (15 ppm) with inhibition zones of 1.00 and 0.81 mm, respectively. At Fe concentration of 20 ppm the inhibition zones exceeded 1 mm, but Bacillus sp. had smaller inhibition zone than that of Pseudomonas sp. (1.15 mm versus 2.86 mm). Growth inhibition of Bacillus sp. was concentration-dependent on Fe exposure but could survive at Fe exposure up to 20 ppm. In conclusion, Bacillus sp. had a resistance against Fe exposure and its bioremediation potential is subjected for future studies.

First Report of Fusarium tricinctum Causing Root Rot on Chinese dwarf cherry (Cerasus humilis) in China.

Chinese dwarf cherry (Cerasus humilis) is a perennial small shrub indigenous to northern China, highly regarded for its calcium-rich fruit known as 'calcium fruit'. These fruits have a remarkable capacity to aid in human calcium absorption. Additionally, they contain beneficial flavonoids that hold promise for applications in the healthcare industry (Wang et al., 2018). In July 2020, a concerning development occurred on the farms in Jingtai County (37.48o N, 103.82o E), Baiyin City, Gansu Province in China. Approximately 20 to 30% of C. humilis at the full culture stage exhibited symptoms of root rot, including brownish leaves, rotten roots, and plant mortality, leading to a decrease of over 30% in calcium fruit yield. To identify the pathogen, the surface of symptomatic roots was sterilized with 3% NaOCl for 2 minutes, followed by 70% ethanol for 30 seconds, and rinsed three times with sterile water. Tissue sections (5×5mm) from the margin of the necrotic lesion were cut and cultured on potato dextrose agar (PDA) medium, and incubated for 7 days at 25℃. Five pure culture isolates were obtained from individual spores. Initially, the isolates exhibited abundant white aerial mycelia that turned light pink on the third day. Macroconidia were falciform, two to five septate, straight or slightly curved, and measuring 20.1 to 32.5×2.2 to 3.8 μm (n=50). Napiform microconidia were oval-ellipsoid, non-septate, and measuring 6.2 to 9.3×4.2 to 5.8 μm (n=50). Based on these morphological characteristics, the fungus was tentatively identified as Fusarium species (Leslie and Summerell, 2006). To confirm the identification, the internal transcribed spacer region (ITS) and the translation elongation factor (EF1α) of the isolate CH-2 were partially amplified and sequenced using the primers ITS1/ITS4 and EF2T/EF3 (Li et al., 2013, Yang et al., 2022). Upon comparison with the sequences in GenBank, 857 bp ITS sequence showed 100% homology to Fusarium tricinctum isolate QY3-1 (GenBank accessions no. MZ572963.1), and 665 bp EF1α sequence showed 99.7% homology to F. tricinctum strain TQC-C2 (GenBank accessions no. KF939493.1). The resulting sequences were deposited into GenBank with accession nos. OQ581576 and OQ848462 respectively. A maximum likelihood (ML) phylogenetic analysis based on combined partial ITS and EF1α data set was conducted via ML bootstrapping using MEGA 11. According to morphology and phylogenetic analysis, the isolate was identified as F. tricinctum (Wang et al., 2022). For a pathogenicity test, a pot experiment was conducted in a greenhouse with a temperature range of 20-27℃ and 60% relative humidity. Roots of C. humilis were immersed in a spore suspension (1×107 conidia/ml) of isolate F. tricinctum CH-2 for approximately 5 minutes. Subsequently the treated roots were planted in pots filled with sterilized field soil, while roots dipped in sterilized water were used as the control. The experiment considered of ten pots for the inoculation treatment and six pots for the control treatment. All pots were maintained in the greenhouse. After 15 days, it was observed that 80% of the inoculated plants displayed symptoms consistent with the field observations, indicating successful infection. In contrast, plants in the control treatment did not exhibit any symptoms. The same fungal pathogen as F. tricinctum CH-2 was reisolated from the diseased root tissue and confirmed through morphological and molecular assays, thereby satisfying Koch's postulates. This is the first documented report of F. tricinctum causing root rot in C. humilis in China. This disease has the potential to become one of the most significant diseases affecting C. humilis in China.

Screening of Fungal Isolates for Biodegradation Potentials of Low-Density Polyethylene from Selected Dumpsites

Polyethylene is the most commonly used synthetic plastic and poorly degraded in natural environments. This study was designed to evaluate biodegradation potentials of low-density polyethylene by fugal isolates from Station Market, Kaduna State, Nigeria. Soil samples were collected at a depth of 20cm using a sterilized hand towel and taken to microbiology laboratory, Kaduna State University. Fungi were isolated and each colony was repeatedly sub-cultured on freshly prepared SDA to obtain pure isolates. Pure separated single colonies were maintained on sterile slants at 4˚C for further investigation. Low-density polyethylene (LDPE) sheets were cut into bits and immersed in xylene and boiled for 15 minutes to dissolve and crushed. The LDPE powder was added to a mineral salt medium (MSM) containing salts in 1liter of distilled water after which the fungal isolates were introduced. Scanning electron microscopy and Fourier- Transform Infrared Spectroscopy (FTIR) analysis were used to determine the changes in the polymer bond of LDPE sheet after the biodegradation. The findings shows that Aspergillus niger, Aspergillus flavus, Brown rot and White rot fungi showed 21.1%, 16.1%, 17.6%, and 18.3% reductions in the polyethylene discs after incubation respective. Therefore, the study concludes that fungal species play a significant role in the degradation of LDPE and can be used in future studies for the degradation of complex plastic materials. Finally, the study recommends that Aspergillus niger and Aspergillus falvus be considered as potential candidates to degrade LDPE.

Isolation, identification, and characterization of resistant bacteria to antibiotics from pharmaceutical effluent and study of their antibiotic resistance.

Pharmaceutical effluents primarily enter aquatic environments through the discharge of treated and untreated wastewater from various sources, including hospitals, pharmaceutical manufacturing facilities, and households. Microbes sourced from pharmaceutical effluents such as Pseudomonas spp. pose a significant public health concern because of their high levels of resistance to multiple drugs and extreme multidrug resistance. Therefore, the present study was conducted for the isolation, identification, and molecular characterization of selected isolates from pharmaceutical effluents and also determined their antibiotic sensitivity patterns. From June 2016 to March 2017, a study was conducted on four well-known pharmaceutical companies specializing in antibiotic production in Dhaka and Gazipur. Four wastewater samples were collected from various origins and then brought to the Bacteriology laboratory for microbiological examination. Twelve pure isolates were obtained and characterized through cultural and biochemical tests while molecular identification of Pseudomonas spp. was performed using the 16S rRNA gene sequence. Twelve commercially available antibiotics were used for antibiotic sensitivity tests using Kirby-Bauer disk diffusion methods. We isolated the most predominant isolates, Pseudomonas aeruginosa (41.67%), followed by Bacillus spp. (33.33%) and Staphylococcus spp. (25%) respectively. Among 12 antibiotics, ciprofloxacin is 100% sensitive against P. aeruginosa, while the remaining 11 antibiotics are 100% resistant. Bacillus spp. showed 100% resistance to all antibiotics while 50% sensitive to vancomycin and 100% to chloramphenicol, respectively. Staphylococcus spp. was 100% resistant to all antibiotics. Our research suggested that P. aeruginosa is the reservoir of antibiotic resistance genes and spreads disease to humans from the environment. The findings of this study, i.e., the isolation, identification, and characterization of antibiotic-resistant bacteria from pharmaceutical effluent have highlighted, comprehended, and mitigated the dissemination of antibiotic resistance and opportunistic bacteria.

Characterization Of Probiotic Lactic Acid Bacteria From Honey And Assessment Of Their Effects On Consumption By Type-2- Diabetes Using Wistar Rat

The relevance of probiotic, especially lactic acid bacteria cannot be over emphasized. In this present study three honey sources were serially diluted and cultured on De Man, Rogosa and Sharpe (MRS) agar among which only one of this sources grew on MRS agar. The pure Lactobacilli isolate were subjected to gram staining, biochemical tests, physiological test, molecular analysis using Polymerase Chain Reaction (PCR) techniques and Deoxyribonucleotide (DNA) sequencing. Only one isolate was obtained known as Enterococcus fecalis. The isolate was subjected to probiotic selection and was found fit for consumption, however their effect when consumed by type-2 diabetic are alarming and based on the outcome of this study, diabetic patient are advised not consume honey.

Kinetic studies and bioremediation potential of chromate reductase-characterized Aspergillus flavus from tannery effluent

Chromium is among the major environmental pollutants endangering the health of all living things, including humans. Conventional methods for chromium detoxification are expensive, so bioremediation using fungi as potential agents for the detoxification of chromium is justified. This study aims to investigate chromium-resistant fungi capable of reducing or transforming Cr (VI) to less toxic Cr (III) from tannery effluents. The fungi were isolated from tannery wastewater and identified using 16S rRNA gene sequencing. Chromate reductase was isolated from pure fungal isolate and purified using molecular weight estimation and gel filtration chromatography. Aspergillus flavus, in an interesting turn of events, demonstrated 97% chromate reduction. The chromate reductase from Aspergillus flavus has KM of 0.015 mM and a Vmax of 0.25 mol/min. Furthermore, the enzyme's activity was constant at its peak at pH 7.0 and 50 oC, with a single band displaying a molecular weight of 44 kDa for a pure protein. The data suggest that A. flavus can be a good candidate for the detoxification of Cr (VI) in industrial effluents. The efficiency of which can be attributed to the action of chromate reductase in the A. flavus.

First Report of Vacuiphoma oculihominis Causing Fruit Rot on Navel Orange (Citrus sinensis) in China.

Newhall navel orange [Citrus sinensis (L.) Osbeck] is an economically important agricultural product in China. In February 2022, a rare lesion symptom was observed on Newhall navel oranges that were harvested from an orchard Ganzhou city, Jiangxi province, China (25.53° N, 114.79° E) and stored for 90 days (18±2℃, 80 to 90% RH) at the Jiangxi Key Laboratory for Postharvest Technology and Non-destructive Testing of Fruits and Vegetables (28.68° N, 115.85° E). Approximately 2% (15/750) of the oranges exhibited symptoms, with normal appearance but ink-black flesh and juice, yellowish lesions on edges of the symptoms, and no unusual odor. To isolate the pathogen, three 5 × 5 mm pieces of symptomatic tissue from a diseased orange were disinfected in 75% ethanol for 30 s, rinsed three times with sterile water, and inoculated on potato dextrose agar (PDA) at 25±1℃ and a 12:12 h photoperiod for 7 days. A pure isolate named ND-hsp was obtained. The colony was light yellow center with pale edge on the top and brown on the bottom. Conidia and pycnidia were observed on PDA medium after 2 months. Conidia were long oval, no septa, 2.9 × 3.4 μm (n = 50), and pycnidia were solitary, 39.4 × 43.9 μm (n = 20), with one or no orifice, brown to dark brown. The morphological characteristics of ND-hsp strain on PDA, oatmeal agar and malt extract agar were similar to those of the Didymellaceae (Aveskamp et al. 2010). Ulteriorly, the genomic DNA of the ND-hsp isolate was extracted from its mycelia using a fungal genomic DNA extraction kit (Solarbio, Beijing, China) for subsequent phylogenetic analyses. Four primer sets, LR0R (Rehner and Samuels 1994) /LR7 (Vilgalys and Hester 1990), V9G (Hoog and Gerrits 1998) /ITS4 (White et al. 1990), Btub2Fd/Btub4Rd (Woudenberg et al. 2009) and RPB2-5F2 (Sung et al. 2007)/RPB2-7cR (Liu et al. 1999) were used to amplify the corresponding DNA fragments of large subunit ribosomal RNA (LSU), internal transcribed spacer region (ITS), beta-tubulin gene (TUB2) and RNA polymerase Ⅱ second largest subunit (RPB2), respectively. The obtained sequences were assigned GenBank accession numbers and showed 99 to 100% identity with their counterparts of Vacuiphoma oculihominis UTHSC DI16-308. A phylogenetic tree was constructed in MEGA 7.0 using the concatenated sequences, placing the isolate within the V. oculihominis clade by 100% bootstrap support. Pathogenicity experiments were performed in triplicate. Ten Newhall navel oranges were surface sterilized with 75% ethanol and inoculated with 15μL of a spore suspension (2×106 spores/ml) into a 3 mm-diameter wound on the equator. The control group received sterile water instead of the spore suspension. Treated and control oranges were incubated at 25±1 ℃ and about 90% relative humidity for 20 days. All oranges were cut longitudinally or transversely through the inoculated wound and examined internally. The oranges inoculated with ND-hsp exhibited ink-black flesh and juice symptoms consistent with the initial oranges. The control oranges remained asymptomatic. Under the Koch's rule, V. oculihominis was reisolated from diseased oranges and kept in Collaborative Innovation Center of Postharvest Key Technology and Quality Safety of Fruits and Vegetables in Jiangxi Province. GenBank database analysis confirms that V. oculihominis has been found in human eye secretions and decayed trees. This is the first report of V. oculihominis as a pathogen on navel oranges in China. Our findings contribute to understanding of citrus fruit pathogens.

Genetic Diversity of Botrytis Cinerea Which Caused Gray Mold Disease in Strawberry and Some Other Vegetable Plants

The study aimed to isolate and identification the fungus Botrytis cinerea that caused gray mold disease, depending on its morphological and molecular characterizations. The results of isolation on PDA showed that 75 pure isolates were obtained from The pathogenic fungus B. cinerea is distributed as follows: Strawberry (45) isolates, eggplant (11) isolates, squash (3) isolates, cucumber (3) isolates, pepper (9) isolates, and tomato (4) isolates. DNA was extracted from 17 isolates of pathogenic fungus B. cinerea, the results of electrophoresis on 2% agarose gel, which included 17 isolates distributed in crops (strawberry 11 isolates, eggplant 4 isolates, squash and tomato one isolate each) was detected at 550 base pair(bp) of all the isolates using ITS1 and ITS4 primers. The results of detection and investigation of genetic variation of pathogenic Fungi isolates were clarified by determining the degree of kinship and drawing a phylogenetic tree An evolutionary and relationship tree was drawn using Snap Gene Viewer, When performing Allayment analysis of the isolates, the results showed that the isolates bearing the symbols (S3,S2,S1) isolated from strawberry fruits were identical to the SMGM003 strain registered in South Korea with a percentage of 100%, and isolates (S6,S5,S4) isolated from strawberry fruits are identical to the strain WMG recorded in South Korea, and isolates (S10,S9,S8) isolated from strawberry fruits are identical to the strain CIM13704 registered in China, and isolates (S11) isolated from strawberry fruits.

Morphological, Biochemical and Molecular Identification Bacteria Streptomyces spp. Causing Common Scab Disease on Potato in Iraq

This study was carried out at the University of Anbar - College of Agriculture - Department of Plant Protection. The study aimed to isolate and diagnose the bacterium that causes scabies on potatoes, Streptomyces spp., depending on the phenotypic characteristics, biochemical interactions, and molecular methods. The results of isolation from different regions and on PDA medium showed that it was obtained On 45 pure isolates of the pathogenic bacteria Streptomyces spp, and the results of growth in different media showed that the best medium for growth was PDA and Nutrient agar. The results of phenotypic diagnosis and biochemical tests revealed that 8 isolates belong to the type Streptomyces scabies and two isolates belong to the type Streptomyces acidiscabies. DNA was extracted from 10 isolates of pathogenic bacteria, and the results of molecular diagnosis using the 16S rRNA gene showed that light bands of 1250 pairs were obtained. The nitrogenous base sequences of these isolates were determined and deposited in GenBank under accession codes (OQ8191, OQ819106, OQ815918, OQ789975, OQ789966, OQ815949, OQ815950).

Skrining fitokimia dan isolasi senyawa steroid/triterpenoid dari ekstrak n-heksana biji nangka (Artocarpus heterophyllus Lam).

Jackfruit (Artocarpus heterophyllus Lam) is a plant that can grow well in tropical regions such as Indonesia. Jackfruit seeds contain steroid/triterpenoid chemical compounds, including several types of steroids that are useful as ingredients in medicines, such as estrogen, progestin, and cardenolides. The research aims to conduct phytochemical testing and isolate steroid/triterpenoid compounds from jackfruit seeds. This research uses experimental methods, including the compound extraction process using the maceration method and the isolation of active compounds via column chromatography. Identification of pure isolates was carried out using UV spectrophotometry and IR spectrophotometry. The results of phytochemical screening showed the presence of steroid/triterpenoid compounds, glycosides, and alkaloids in the samples. The isolate has a purple color with an RF value of 0.25, and the maximum absorption peak is located at a wavelength of 208.20 nm, indicating the presence of a chromophore group. IR spectrophotometry results show the presence of OH, aliphatic -CH, CH2, CH3, C=O, C-O, and C=C. This research concludes that the isolate obtained belongs to the steroid/triterpenoid compound group.

Optimisation of lutein production potential in marine biflagellate Proteomonas sulcata isolated from Indian coastal waters

The present study was conducted to isolate, identify, and extract the lutein from a biflagellate Proteomonas sulcata from the coastal waters of the Gulf of Mannar, Southern east coast of India. The targeted unicellular biflagellate P. sulcata belonging to the group Geminigeraceae was collected and isolated using 96 well plates followed by the serial dilution method and morphologically confirmed. However, the pure isolate was also confirmed based on molecular identification by PCR and sequence analysis of 18 S rRNA. The resultant sequence was analysed and reflected in phylogenetic tree. Optimised the environmental parameters to obtain the maximum cell density of 50.36 ± 0.81 × 105 cells/ml. The unique characteristic feature of the isolate was the change in colour during the culture phases. ie., red colour in growing phase up to 6th day in haffkine flask and changed into green during the stationary phase until the death phase. The reason for the colour change might be due to phycobiliprotein present in the microalga and also confirmed by FTIR analysis. The maximum chlorophyll content and carotenoid of 4.59 ± 0.38 mg/g 2.55 ± 0.12 mg/g was estimated. The conventional lutein extraction process such as saponification and solvent extraction methods was followed. The process was optimized and extracted crude lutein was analysed by using HPLC. The present study concluded that, the maximum lutein yield of 3.96 ± 0.1 mg/g was obtained at a 6 M concentration of KOH and also found that the highest amount of lutein was obtained from wet biomass (red-coloured biomass) of 4.72 ± 0.38 mg/g which is significantly higher than that of freeze-dried biomass.

First report of walnut anthracnose caused by Colletotrichum orientalis in South Korea.

Walnut (Juglans regia L.) is known as "hodu" in South Korea. It is cultivated as a cash crop and an ornamental plant across South Korea. Walnut anthracnose is one of the main limiting factors for walnut production. In June 2022, severe anthracnose was observed in several walnut fields in Gimcheon, South Korea. The disease incidence was ~35% and symptoms on affected fruits consist of necrotic and circular lesions with or without conidial mass. Six pure isolates with similar morphological characteristics were obtained from the tissue of six affected fruits. Diseased tissues were cut into 3-to-4 mm2 pieces, surface sterilized by 1% sodium hypochlorite (NaOCl) for 2 minutes, and washed three times in sterile distilled water, then dried by blotting. Four pieces/plates were placed onto potato dextrose agar (PDA; Difco Becton Dickinson) and incubated at 25°C in the dark for 2-3 days. To obtain pure isolates, hyphal tips were transferred onto fresh PDA. The colonies of all isolates on the PDA were light gray to pinkish on the upper side and pink to vinaceous on the reverse after 7 days of incubation at 25°C in the dark. The conidia were hyaline, fusiform, aseptate, and 11.3 to 20.1 µm × 3.6 to 6.9 µm (n =100). Appressoria were single, cylendrical, smooth-walled, dark brown, and 8.1 to 14.3 × 5.4 to 10.5 μm (n =30). The morphological characteristics of the isolates were comparable with those of Colletotrichum orientalis (Chen et al. 2022). Three isolates (WN23, WN28 and WN30P) were selected based on morphological characteristics for molecular identification. The beta-tubulin (TUB2), actin (ACT), chitin synthase 1 (CHS-1), and histone 3 (HIS3) genes of selected isolates were amplified and sequenced using the primers Bt2a/Bt2b, ACT512F/ACT783R, CHS-79F/CHS-345R and CYLH3F + CYLH3R, respectively (Damm et al. 2012). The resulting consensus sequences of each gene were deposited in GenBank with the accession numbers (TUB2: LC742910 to LC742912; CHS-1: LC742916 to LC742918; ACT: LC742919 to LC742921, HIS3: LC742922 to LC742924). The constructed maximum likelihood tree based on TUB2, ACT, CHS-1, and HIS3 sequence data revealed that the three isolates formed a clade with C. orientalis reference strain CBS 128532 (ex-type). The pathogenicity of two representative isolates (WN28 and WN30P) was confirmed on healthy, surface-sterilized, and detached walnut fruits. Both wounded and nonwounded fruits (10/isolate) were inoculated with the conidial suspension (106 spores/ml). The wounded and unwounded control fruits were inoculated with sterile distilled water. All the fruits (inoculated and control) were incubated in containers at 25°C with a 12-h/12-h light/dark cycle. This experiment was repeated twice. Necrotic lesions were observed in all inoculated wounded fruits, while unwounded and control fruits remained asymptomatic. The pathogen was re-isolated from inoculated fruits and identified as C. orientalis by morphological characteristics and ITS and TUB2 sequences. Damm et al. (2012) has identified C. fioriniae as species belongs to two well separated clades. However, Chen et al. (2022) split the C. fioriniae species into two species, with the new species called C. orientalis, as a causal agent of apple bitter rot. In previous studies, C. fioriniae has been reported as a causal of walnut anthracnose (Luongo et al. 2021; Zhu et al. 2015; Varjas et al. 2019). To our knowledge, this is the first report of C. orientalis as the causal agent of walnut anthracnose in South Korea. Since this disease reduces the commercial value and quality of walnut fruits, effective management practices should be developed to deal with C. orientalis.