Sterile Petri Dishes Research Articles

Identification and control of toxin-producing fungus in wheat grain stores using environment friendly factors

Background: The problem of mycotoxins in wheat crops can lead to health issues for humans and animals. It is very important to find safe and efficient methods to prevent the growth of toxin-producing fungi or reduce and remove them from food to prevent their risks to humans, animals, and agricultural crops. The paper focuses on finding environmentally friendly materials to protect wheat grains from toxin-secreting fungi, particularly Aspergillus niger, reducing the production of aflatoxin B1.Methods: Eight fungal genera were isolated from wheat grain samples, and the most frequent fungus was A. niger. Plant extracts were used to reduce the growth of A. niger, and the culture media was used to test the ability of A. niger fungus isolates to produce aflatoxin toxins. The media was also used to sow wheat grains for the purpose of isolating the fungi associated with them, growing the fungi, and purifying it. The culture media was poured into sterile petri dishes with a diameter of 9 cm, with three replicates for each concentration, and the comparison treatment in which an untreated food media was used. Results: The paper found that A. niger was the frequent fungus among the isolated fungal genera in wheat grain samples. The plant extracts, particularly citrullus extract, showed a clear effect in inhibiting the growth of A. niger and reduced the AFB1 toxin that it produces. The paper recommends continuous examination of stored wheat grains to determine the amount of fungal contamination and to ensure good storage conditions before receiving the grains in grain stores.Conclusion: The paper aimed to detect fungi associated with local and imported wheat grains stored in the stores of Al-Muthanna Governorate and the possibility of reducing them by using some plant extracts. Keywords: Citrullus; Phylex; Aspergillus niger; AFB1

Antimicrobial efficacy of calcium silicate-based bioceramic sealers against Enterococcus faecalis and Staphylococcus aureus - An in vitro study.

The main objective of endodontic therapy is to achieve a sterile environment and three-dimensional filling of the root canals. Endodontic sealers play an important role in the obturation of the root canal system as they may provide a seal, which prevents the penetration of bacteria. Sealers are meant to entomb residual bacteria, prevent leakage of nutrients, and ideally possess antibacterial properties. The aim of the study was to evaluate and compare the antimicrobial effectiveness of calcium silicate-based bioceramic root canal sealers against Enterococcus faecalis and Staphylococcus aureus using the agar diffusion method. The materials were divided into four groups of nine samples each for both E. faecalis and S. aureus. Group I (control group) - MTA-Fillapex (Angelus), Group II - BioRoot RCS (Septodont), Group III - Bio-C Sealer (Angelus), and Group IV - Dia-Root Bio Sealer (Diadent). To evaluate the antibacterial activity of the samples, the agar diffusion method was utilized. To begin, a bacterial suspension was evenly spread onto sterile Petri dishes using sterile swabs. Four wells of 4 mm diameter were then punched, and the sealers were mixed following the manufacturer's instructions and placed onto the prepared wells. The Petri dishes were then incubated at 37°C for 72 h. After incubation, the zones of inhibition around each well were measured at 24 h, 48 h, and 72 h, and the resulting values were statistically analyzed. The data were analyzed using the ANOVA and post hoc Tukey's test. Calcium silicate-based root canal sealers showed significantly better efficacy than the control group. BioRoot RCS (3.44 ± 1.13 mm) showed maximum antibacterial effect against E. faecalis at 24 h, 48 h, and 72 h time intervals. Bio-C Sealer (16.44 ± 1.42 mm) showed maximum antibacterial effect against S. aureus at 24 h, 48 h, and 72 h time intervals. The effectiveness of root canal sealants eventually decreased over a period. Based on analysis using the ANOVA test, a statistically significant difference (P ≤ 0.05) was observed between the mean values of antimicrobial efficacy of sealers against both E. faeca lis and S. aureus. The study's findings lead to the conclusion that BioRoot RCS showed a maximum antibacterial effect against E . faecal is and Bio-C Sealer showed maximum antibacterial effect against S. aureus at 24 h, 48 h, and 72 h.

Comparison of Antibacterial Activity of Ethanol Extract and Nanoparticles of Crassocephallum Crepidioides S.Moore Against Staphylococcus Aureus

This research seeks to examine the width of the inhibitory zone of extracts and nanoparticles of Crassocephalum crepidioides leaf simplisia, which have many advantages such as wound therapy, diarrhea relief, anti-inflammatory properties, and antibacterial effects. The antibacterial activity of Crassochepallum crepidioides (Benth) was evaluated by using the ethanol extract and nanoparticles. S. Moore departs. The agar diffusion technique was used to test it on paper plates. 0.1 ml of bacterial inoculum was introduced into a sterile petri dish, followed by the addition of 15 ml of Mueller Hinton agar media at a temperature range of 45-50ºC. Subsequently, multiple paper plates were immersed in a test solution containing a strong leaf ethanol extract and nanoparticles with different concentrations. Characterization reveals that Crassocephalum crepidioides is composed of many bioactive components, including alkaloids, flavonoids, saponins, tannins, and steroids. The analysis of the extract revealed that it contained 5.92% water, 12.86% total ash, 1.55% acid-insoluble ash, 14.33% water-soluble essence, and 9.34% ethanol-soluble essence. The nanoparticles had a size of 64.37 nm and exhibited antibacterial activity of extract ethanol at a concentration of 50% with a diameter of 12,00 mm. The minor concentration of the extract had a diameter of 3,166 mm, while the nanoparticles showed antibacterial activity at a concentration of 50% with a diameter of 10,53 mm. The minor concentration of the nanoparticle had a diameter of 3,00 mm The activity test results of the ethanol extract of Crassochepallum crepidioides with nanoparticles demonstrated that the inhibitory diameter of the ethanol extraction results remained more strong compared to the nanoparticles of simplisia powder.

Mycotic flora characteristics of the endontic flora in patients affected by COVID-19

BACKGROUND: Owing to the fact that, presently, at a dental appointment, it is possible to observe a wide range of consequences arising after COVID-19, among which are observed lesions of the pulp and periodontium of the tooth, additional diagnostic methods should be introduced before choosing treatment tactics. In our previous study, we analyzed the contents of root canals of teeth in patients who had COVID-19 using bacterioscopy. Coccus flora was detected in all cases and elements of Candida yeast-like fungi were recorded in 89 cases (76.1%). Based on the obtained data, it was advantageous to perform microbiological examination of the endodontium in this group of patients to further study their physical and chemical properties and correct the treatment regimen in gangrenous form of chronic pulpitis and aggravated forms of periodontitis, which determined the relevance of the present study. AIM: To reveal the characteristics of the mycotic flora of the endodontium in patients who previously had COVID-19. MATERIALS AND METHODS: A bacteriologic study of root canal contents in patients who had previously undergone a new coronavirus infection at different times, diagnosed as “exacerbation of chronic periodontitis”, “chronic gangrenous pulpitis”, and “acute suppurative periodontitis”, was conducted to detect fungi of the genus Candida in the tooth root canal system, which was obtained during mechanical treatment with a sterile endodontic instrument. The patients (n=49) were divided into groups according to their final diagnosis: group 1, chronic gangrenous pulpitis (27 patients); group 2, acute purulent periodontitis (9 patients); and group 3, exacerbation of chronic periodontitis (13 patients). Then, the collected material was placed in a tube with Amies transport medium and sent to the laboratory. Seeding of the material was performed in sterile Petri dishes on Sabouraud agar by rubbing with a plastic spatula. Further, Petri dishes were placed in the thermostat for 24–48 hours of incubation at 37±10 °C. Then, the study was carried out according to the generally accepted scheme: the obtained cultures were identified to species by the character of growth on dense medium. RESULTS: The given bacteriological study of the patients’ endontic contents confirmed the data of bacterioscopy and concretized the previously obtained results. On final examination, black colonies of Candida albicans were observed in 95.92% of cases. No growth of Candida albicans colonies was observed in two cases (4.08%). CONCLUSION: The bacteriologic study performed after bacterioscopy of diagnosis confirmed the presence of fungi of the genus Candida in the root canals of teeth in patients who previously had COVID-19. The study showed that when treating similar patients, the high risk of infection of periodontal complex tissues by Candida fungi should be considered. It is advisable to supplement the standard protocols of endodontic treatment with physical exposure, for example, laser radiation, or additional medication with antifungal drugs, such as fluconazole.

First Report of Kiwifruit Root Rot Caused by Globisporangium spinosum in China.

Kiwifruit is widely cultivated for its high vitamin C content and nutritional value. In January 2022, root rot symptoms were found in about 30% of Actinidia chinensis cv. Jinyan plants grafted on A. deliciosa rootstocks in an orchard located in Sanming (26.32°N, 117.23°E), Fujian Province of China. The affected plants appeared stunted, with brown and decaying roots, some of which were covered with white hyphae. To isolate the pathogen, the surfaces of typical symptomatic roots were sterilized for 30 s using 75% ethanol, followed by four rinses in sterile water, placing on potato dextrose agar (PDA), and incubating away from light at 25°C for 7 days. 16 Globisporangium-like isolates were obtained through hyphal tip isolation, displaying a milky-white appearance with irregular protuberances on the surface, and yellow-white backs with radial fold lines. The isolates were then cultured on corn meal agar for 5 days at 25°C in dark for morphological characteristics. Under microscope, the hyphae appeared as long strips without septa and 4.1 to 8.2 µm wide (average 6.7 µm), containing irregularly sized spherical droplets. Both terminal and intercalary hyphae swellings were observed; these appeared either spherical or subspherical, with some having projections. Their dimensions were 12.3 to 27.6 µm (average 17.3 µm). The oospores were mostly spherical, either plerotic or aplerotic, 11.8 to 22.3 µm wide (average 18.9 µm), with occasional projections. The antheridia were rod-shaped and curved, with one end attached to the oogonia. Amplification of the sequences of internal transcribed spacer (ITS) regions and cytochrome c oxidase subunit I (COI) were conducted using the primers ITS1/ITS4 (White et al. 1990) and OomCoxI-Levlo/OomCoxI-Levup (Robideau et al. 2011), respectively. The sequencing results revealed identical ITS and COI sequences in all 16 isolates. BLASTn analysis of the 969-bp ITS sequence ON202808 showed 99.38-99.59% similarity (965/971bp, 967/971bp) with the KJ162353 and AY598701 sequences from Globisporangium spinosum isolates, while the 700-bp COI sequence ON075783 showed 100% and 99.41% identity (680/680bp, 676/680bp) with the GenBank sequences HQ708835 and HQ708832, respectively, from G. spinosum. Phylogenetic analysis also showed that the obtained isolate (termed MA16) clustered with isolates from G. spinosum on the same evolutionary branch. For pathogenicity testing, four-month-old healthy Jinyan (A. chinensis) plants grown in sterilized media were transferred to sterile petri dishes covered with wet filter paper, and their roots were inoculated with a 5-mm-wide disk of MA16 when cultivated on PDA medium for 5 days. Miliang-1 (A. deliciosa) and Hongyang (A. chinensis) plants were treated similarly. The control groups each included three plants that were inoculated with non-colonized PDA. The plants were kept at 25 °C with a 12-/12-h light/dark cycle for 10 days when the inoculated plants exhibited root rot symptoms similar to those seen in the field, together with rotting and browning of the leaves. The control plants appeared healthy with no symptoms. After re-isolated from infected tissues, the pathogen was verified to be G. spinosum according to its ITS sequence, thus fulfilling the Koch's postulates. Recently, Pythium spinosum has been classified as G. spinosum according to whole-genome sequencing and phylogenomic analysis (Nguyen et al. 2022). Based on the morphological features and pathogenicity results, MA16 was identified as G. spinosum (van der Plaats-Niterink 1981; Huo et al. 2023). This report appears to be the first description of kiwifruit root rots caused by G. spinosum in China, and its identification will assist the development of strategies to counteract the disease.

An inexpensive moist chamber culture technique for finding microbiota on live tree bark.

Traditional moist chamber cultures (MCs) prepared in aseptic laboratory environments using sterile Petri dishes are commonly used to quantify the microbiota of rough-bark tree species and woody vines. MCs are typically expensive and may be difficult to make, so a less expensive option made from easily available supplies was developed. These cost-friendly MCs were compared with standard laboratory methods to demonstrate their efficacy. Modified MCs were made using inexpensive, store-bought supplies; compared to a standard laboratory setting, the modified MCs are shown to be less expensive with a faster setup time and larger size that facilitates a variety of tree and woody vine species. MC use resulted in the discovery of new species of fungi and myxomycetes with associated locality records. We provide detailed instructions for creating modified MCs, as well as a list of myxomycete species and their associated bark characteristics, pH values, and water-holding capacity. This new, low-cost MC technique makes the study of microbiota more inclusive and accessible for those in research laboratories, classrooms, and homes, including both amateurs and professionals. MCs are easy to prepare, versatile, and applicable for many areas of botany and the biological sciences, potentially allowing exploration into unexplored areas in urban ecosystems.

Toxicological assessment of synthetic pesticides on physiology of Phaseolus vulgaris L. and Pisum sativum L. along with their correlation to health hazards: A case study in south-west Nagaland, India

Pesticide toxicity has been a major concern in the recent time. Higher concentrations of pesticides affect the organisms and environmental health. The present study aims to assess the toxicological effect of some commonly used pesticides in southwest Nagaland of India and their impact on the physiology of Phaseolus vulgaris and Pisum sativum along with their correlation to health hazards. The study area is a hilly region where the overall temperature ranges in between 2 °C and 35 °C. The annual rainfall received in the region is between 1000 and 2000 mm. The region shares a subtropical to tropical climate. Agriculture is the main activity of the region. Seeds of Phaseolus vulgaris and Pisum sativum were collected from the local market and sterilized with 2 % sodium hypochlorite for 15 min. The solution of monocrotophos, chlorpyrifos, dimethoate, dichlorvos, and cypermethrin was prepared with 1 ppm, 10 ppm, 50 ppm, and 100 ppm concentrations. The seeds were soaked for 24 h in the test solutions and kept uniformly in sterilized petri dishes in 10 ml of test solutions. For every experiment, three replicates of each sample were taken including the control. The control sample was run with tap water. For each replica 10 seeds were taken, so one sample contained 30 seeds. The seeds of the above two vegetable species were treated with these pesticides and analyzed for germination rate, phytotoxicity, dry biomass, and seedling vigor index. The result has shown the negative impact of higher concentrations of pesticides on plant physiology. A questionnaire and interview-based field survey were also conducted among the users to find out exposure-related health hazards of pesticides. The average time utilized for a single interview was 30 min. According to the self-assessed reports of 323 pesticide users, 17 physical disorders were recorded from the study. Eye irritation (49.12), excessive sweating (46.68), and excessive tiredness were some of the highly observed signs and symptoms among the users. For the statistical analysis, analysis of variance (ANOVA) for the biomass production in radicle and plumule of the studied species was done with OriginPro 8.5 software. A chi-square (χ2) test was performed at the significance level of 5 % for the comparative study among male and female farmers for exposure-related disorders. The correlation analysis was done with MS-EXCEL software. A coefficient correlation value of 0.54 was recorded, which indicates a positive correlation between the studied factors.

Prevalence of Tinea capitis in Schoolchildren in Three Villages of Woleu-Ntem Province Haut Ntem Department, Gabon

Introduction: Tinea capitis is a benign alopecising fungal infection mainly affecting schoolchildren with a tendency to spontaneous healing at puberty, and caused by dermatophytes. Objective: to determine the prevalence of Tinea capitis in schoolchildren in the Haut Ntem department. Materials and Methods: This was a descriptive cross-sectional study conducted from November 11, 2021 to April 7, 2022 in 3 villages in the Haut Ntem department. The ringworm lesions were taken from schoolchildren head using a scalpel and collected in a sterile Petri dish. Each sample was examined directly and cultured on Sabouraud Chloramphenicol Actidione medium. Tinea capitis was diagnosed if either direct examination or culture was positive. Results: Among the 128 children, included, median age was 9 years, Tinea capitis with small plaques (66.4%) were more frequent than those with large plaques (33.6%). The frequency of Tinea capitis was higher on direct examination (23.6%) than on culture (9.4%). The age group most affected by both direct examination and culture was [6-9 years] (p=0.27). After culture, Tinea capitis of the scalp was more frequent in Gabon-Ville (41.7%); whereas its frequency (25.0%) was similar in Akok and Gabon-Ville on direct examination. Endothrix parasitism was significantly more frequent (p<0.0001). Tinea capitis was mainly due to the Trichophyton genus (100%, p<0.0001). Conclusion: Tinea capitis are a public health problem in rural Gabon. They are dominated by small-plate ringworm caused by fungi of the Trichophyton genus.

Superfície de resposta para quantificação dos efeitos de NaCl e pH em alface

Abstract The objective of this work was to evaluate the response surface methodology (RSM), with a large sample size, to investigate the effects of NaCl and pH on the root elongation of lettuce (Lactuca sativa) in vitro. For this, 20 lettuce seeds were placed in sterile Petri dishes with two filter papers at the bottom and 3.0 mL of the sample being tested. A rotatable 2x2 factorial arrangement was used, with two factors and a two-level matrix, incorporating two center points and ±1.41 α axial points. Each treatment group consisted of 40 replicates, resulting in 400 experimental observations. Increasing NaCl concentration from 0.0065 to 2,700 g L-1 caused a 19.3% decrease in lettuce root elongation. When pH decreased from 7.0 to 1.5, there was a 68.8% decrease in root elongation, but, when it increased from 7.0 to 12.5, there was a 44.9% decrease. The analysis of combined effects showed that the increase in NaCl concentration reduced the ability of the plant to resist changes in pH. The RSM was effective in evaluating the statistical significance of the NaCl and pH factors, including their second-order effects and interactions, as well as in quantifying their effects on the root elongation of lettuce under the experimental conditions. Therefore, the RSM is a valuable tool for interpreting the results and evaluating the sensitivity of multi-factor research, even when dealing with a large sample size.

Bioactive potential of L. camara and O. gratissimum extracts in the control of mosquito larvae: An ecologically friendly approach

In the present study, the larvicidal efficacy of the extracts of the weeds Lantana camara Linn (L. camara) and Ocimum gratissimum Linn (O. gratissimum) was evaluated against the larvae of the malaria vectors A. aegypti, An. subpictus, and C. quinquefasciatus. The ethanol, n-hexane and diethyl ether crude leaf extracts were evaluated in a preliminary screening for larvicidal activity at 20,40,80,160,320 and 640 mg/l concentrations. 20 larvae of each species were introduced in different sterile Petri dishes in aqueous media under a controlled environment to assess biological activity. The larvicidal activity of both extracts was evaluated at 6 and 12-h post-exposure time points by observing the movement of each larva. The obtained data were subjected to probit analysis to determine the lethal concentrations that kill 50% and 90% (LC50 and LC90) of the treated larvae. The results revealed a noticeable larvicidal activity following 12 h of exposure. The extract of L. camara leaves exhibited an LC50 range between 18.66 and 51.35 mg/l and an LC90 range between 87.78 and 312.21 mg/l. Moreover, for the extract of O. gratissimum leaves, the LC50 range was 52.26 to 59.09 mg/l and the LC90 range was 260.22 to 348.23 mg/l. Taken together, the results indicate that the extracts of L. camara and O. gratissimum leaves may be useful as effective, economical, and eco-friendly larvicidal agents. However, additional studies are needed to explore the bioactive components of the weeds that exhibit larvicidal activity along with their mode of action.

Antibacterial Activities of Heterotrigona itama Honey Farmed in Ex-Mining Area

Honey is considered to be a valuable non-timber forest product due to its numerous preventative and therapeutic properties. The nutritional content in honey is the most complete so it can boost the immune system. This study aims to determine the potential inhibition of Heterotrigona itama stingless bee honey against the activity of Staphylococcus sp bacteria and Propionibacterium sp bacteria. This research adopted the agar diffusion method to conduct antibacterial testing. The nutrient agar media was aseptically put into a sterile petri dish and thereafter left to air-dry. Following this, the bacterial suspension was reconstituted and subsequently inoculated onto the solidified agar surface by creating perforations. The experiment involved conducting antibacterial tests using many concentrations of the samples. The experimental setup included a positive control using chloramphenicol and a negative control using distilled water. The test results showed that the activity of the inhibition of Heterotrigona itama kelulut honey against Staphylococcus sp bacteria and Propionibacterium sp bacteria belonged to the strong antibacterial category. The antibacterial activity of honey exhibits an enhanced potency against both types of bacteria in direct proportion to the content of the honey sample.

353 Evaluation of Native and Invasive Seed Germination Ability After in Situ Incubation in the Rumen for Rangeland Restoration Through Targeted Seed Supplementation

Abstract Changes to native plant compositions of drylands are often associated to loss of wildlife, increased frequency, and severity of wildfires; further, it complicates the grazing permits and allocation during periods of restoration. The goals herein investigate the germination rates of native seed species after in situ incubation to evaluate their potential dispersal as supplements for grazing cattle. Native: Indian ricegrass (Achnatherum hymenoides; ACHY), white yarrow (Achillea millefolium; ACMI), crested wheatgrass (Agropyron cristatum; AGCR), sagebrush (Artemisia tridentata; ARTR), fourwing saltbrush (Atriplex canascens; ATCA), bottlebrush squirreltail (Elymus elimoides; ELEL), bluebunch wheatgrass (Pseudoregneria spicata; PSSP), and gooseberryleaf globemallow (Sphaeralcea grossulariifolia; SPRT) and nonnative: cheatgrass (Bromus tecorum, BRTE; fall BRTEf and spring seeds BRTEs) seeds were examined. Four ruminally canulated Angus x Hereford steers adapted to a forage kochia-alfalfa diet were utilized for seed incubations. Seeds were weighed into 84 bags at a rate of 5 g per bag (duplicated within and in different animals). Seeds were incubated sequentially (0, 6, 12, 24, 36, 48, and 96 h) and removed at the 96-h point when seeds were thoroughly washed and immediately counted (50) on square 80 mm 80 mm commercial sterile petri dishes with moisture pads and 5 mL of water. Seeds were incubated at 20 °C with 12 h of light/no-light intervals. Germination plates were incubated for 50 d and counted every 10 d for germinations. Generalized linear models assuming a Poisson distribution with separation of means through Dunnett’s multiple comparisons test. Statistical significance was declared at P < 0.05. All statistical analyses were performed on R (R Core Team 2022). Incubation times inside of the rumen linearly decreased germination rates across most seed species. For BRTEs, germination completely stopped at 48 h with a steep decrease observed after 24 h incubation. The BRTEf were also incubated but showed low germination both before and after rumen exposure (winter dormancy). Though ARTR is very important in rangeland restoration in the intermountain west, its germination declined more than 85% within just a 6 h incubation in situ (P < 0.001). A significant germination decrease was observed for AGCR at 36 h; however, germination was full at 48 h and 50% of the original at 96 h (P < 0.05). For ACMI, germination rates significantly declined after 36 h incubation, and for PSSP after 24 h. Low germinations were observed for ACHY, SPRT, and ATCA even with high reported in-bag tetrazolium chloride viabilities. Our results highlight that cattle grazing of cheatgrass does not spread seeds through fecal pats. Additional tests are required to determine seed viability of the dormant (low germination) BRTEf. Seed toleration to ruminal degradation highlights the potential targeted seed supplementation during grazing for rangeland restoration.

First Report of Pantoea agglomerans Causing a Leaf Blight and Bulb Rot of Onion in Chile.

Onion (Allium cepa L) is an important vegetable crop in Chile with 3.675 ha (ODEPA, www.odepa.gob.cl). Since 2017, a series of symptoms has been increasingly observed, mainly in mature bulbs which include water-soaked lesions on mature leaves and external and central cataphylls, which evolve to necrotic lesions and rot, reducing yields and affecting the postharvest life. As the season progresses over 90% incidence has been estimated in severely affected fields. Symptomatic plants were collected for disease identification in summer (February 2022) in Santa Cruz, O´Higgins, Chile, (34º41'32"S and 71º27'17"W). Isolations were done on semi-selective OEM medium (Zaid et al. 2012) and isolated colonies were transferred and maintained on King's B (KB) medium. Colonies on OEM medium were yellow slightly green in the center, circular to irregular shape, elevated, smooth margin, and mucoid texture (Tho et al. 2015). On KB medium colonies were yellow cream, slightly darker in the center, with smooth margins and circular or slightly irregular. Two isolates (CHS B3, CHS B1), obtaining in three different onion fields, were further identified by PCR amplification of partial 16S rRNA using primers 27F/1492R (Frank et al., 2008). The resulting sequencing were deposited in GenBank under accession numbers OP934962.1 and OP934961.1. A BLAST search of GenBank showed that these sequences exhibited 99,89 to 100 % nucleotides similarities with multiple Pantoea agglomerans sequences. Three isolates were used to test pathogenicity on detached cataphylls and onion plants tests in the laboratory and greenhouse, respectively. Cataphylls (n=10) were surface disinfected with 2% NaOCl for 2 min, placed in sterile Petri dishes, and injected with 50 µL (106 UFC/mL) inoculum suspension with the aid of a hypodermic syringe. Cataphylls were incubated in humid chamber at 22 + 2ºC for 14 days. Additionally, three-month-old onion plants (n=10), growing in pod, were inoculated by injecting leaves with 1 mL inoculum suspension (106 UFC/mL). Plant were incubated under greenhouse for 30 days to 28ºC. An equal number of cataphylls and onion plants, injected with sterile water, were left as controls. Water soaked lesions on inoculated cataphylls appeared five days after inoculation. While onions plants develop water soaked lesion 1- 4 days after inoculation. Blight and bulb rot were observed only on inoculated plants. Symptoms were identical to those previously observed in the field. Reisolations were accomplished for 100% of the inoculated cataphylls and leaves fulfilling Koch's postulates (Biochemical characterization and PCR amplification of partial 16S rRNA). Previously, Pectobacterium carotovorum was reported causing a similar disease in Chile (Acuña 2008). However, to our knowledge this is the first report of P. agglomerans causing onion leaf blight and bulb rot in O'Higgins Region, Chile. In agreement with other reports (De Armas et al. 2015, Tho et al. 2015), leaf blight and bulb has been a very aggressive disease in Chile. These results do not exclude the possibility that other Pantoea species (Armas et al. 2015) may be involved in the leaf blight and bulb rot of onions.

Analysis of the New York Subway System using an Adaptive Network Model

Through the use of a Physarum Polycephalum organism and a scaled-down model of the New York Subway System, this study created and analyzed a system of "maximum efficiency". By taking advantage of the capabilities of a Physarum organism, a system reminiscent of the existing subway system was created, with deviations analyzed to find differences in both track usage and overall costs. Previous research regarding Physarum Polycephalum and infrastructure development were relatively separate. This study holds similarities to research done by Atsushi Tero of Kyoto University, but while his team sought perfect replication, this study sought to use the growth patterns of Physarum to create a more efficient system. Using a geographically accurate mapping of the New York Subway System, a scaled-down model of the Subway System was created, with major subway stations represented as oat nodes atop an agar solution in a sterile petri dish. The Physarum was placed within and allowed to grow for roughly 6 days. Following the full connection of all oat nodes, an analysis of the Physarum system was conducted using a ModaCam system. This analyzed system was then scaled up to that of the existing Subway System, and a comparison of the two was conducted. This study concluded that the Physarum system was significantly more efficient than the existing system, using fewer subway tracks and consequentially a lower maintenance cost.

Non-invasive detection of Orthohalarachne attenuata (Banks, 1910) and Orthohalarachne diminuata (Doetschman, 1944) (Acari: Halarachnidae) in free-ranging synanthropic South American sea lions Otaria flavescens (Shaw, 1800).

Respiratory mites of the genera Orthohalarachne and Halarachne (Acari: Halarachnidae) are causative agents of nasopharyngeal/nasopulmonary acariasis in pinnipeds and sea otters. Until now, these endoparasitic mites were mainly diagnosed via necropsies and invasive procedures. So far, non-invasive diagnostic techniques have neither been developed nor applied in free-ranging pinnipeds. In the current study, we aimed to evaluate the halarachnid mite infestation status of free-ranging "urban" South American sea lions Otaria flavescens in the city of Valdivia, Chile. Therefore, non-invasive sampling methods were used in the current study, e. g. by observation-based sampling of freshly expectorated nasal mucus in the animal environment. Further, collection devices were developed for target-oriented sampling of sneezed nasal mucus, including sterile petri dishes and stretched clingfilms mounted on telescopic rods. Applying these techniques, 26 individual sputum samples were collected. 11.5% of sputum samples proved positive for halarachnid larvae (in total, n=7), which were morphologically identified as Orthohalarachne attenuata (n=2) or Orthohalarachne diminuata (n=5). In one of the individual sea lion mucus samples, both Orthohalarachne species were detected, thereby confirming a patent co-infestation in vivo. 16S rDNA-based molecular identification of individual Orthohalarachne spp. larvae confirmed morphological findings. For the first time, we here molecularly characterized Orthohalarachne spp. on the basis of three gene regions [18S, 28S and the internal transcribed spacer 1 (ITS1)]. Overall, current data include the successful application of non-invasive techniques to sample halarachnid mites from free-ranging synanthropic pinnipeds and contribute to the current knowledge on respiratory mites infesting South American sea lions by combining morphological and molecular methods to overcome challenges in species identification. This study should further serve as baseline study and calls for more research on occurrence, biology and health implications of orthohalarachnosis in free-living as well as captive pinnipeds.

First Report of Brown Rot Disease on Cultivated Auricularia cornea Caused by Trichoderma pleuroticola in Sichuan Province, China.

First Report of Brown Rot Disease on Cultivated Auricularia cornea Caused by Trichoderma pleuroticola in Sichuan Province, China.

First Report of Green Mold Disease Caused by Penicillium ochrochloron on Medicinal Plant Pseudostellaria heterophylla in China.

Pseudostellaria heterophylla is one of the Chinese herbal medicines with high medicinal and economic values. From 2019 to 2021, postharvest green mold disease was observed with an incidence of 2~5% on the tuberous roots of the harvested P. heterophylla at eight locations in Zherong county, Fujian Province, China. The symptoms were as follows: white mycelial growth on the tuberous roots surface initially, then green mold layers forming, and the tuberous roots decaying finally. To identify the causal agent, a total of 20 symptomatic tuberous roots were collected. Small pieces (5 mm×5 mm) were treated by surface disinfestion with 75% ethanol and 1% NaOCl, then rinsed 3 times with sterile distilled water. These treated pieces were transferred onto potato dextrose agar (PDA) and incubated at 25°C in the dark for 7 days. Twenty pure cultures were obtained by single-spore isolation method. Colonies on PDA medium initially appeared as white mycelium that developed grayish-green conidia with white margins. Mycelium was septate and colorless. Conidiophores were predominantly monoverticillate, occasionally biverticillate. Stipes was long and slender. Phialides were ampulliform to almost cylindrical with collula, 11.25 (7.80-23.50) µm long (n=50). Conidia were smooth walled and pale green, with globose to ellipsoidal shape, 2.75 (2.37-3.27)× 2.47 (2.18-3.13) µm (n=50). Based on these morphological characteristics, the isolates matched the description of the genus Penicillium. Genomic DNAs from two representative isolates (FJAT-32578 and FJAT-32579) were extracted with a fungal genomic DNA extraction kit. The rDNA ITS region and partial β-tubulin gene (BenA) were amplified using the primers ITS1/ITS4 (White et al. 1990) and Bt2a/Bt2b (Glass and Donaldson 1995), respectively. The sequences of isolate FJAT-32578 and FJAT-32579 were deposited in GenBank (ITS, OM920986 and OM920987; BenA, OM953825 and OM953826). All sequences showed above 99% similarity to P. ochrochloron type strain CBS357.48 (ITS, NR111509; BenA, GU981672). In multilocus phylogenetic analysis (ITS + BenA), the two isolates from this study clustered together with other strains of P. ochrochloron with 100% bootstrap support. The two isolates were thus identified as P. ochrochloron based on both morphological and molecular characteristics. Pathogenicity tests were conducted in triplicate by inoculating the aseptic wounds with 10 µl of conidial suspension (1×106 conidia/ml) of the two isolates in the each healthy tuberous root (cv. Zheshen No.1). The experiment was conducted twice. All the inoculated tuberous roots were placed in sterilized Petri dishes with moistened filter paper, and incubated at 25 ± 2 °C. Fifteen days after inoculation, all inoculated tuberous roots demonstrated the same symptoms as those observed in the field conditions. The re-isolated fungi from the artificially infected tuberous roots were confirmed as P. ochrochloron using the method described above, while the control tuberous roots treated with sterile water did not develop symptoms, fulfilling Koch's postulates. To our knowledge, this is the first report of P. ochrochloron causing green mold disease on P. heterophylla in China, which would be a potentially new threat to the medicinal plant.

Method of selective isolation of <i>Acinetobacter baylyi</i> from river water

The aim of the study is to increase the selectivity of isolating Acinetobacter baylyi from river water. For developing a selective culture medium, we used 5 strains of A. baylyi, 4 of which were isolated from the water of the river Neva, 1 from clinical material. A. baylyi strains were identified by a combination of phenotypic features and/or using the mass spectrometric method (MALDI-ToF MS). In preliminary studies we found that the bacteria A. baylyi utilizes no L-phenylalanine as a single source of carbon and nitrogen, as well as the only source of carbon in a medium with mineral nitrogen sources, but utilizes L-phenylalanine as the only source of nitrogen only if it is supplemented with ethanol as the only source of carbon. Based on this A. baylyi property, a selective medium was developed for the isolation of these bacteria from river water. Composition and preparation of liquid selective medium were as follows: 1 liter of distilled water added with L-phenylalanine (CAS 63-91-2) 2.0 (g/l); NaCI 5.0; Na2SO4 2.0; KH2PO4 1.0; K2HPO4 2.5; MgSO4 0.1; dissolved while heating, boiled for 5 minutes, 4 ml of 96-degree ethanol was added to a warm medium, pH 7.20.2 assessed; 40 ml were poured into sterile vials. Dense selective medium: the same ingredients and extra 15.0 g of agar are added to 1 liter of distilled water; with similar preparation technique, the medium is poured into sterile Petri dishes. Method of application of selective nutrient medium for isolation of A. baylyi from river water: water from the river Neva is seeded in a volume of 10 ml into a vial with 40 ml of liquid selective medium, incubated in aerobic conditions at 28С for 48 h, then the enriched material from the flask is placed with a bacteriological loop onto the surface of same dense selective medium in two flasks; incubated in aerobic conditions at 28С for 48 h. 10 isolated colonies are selected for subsequent identification, they are transplanted into sectors of nutrient agar, incubated at 28С 24 h and isolates are identified by a complex of phenotypic characteristic of A. baylyi, and/or by the MALDI-ToF MS method. In this case, 19 isolates from 20 were identified as A. baylyi (95% of isolates). A study using this technique of three more water samples from the river Neva showed the results of the same narrowly selective orientation 9095% of the isolates were bacteria A. baylyi.

Evaluation of the mosquito larvicidal potential and comparative assessment of the juice of Lantana camara Linn and Ocimum gratissimum Linn

In the present study, the larvicidal efficacy of the juices of the weeds Lantana camara Linn (L. camara) and Ocimum gratissimum Linn (O. gratissimum) was evaluated against the larvae of the malaria vectors Aedes aegypti, Anopheles subpictus and Culex quinquefasciatus. The freshly prepared juices of leaves were prepared by grinding them and diluting them at concentrations of 25, 50, 75, and 100 ppm. Twenty larvae of each species were introduced in different sterile Petri dishes in aqueous media under a controlled environment for the assessment of biological activity. The larvicidal activity of both juices was evaluated at 6, 12 and 24 h post-exposure time points by observing the movement of each larva. The obtained data were subjected to probit analysis to determine the lethal concentrations that kill 50% and 90% (LC50 and LC90) of the treated larvae. The results revealed a noticeable larvicidal activity following 24 h of exposure. The juice of L. camara leaves exhibited an LC50 range of 47.47–52.06 ppm and an LC90 range of 104.33–106.70 ppm. Moreover, for the juice of O. gratissimum leaves, the LC50 range was 42.94–44.91 ppm and the LC90 range was 105.11–108.66 ppm. Taken together, the results indicate that the juices of L. camara and O. gratissimum leaves may be useful as effective, economical and eco-friendly larvicidal agents. However, additional studies are needed to explore the bioactive components of the weeds that exhibit larvicidal activity along with their mode of action.

Evaluating the effectiveness of isolated fungi against Fall armyworm (Spodoptera frugiperda)

Spodoptera frugiperda, also known as the fall armyworm (FAW), is a polyphagous, destructive, and internationally dispersed insect pest that poses a serious danger to the production of cereals in Africa. Entomopathogens are a safe and environmentally beneficial way to control insect infestations when the all types of insecticides being used to control FAW are linked to a range of serious human health problems from respiratory to cancer. The objective of this study was to identify fungi from local soil that were effective against S. frugiperda larvae. Aluminum foil was used to gather soil samples from various maize growing regions in Ethiopia. Fungi isolated from soil samples and suspended conidia preparation was done in the mycology lab of Addis Ababa University in Ethiopia. At Melkasa Agricultural Research Center a bioassay test was conducted on S. frugiperda larvae. Three replications and a fully randomized design was used to set up the six treatments for the experiment. Twenty FAW larvae (3rd instar) were put into sterile Petri dishes with a diameter of 9 cm and a filter paper lining. A new, unopened syringe was used to inject 3 ml of the suspended solution into each treatment. As a negative control, distilled and sterilized water was used. FAW larval mortality was measured and examined independently for each treatment using GLM, followed by a Tukey's HSD post-hoc test. The cumulative mortality rate (P = 0.0001) and second-day mortality rate (P = 0.001) showed a significant difference between regimens. These noteworthy variations were seen in third instar larvae. S. frugiperda larvae from isolation fungi F1 and F5 had the highest (96.67%) and lowest (80.0%) mean cumulative death rates. Under negative control, the mean cumulative mortality rate was 8.33%. The outcomes showed that S. frugiperda could potentially be controlled by fungal isolates found in maize fields. Conidial concentration, as well as field evaluation, characterisation, and species identification of isolate fungi, all require more study.