Bacterial Culture Research Articles

Environmental dependency of ectomycorrhizal fungi as soil organic matter oxidizers.

Forest soils play a pivotal role as global carbon (C) sinks, where the dynamics of soil organic matter (SOM) are significantly influenced by ectomycorrhizal (ECM) fungi. While correlations between ECM fungal community composition and soil C storage have been documented, the underlying mechanisms behind this remain unclear. Here, we conducted controlled experiments using pure cultures growing on naturally complex SOM extracts to test how ECM fungi regulate soil C and nitrogen (N) dynamics in response to varying inorganic N availability, in both monoculture and mixed culture conditions. ECM species dominant in N-poor soils exhibited superior SOM decay capabilities compared with those prevalent in N-rich soils. Inorganic N addition alleviated N limitation for ECM species but exacerbated their C limitation, reflected by reduced N compound decomposition and increased C compound decomposition. In mixed cultures without inorganic N supplementation, ECM species with greater SOM decomposition potential facilitated the persistence of less proficient SOM decomposers. Regardless of inorganic N availability, ECM species in mixed cultures demonstrated a preference for C over N, intensifying relatively labile C compound decomposition. This study highlights the complex interactions between ECM species, their nutritional requirements, the nutritional environment of their habitat, and their role in modifying SOM.

Isotope Reverse-Labeled Infrared Spectroscopy as a Probe of In-Cell Protein Structure.

While recent years have seen great progress in determining the three-dimensional structure of isolated proteins, monitoring protein structure inside live cells remains extremely difficult. Here, we examine the utility of Fourier transform infrared (FTIR) spectroscopy as a probe of protein structure in live bacterial cells. Selective isotope enrichment is used both to distinguish recombinantly expressed NuG2b protein from the cellular background and to examine the conformation of specific residues in the protein. To maximize labeling flexibility and to improve spectral resolution between label and main-band peaks, we carry out isotope-labeling experiments in "reverse-labeling" mode: cells are initially grown in 13C-enriched media, with specific 12C-labeled amino acids added when protein expression is induced. 1 Because FTIR measurements require only around 20 μL of sample and each measurement takes only a few minutes to complete, isotope-labeling costs are minimal, allowing us to label multiple different residues in parallel in simultaneously grown cultures. For the stable NuG2b protein, isotope difference spectra from live bacterial cultures are nearly identical to spectra from isolated proteins, confirming that the structure of the protein is unperturbed by the cellular environment. By combining such measurements with site-directed mutagenesis, we further demonstrate that the local conformation of individual amino acids can be monitored, allowing us to determine, for example, whether a specific site in the protein contributes to α-helix or β-sheet structures.

A new leaf spot caused by Alternaria alternata on Atractylodes lancea in Jiangsu, China.

Atractylodes lancea is an important Chinese herbal medicine, which is mainly produced in Jiangsu province, China. In June 2022, leaf spots symptom were observed on some A. lancea seedlingsgrowing in a Chinese herbal medicine resource garden of NanjingBotanical Garden, Jiangsu. Approximately 75% of 100 A. lancea seedlings suffered from the disease. Initially, gray to black spots appeared at the tip of the blade, then spread deep into the petiole, finally causing the blade to wither and fall. To isolate the pathogen, five diseased leaves were collected from five different seedlings. Leaf sections (3 to 4 mm) were excised from the margins between healthy and diseased tissues, surface sterilized in 75% alcohol for 30 s, then in 1.5% NaClO for 90 s, rinsed three times in sterilized distilled water, plated on potato dextrose agar (PDA) and incubated at 25℃in darkness. Pure cultures were obtained by monosporic isolation. Eighteen isolates were obtained, and 77.8% of isolates was identified as Alternaria spp. A representative isolate, CS4-1 was used for further investigation. The colony of CS4-1, growing on PDA was cotton-like and black to brown with gray-white aerial hyphae on their surfaces, and dark gray on the back. The conidia were solitary on conidiophores and were oval to pear-shaped, brown in color, with 1 to 4 transverse septa and 0 to 1 oblique septa, parietal cells extending into the beak, and measured 8.9 to 39.5×6.0 to 13.5 µm (n=35). These characteristics were consistent with the description of Alternaria spp. (Simmons 2007). Six DNA regions, i.e.,internal transcribed spacer (ITS),large subunit ribosomal RNA (LSU), small subunit ribosomal RNA (SSU), anonymous region OPA10-2, Alt a 1 major allergen (Alta1), glyceraldehyde 3-phosphate dehydrogenase (GAPDH)and translation elongation factor 1-alpha (TEF1)with the respective GenBank Accession No. OP836052, OP836054, OP836051, OP851487, OP851488, OP851489, and OP851490, were amplified and sequenced with the primer pairs described by White et al. (1990) and Woudenberg et al. (2015). A neighbor-joining phylogenetic tree was generated by combining all sequenced loci in MEGA7, and CS4-1 clustered in the A. alternata clade. To test pathogenicity, 12 leaves, on three one-month-old A. lancea seedlings (four leaves from each seedling) were wounded with a sterile needle and inoculated with 20 μL of conidial suspension (1×106 spores/mL) on the left sides of leaves. The right sides of the leaves were inoculated with 20 µL sterile water and used as the control. All inoculated detached leaves and seedlings were covered with clear polyethylene bags to keep moisture and were incubated in a greenhouse at 25℃, 80% relative humidity, and a 12-h light/dark cycle. The experiment was repeated three times. After 4 days, typical gray to black spots were visible on the left sides of all inoculated leaves and the inoculated seedlings, and the right sides remained asymptomatic. Subsequently, the same fungus was reisolated and identified based on morphological and molecular traits. A. alternata has a very wide host range but has never been reported on A. lancea worldwide (nt.ars-grin.gov). Because of the medicinal value of A. lancea, further studies should be directed toward control of this disease.

Cyclic Isothiocyanate Goitrin Impairs Lotus japonicus Nodulation, Affects the Proteomes of Nodules and Free Mesorhizobium loti, and Induces the Formation of Caffeic Acid Derivatives in Bacterial Cultures.

The continuous release of glucosinolates into the soil by Brassicaceae root exudation is a prerequisite to maintaining toxic levels of breakdown products such as isothiocyanates (ITCs). ITCs influence plant and microbial diversity in ecosystems, while fungi and Rhizobiaceae are particularly injured. Studies explaining the molecular mechanisms of the negative effects are presently limited. Therefore, we investigated the early effects of cyclic ITC goitrin on proteomes of the host and symbiotic Mesorhizobium loti in the nodules of Lotus japonicus and of free-living bacteria. In the nodules, many host proteins had a higher abundance, among them, peroxidases and pathogenesis-related PR-10 proteins functioning in the abscisic-acid-activated signaling pathway. In the microsymbiont, transporter proteins as a prominent group are enhanced; some proteins involved in N-fixation decreased. The proteomes give a report about the loss of immunity suppression resulting in the termination of symbiosis, which initiates nodule senescence. Free-living M. loti are severely damaged, indicated, i.a., by a decrease in transporter proteins, the assumed candidates for goitrin protein complex formation, and high proteolysis. The production of chicoric acid by the accompanying bacteria is inhibitory for M. loti but connected to goitrin elimination, as confirmed by mass spectrometric (MS) analysis. In summary, the nodulation process is severely affected by goitrin, causing nodule dysfunction and failed nodule development. N deficiency conditions leads to yellowish leaves and leaf abscission.

Cassava for thefuture: embryogenic liquid cultures suitable for new biotech techniques.

Cassava, a crop of importance for subsistence farming in Africa, Asia, and Latin America, has the potential to benefit from global economic integration as a versatile industrial resource. Enhancing cassava productivity is not just a matter of agricultural competitiveness but a crucial step toward ensuring many communities' food security and livelihoods. Given its high performance in marginal environments, where climate change poses threats, ensuring food security and livelihoods relies on rapidly adapting cassava. This study aimed to develop a protocol that swiftly transitions cassava embryogenic short-period liquid suspension cultures, facilitating the regeneration of genetically stable in vitro plants. The resulting protocol, with its potential to be a foundational component in future technologies employing various genome editing or genetic modification techniques, holds promise for the advancement of cassava biotechnology.

Erinacenones A–L: Twelve New Isoindolinone Alkaloids from the Edible and Medicinal Mushroom Hericium erinaceus

A total of twelve previously unreported isoindolin-1-one compounds, erinacenones A–L (1–12), were isolated from liquid cultures of the medicinal fungus Hericium erinaceus. Their structures were elucidated based on spectroscopic data analysis. The absolute configuration of 12 was determined by comparing its optical rotations with values reported in the literature. The most distinctive feature of these compounds is that their nitrogen atoms are connected to different parts of the special structure moieties. Among them, compounds 3 and 4, as well as 10 and 11, are two pairs of isomers differing only by a small change in the position of one double bond. Compounds 4 and 5 were found to show cytotoxic activities, with IC50 values of 24.7 and 18.4 μM, respectively, against MCF-7 cell lines.

Medical-grade honey has superior antibacterial properties against common bacterial isolates in wound cultures of dogs and cats in comparison to non-medical-grade honey types.

To compare the antibacterial activities of different types of honey against common bacterial isolates cultured from wounds of dogs and cats. 4 types of honey were used including a medical-grade manuka honey, a non-medical-grade manuka honey, a locally sourced non-medical-grade honey (non-MGH), and a commercially sourced non-MGH. Bacterial isolates were obtained from clinical wound cultures of dogs and cats including Staphylococcus pseudintermedius, Escherichia coli, Enterococcus faecalis, and Pseudomonas aeruginosa. The macro-broth dilution method was used to analyze the MIC and minimum bactericidal concentration. The percentage of growth inhibition was assessed for different types of honey at different concentrations using a generalized linear regression model. Medical-grade honey exhibited the lowest minimum bactericidal concentration against S pseudintermedius, E faecalis, and P aeruginosa, alongside the lowest MIC at 90% with statistically significant higher bacterial growth inhibition in medium and low concentrations. Non-medical-grade manuka honey had a similar bactericidal activity against S pseudintermedius and P aeruginosa compared to locally and commercially sourced non-MGH. In this in vitro study, MGH exhibited superior antibacterial activity against all bacterial isolates compared to other types of honey. Medical-grade honey displayed the greatest antibacterial activity against common wound pathogens and could be considered over other types of honey for wound management in cats and dogs. Locally and commercially sourced non-MGH appears to have a comparative efficacy against certain bacteria compared to non-medical-grade manuka honey and is more cost effective. Further in vivo studies are needed to confirm these findings.

Dural tears with cauda equina herniation following percutaneous endoscopic lumbar discectomy: a case report and literature review

Lumbar disc herniation (LDH) is a prevalent condition that severely impacts patients' quality of life and work capacity. Traditional surgical treatments like laminectomy, while effective, involve significant invasiveness and potential complications, including long-term spinal instability and recurrent symptoms. With the advancement of minimally invasive techniques, percutaneous endoscopic lumbar discectomy (PELD) has become a popular option due to its reduced trauma and faster recovery. However, PELD, while beneficial, carries risks, including complications that may not be immediately evident. This report presents the case of a 60-year-old female patient who underwent PELD for L4/5 disc herniation but experienced significant postoperative complications, including increased pain and neurological symptoms. Initial conservative management failed, and further investigations suggested possible postoperative infection, though this was later ruled out through surgical exploration and bacterial cultures. The patient subsequently underwent open surgical exploration, which revealed extensive tissue damage and required additional interventions, including a minimally invasive lateral anterior approach for stabilization and fusion (MIS-OLIF). Postoperative recovery was successful, with complete symptom resolution and stable spine alignment at a six-month follow-up. This case highlights the complexity of managing PELD-related complications and underscores the importance of thorough diagnostic evaluation and the potential need for additional surgical interventions to ensure long-term patient outcomes.

Establishment, Multiplication, and Biochemical Analysis of Embryogenic Lines of the Amazonian Palm Euterpe precatoria Mart. under Suspension Culture

The palm Euterpe precatoria holds great social, cultural, and environmental importance. The heart of palm and the fruit are the main products used for industrialization due to their energetic properties. Thus, the aim of this study was to establish a suspension cultivation protocol for the species using different explant sources. For this, eight lineages of E. precatoria embryogenic calluses were tested, with five in liquid medium Murashige and Skoog (MS) with 5 μM Picloram and three for comparison in semisolid medium MS with 20 μM Picloram and 5 μM 2iP. The growth curve was obtained by weighing the calli from 60 to 180 days of cultivation. The Gompertz model was applied, and growth kinetics were evaluated. At 100 days, the contents of total soluble sugars (TSSs) and total soluble proteins (TSPs) were determined. Principal components (PCA) were measured. According to the analysis of the data, the cultivation of E. precatoria lineages in liquid medium was successfully carried out, and the establishment was achieved. The model can be considered adequate since the R2 values found describe more than 90% of the growth kinetics of the lineages. In the liquid system, lineages L1 (from leaf explants and multiplied in semisolid medium—SM), L2 (from leaf explants and multiplied in SM), and L6 (from zygotic embryo explants and multiplied in liquid medium—LM) showed the shortest time to double the biomass accumulation. Multivariate analysis reveals a significant increase in masses in liquid cultures, represented by lineages L6 and L2. There was statistical difference in the amount of TSSs extracted; the highest TSS levels were observed in lineages cultivated in LM. The protein content found was very low, showing statistical differences among the lineages. In this work, the establishment and multiplication of embryogenic calli of E. precatoria are described for the first time, and they emerge as viable alternatives for the vegetative propagation of the species.

Clinical Utility of Broad Range PCR for Infectious Organisms

Abstract Broad range PCR is used to identify infectious organisms when bacterial, fungal, and mycobacterial cultures are negative despite clinical evidence of infection. We performed a 6-month retrospective chart review to assess the actionability of broad range PCR and identify opportunities to improve the utility of this referral test at our institution. One hundred eleven broad range PCR tests were ordered from July to December 2023. Seventy-five (67.6%) results did not affect patient management, 18 (16.2%) results affected patient management, and the treatment impact of 18 (16.2%) results is unknown because they were from outside consult cases. There were no instances in which antibiotic therapy was completely discontinued due to a negative broad range PCR result. Concurrent in-house microbiology testing yielded a positive result in 15 (13.5%) cases, of which 8 (53%) had a negative broad range PCR result and 2 (13.3%) had a conflicting broad range PCR result. Thirty-eight (34.2%) specimens were formalin fixed paraffin embedded tissue, 29 (26.1%) were fresh tissue, 13 (11.7%) were ESWABS, and the remaining 31 specimens were various fluid types (27.9%). While fresh tissue is the preferred specimen, broad range PCR detected an organism in 20 (53%) formalin fixed paraffin embedded tissue specimens, 11 (37.9%) fresh tissue specimens, 3 (23.1%) ESWAB specimens, and 2 (6.5%) fluid specimens (abscess aspirate and cerebrospinal fluid). Improvements in physician communication and laboratory workflow are necessary to improve the specimen quality and thus the utility of this referral test at our institution.

Lack of Diagnostic Value from Fungal and Mycobacterial Cultures in Cases of Suspected Prosthetic Joint Infection

Abstract Introduction/Objective Infections of prosthetic joints cause significant morbidity and often result in removal of the infected joint. In many cases, an infectious etiology can be difficult to definitively identify. As a result, providers suspecting prosthetic joint infections (PJI) often place large order sets of microbiology tests, including fungal and mycobacterial cultures. Fungal and mycobacterial cultures are labor-intensive for the laboratory taking up to 28 days and 42 days, respectively, to complete. As a potential area for a diagnostic stewardship intervention, we wanted to evaluate how often a fungal or mycobacterial culture produced actionable information in cases of suspected PJI. Methods/Case Report Data reports were retrieved from both the Electronic Health Record (EHR) and the Laboratory Information System (LIS) from May 2022 to May 2023. As sources submitted by the provider often do not specify that the joint is prosthetic, data from LIS was evaluated to identify microbiology culture test sets ordered on joint sources, while data from EHR was examined to isolate patients with implanted joints. Using Microsoft Excel, data from both systems was compared in order to match microbiology culture test sets to the known implanted joints. LIS data from implanted joint sources was assessed to record negative (no growth) and positive (growth) cultures, and to register all organisms isolated. Results (if a Case Study enter NA) Of 794 order sets for cultures collected from prosthetic joint sources, 430 included requests for fungal culture and 79 included requests for mycobacterial culture. Of these 79 requests for mycobacterial culture, 0 organisms were isolated (0%). Of these 430 requests for fungal culture, 7 grew yeast (1.6%) and 7 out of 7 (100%) of these yeasts were also isolated on general (aerobic bacterial) culture. No molds were isolated. Conclusion Discontinuing the routine practice of ordering fungal and mycobacterial culture for prosthetic joint sources would have no negative impact on patient care but would reduce unnecessary work in the microbiology laboratory and potentially avoid additional costs to the patient. Education to the orthopedic provider group as well as best practice alerts in the EHR will be pursued, based on the findings from this study, in order to reduce unnecessary culture orders.

Outcomes of a 3-day transparent film dressing protocol after hypospadias repair

This study aimed to evaluate a 3-day transparent film dressing protocol after hypospadias repair. A retrospective observational study was conducted in boys with hypospadias who were operated in our institution between 2022 and 2023. Postoperatively, the penis was wrapped with a transparent film dressing, which was removed after 3 days. Postoperative complications were observed until postoperative day 14. The associations of age, meatal location, and type of procedure were analyzed using Chi square, Fisher exact, Mann Whitney, and Kruskall Wallis test (p < 0.05 = significant). Sixty-five patients were studied. Median age was five years, the majority had proximal meatus (58.5%), and underwent urethroplasty (76.9%). After dressing removal, positive bacterial culture was found in 43.1%, mild penile edema in 33.8%, bleeding in 10.8%, and SSI in 49.2% of cases, with pus formation (10.8%), dehiscence (9.2%), and urethrocutaneous fistula (10% after urethroplasty procedure). Surgical site infection and positive culture were significantly higher in patients with proximal meatus compared to distal (p = 0.031, p = 0.019; respectively). A 3-day transparent film dressing prevented penile edema and bleeding in most cases. However, the rate of SSI and positive wound culture was high, and was associated with proximal meatal location.

Decomposition of Polythene by Bacteria Isolated from Waste Disposal Sites in Gusau City

The accumulation of polythene can be hazardous and can cause various environmental problems. This work was aimed at studying the biodegradation of polythene by bacteria isolated from waste disposal areas in North West, (Zamfara state) Nigeria. A total of twenty soil samples were collected from four different locations, such as waste disposal areas, hospitals, roadsides, and petrol pumps station. Spread plate method was carried out on Nutrient agar for the growth of bacteria. A total of 13 bacteria were isolated from different areas. The total bacteria count in hospital ranged from 80 × 10-4 - 248 × 10-4 cfu/mL, roadsides ranged from 116 × 10-4 - 208 × 10-4 cfu/mL, petrol pump ranged from 88 × 10-4 - 184 × 10-4 cfu/mL and waste disposal areas ranged from 116 × 10-4 - 264 × 10-4 cfu/mL. The microbial species associated with the polythene materials were identified as Pseudomonas sp, Bacillus sp, Staphylococcus aureus, Providencia stuartii, Clostridium perfringes, Corynebacterium diphtheria, Listeria monocytogen. The bacteria isolates were screened for polyethylene degradation using mineral salt medium containing polyethylene as sole source of carbon and nitrogen for their ability to degrade polyethylene for the period of 2 months. The efficacy of the microbes in the degradation of polythene were analyzed using liquid culture method, among the bacteria Pseudomonas aeruginosa degraded 75% of polythene, Bacillus subtilis 71.4% in a period of 2 months.

Assessing the Pathogenicity of Berkeleyomyces rouxiae and Fusarium oxysporum f. sp. vasinfectum on Cotton (Gossypium hirsutum) Using a Rapid and Robust Seedling Screening Method.

Cotton (Gossypium spp.) is the most important fibre crop worldwide. Black root rot and Fusarium wilt are two major diseases of cotton caused by soil-borne Berkeleyomyces rouxiae and Fusarium oxysporum f. sp. vasinfectum (Fov), respectively. Phenotyping plant symptoms caused by soil-borne pathogens has always been a challenge. To increase the uniformity of infection, we adapted a seedling screening method that directly uses liquid cultures to inoculate the plant roots and the soil. Four isolates, each of B. rouxiae and Fov, were collected from cotton fields in Australia and were characterised for virulence on cotton under controlled plant growth conditions. While the identities of all four B. rouxiae isolates were confirmed by multilocus sequencing, only two of them were found to be pathogenic on cotton, suggesting variability in the ability of isolates of this species to cause disease. The four Fov isolates were phylogenetically clustered together with the other Australian Fov isolates and displayed both external and internal symptoms characteristic of Fusarium wilt on cotton plants. Furthermore, the isolates appeared to induce varied levels of plant disease severity indicating differences in their virulence on cotton. To contrast the virulence of the Fov isolates, four putatively non-pathogenic Fusarium oxysporum (Fo) isolates collected from cotton seedlings exhibiting atypical wilt symptoms were assessed for their ability to colonise cotton host. Despite the absence of Secreted in Xylem genes (SIX6, SIX11, SIX13 and SIX14) characteristic of Fov, all four Fo isolates retained the ability to colonise cotton and induce wilt symptoms. This suggests that slightly virulent strains of Fo may contribute to the overall occurrence of Fusarium wilt in cotton fields. Findings from this study will allow better distinction to be made between plant pathogens and endophytes and allow fungal effectors underpinning pathogenicity to be explored.

Enhancing the biodegradation of hydrophobic volatile organic compounds: A study on microbial consortia adaptation and the role of surfactants

The emission of hydrophobic Volatile Organic Compounds (VOCs) is a serious environmental issue. Typically, biofilters (BFs) are employed for their treatment, with the potential enhancement of mass transfer through the addition of surfactants. However, disparate results in previous studies have been observed, attributed to uncontrolled conditions during the introduction of surfactants to BFs. Additionally, there has been limited exploration of microbial consortium adaptation to surfactants. To address these gaps, this study followed two approaches. First, the long-term (247 days) removal of cyclohexane was studied in a stirred tank bioreactor (STBR) inoculated with Rhodococcus erythropolys E1 and using Tween 80 at three times the critical micelle concentration (CMC). Second, the short-term (9 days) impact of two (bio)surfactants [Tween 80 (1 × CMC) and Quillaja Saponin (QS, 1 × CMC)] on the removal of cyclohexane, hexane and toluene was investigated in batch tests using three types of inocula: a pure culture of Rhodococcus erythropolys E1 (X0), a microbial consortium adapted to cyclohexane (X1), and a microbial consortium adapted to cyclohexane with Tween 80 (X2). For long-term operation, the addition of Tween 80 at 3 × CMC improved cyclohexane removal efficiency (RE) to 87 ± 1% (elimination capacity, EC = 145 ± 25 mg m−3 h−1, gas residence time, GRT = 20 min, inlet concentration, Cin = 14.9 ± 2.5 ppmv), compared to a RE of 32 ± 9% (EC = 44 ± 8 mg m−3 h−1, GRT = 20 min, Cin = 15.1 ± 0.7 ppmv) under similar conditions without surfactants. For short-term operation, the addition of QS at 1 × CMC significantly increased biomass growth, resulting in lower maximum specific consumption rates for X1 and X2 compared to scenarios without surfactants or 1 × CMC Tween 80. The most abundant genera in X1 and X2 were Paludisphaera (26-23%), 67-14 genus (17–23%), and Rhodococcus (9–18%), respectively.

Mucormycosis in the nasal cavity of a canary (Serinus canaria) caused by Lichtheimia corymbifera

A canary (Serinus canaria) that was presented for consultation due to alterations in its beak and slight deterioration in its general condition, revealed during clinical examination the presence of a large rhinolith in the left nasal opening, which also had its margins markedly distended due to chronic lysis of its edges. The rhinolith was extracted but the bird died days later. Cytological evaluations and isolation in pure culture were carried out from the extracted rhinolith, all of whom supported the diagnosis of mucormycosis, which was later confirmed by molecular techniques. The product obtained by PCR was subsequently sequenced, resulting in a 98 % homology with Lichteimia corymbifera. Although mucormycosis affecting the nasal cavity has been diagnosed with some frequency in humans, it is reported for the first time in birds in the present work.

First Report of Fusarium oxysporum f. sp. fragariae Causing Fusarium Wilt on Strawberry (Fragaria × ananassa) in Saudi Arabia.

Recently, Saudi growers have expanded their production of organic, soilless-grown strawberries (Fragaria × ananassa Duch.), but their production shows many difficulties associated with disease susceptibility. In October 2021, 45% of strawberry plants cv. "Festival" organically cultivated in Dammam city, Saudi Arabia (26°31'34.5"N 50°00'51.0"E) showed wilting symptoms. Typical symptoms were yellowing, rapid wilting, death of older leaves, stunting, and decreased roots. Vascular bundle necrosis and crown and root rot were also observed; plants eventually collapsed and died. Twenty symptomatic strawberry plants were sampled to isolate the pathogen. Pieces (4 × 4 mm) of the symptomatic tissues from crowns and roots were sanitized with 1% NaOCl (90 s), submerged in 70% alcohol (20 s), rinsed with sterile water (2x 30 s), placed on potato dextrose agar (PDA; Scharlau Chemie, Spain) and incubated at 25°C for 6 days. Next, we prepared single-spore cultures on PDA and synthetic nutrient-poor agar (SNA). On PDA media, pure cultures produced abundant aerial mycelium, with light pink or purple pigmentation in the medium after incubation at 25°C for 7 days. On SNA media, aerial microconidia were abundant cylindrical to ellipsoid hyaline with zero to one septate (3.8 - 5.9 × 1.3 - 2.5 μm, n = 50). Macroconidia were few, hyaline and falcate, with slightly curved apexes and 2 to 4 septate (18.9 - 27.5 × 3.3 - 4.6 μm, n = 50). Chlamydospores were roundish and terminal or intercalary. As Leslie and Summerell (2006) described, such morphological characteristics are typical of F. oxysporum. The isolates' identities were established by extracting DNA using the DNeasy Plant Mini kit (QIAGEN, Hilden, Germany). This was followed by amplification and sequencing of the internal transcribed spacer (ITS) (White et al., 1990), elongation factor 1-α (TEF1-α) (O'Donnell et al., 1998), and the ribosomal RNA intergenic spacer (IGS) (Canizares et al., 2015). The ITS, TEF1-α, and IGS sequences of an isolate Fof-10 were submitted to GenBank (PP564462, PP703242, and PP784894, respectively). BLAST analysis confirmed 99.71 and 100% identities to the ITS, TEF-1α, and IGS sequences of F. oxysporum (KU931552.1, OR640020.1, and FJ985519.1), respectively. All isolates tested were confirmed at the forma specialis fragariae, level using the specific primers FofraF/FofraR (Suga et al. 2013). The ∼239 bp amplicon was sequenced and submitted to GenBank (PP703243). Two-month-old healthy strawberry plants of cultivars "Festival," "Marquis," and "Monterey" were inoculated by dipping the roots in the spore suspension (107 conidia ml-1) for 15 min (Henry et al. 2017). There were five replicates for each cultivar. Plants dipped in water were used as a control treatment. The plants were transplanted in sterilized soil and placed in a greenhouse at 30/26°C (day/night). Within 4 to 6 weeks, inoculated plants showed severe wilting and discoloration of the internal crown tissue, while control plants were symptomless. The pathogen was re-isolated from the discolored vascular tissue onto PDA and identified morphologically and molecularly as the original one, thus fulfilling Koch's postulates. The test was repeated twice. This report confirms F. oxysporum f. sp. fragariae as a causal agent of Fusarium wilt of strawberries in Saudi Arabia. This pathogen was previously reported to cause the Fusarium wilt of strawberries in California (Dilla-Ermita et al., 2023). This disease has been observed in several hydroponic strawberry greenhouses in Saudi Arabia, with incidence ranging from 25% to 45% across multiple locations. Given this, proper strategies are needed to manage this disease and to be compatible with organic farming.

Spatial–Temporal Patterns in the Enteric Pathogen Contamination of Soil in the Public Environments of Low- and Middle-Income Neighborhoods in Nairobi, Kenya

Public spaces in countries with limited societal development can be contaminated with feces containing pathogenic microbes from animals and people. Data on contamination levels, spatial distribution, and the diversity of enteric pathogens in the public settings of low- and middle-income neighborhoods are crucial for devising strategies that minimize the enteric infection burden. The objective of this study was to compare spatial–temporal differences in the detection rate and diversity of enteric pathogens in the public spaces of low- and middle-income neighborhoods of Nairobi, Kenya. TaqMan array card (TAC) molecular assays were employed to analyze soil samples for 19 enteropathogens, along with a selective bacterial culture for pathogenic Enterobacteriaceae. An observational assessment was conducted during every site visit to document the hygienic infrastructure and sanitation conditions at the sites. We detected at least one pathogen in 79% (127/160) and ≥2 pathogens in 67.5% (108/160) of the soil samples tested. The four most frequently detected pathogens were EAEC (67.5%), ETEC (59%), EPEC (57.5%), and STEC (31%). The detection rate (91% vs. 66%) and mean number of enteric pathogens (5 vs. 4.7) were higher in low-income Kibera than in middle-income Jericho. The more extensive spatial distribution of pathogens in Kibera resulted in increases in the detection of different enteric pathogens from within-site (area &lt; 50 m2) and across-site (across-neighborhood) movements compared to Jericho. The pathogen detection rates fluctuated seasonally in Jericho but remained at sustained high levels in Kibera. While better neighborhood conditions were linked with lower pathogen detection rates, pathogenic E. coli remained prevalent in the public environment across both neighborhoods. Future studies should focus on identifying how the sources of pathogen contamination are modified by improved environmental sanitation and hygiene and the role of these contaminated public environments in enteric infections in children.

Influence of Storage Methods on the Vitality and Growth Rate of Macrofungi.

The work contains a comparative analysis of methods for storing pure cultures of macrofungi. The study used 20 species of macrofungi from various taxonomic and ecological-trophic groups. Storage was carried out using five methods: serial subculturing, storage under a layer of distilled water and three cryopreservation protocols: a protocol using blocks of agar medium, a "perlite protocol," and a "grain protocol." For the selected cryostorage methods, various cryoprotective compounds (glycerol, trehalose) were used. Radial growth rate was used as a criterion for the state of crops. The values of the radial growth rate obtained immediately after isolation of the pure culture were chosen as the control. It has been shown that the most favorable for preserving the physiological activity of cultures are the storage method under a layer of distilled water, "perlite" and "grain" cryopreservation protocols.

Stevia rebaudiana Is a New Host of Stagonosporopsis pogostemonis Associated with Leaf Spot Disease in China.

Stevia rebaudiana is a promising medicinal and edible plant, widely cultivated in China. In 2022-2023, a new leaf spot disease occurred in S. rebaudiana in Hongxing country (32°34'55″N, 118°2'12″E), Dingyuan city, Anhui province. Symptoms were observed on 10 to 15% of plants in three S. rebaudiana nursery beds (0.1 ha in total). The typical symptoms included dark brown spots on the leaves and foliar wilting, the development of brown stems with dieback of top buds, and occasional plant death (Fig. 1a). To identify the pathogen, twenty diseased leaves were collected, cut into small pieces, surface sterilized with 75% ethanol for 30 s, in 0.5% sodium hypochlorite for 2 min, washed three times in sterile water, placed on PDA, and incubated at 25℃ for 5 days. Pure cultures were prepared by subculturing hyphal tips. Twenty-five Stagonosporopsis-like isolates with similar morphology were obtained. After 7 days growth on PDA, colonies had a regular margin, were cottony, and formed concentric circles on the surface that were gray-green. The reverse side of the culture was dark brown with a creme-orange and white, margin. The growth rate was 9.5 mm/day on PDA. Pycnidia were mostly solitary, globose or subglobose, pale to dark brown, thin-walled, glabrous, ostiolate, 95.735~250.851×90.93~266.32 μm (n=50). Conidia were oblong, cylindrical to ellipsoidal, smooth-walled, aseptate, with rounded ends and two polar guttules and measured 3.41 to 5.83 × 1.78 to 3.07 μm (n = 50) (Fig.1 b-d). For molecular identification, the internal transcribed spacer (ITS) rDNA, large ribosomal subunit (LSU) gene, β-tubulin (TUB2) gene and RNA polymerase II (RPB2) gene sequences of two representative isolates (TYJ-SP1 and TYJ-SP2) were amplified by PCR (Woudenberg et al. 2009; Dong et al. 2021). The sequences were deposited in GenBank (accession nos.: OR506193 and OR506194 for ITS, OR533526 and OR533527 for LSU, OR545221and OR545222 for TUB; OR545223 and OR545224 for RPB2) and showed 99.60% to 99.2% similarity to ITS (502/504 bp and 507/511 bp; MZ156571), 100% similarity to LSU (857/857 bp and 857/857 bp; MZ191532), 98.67% to 99.3% similarity to TUB2 (296/300 bp and 298/300 bp; MZ203132) and 99.78% (888/890 bp and 868/870 bp; MZ203135) of S. pogostemonis strain ZHKUCC 21-0001. A maximum likelihood phylogenetic analysis based on the concatenated sequences of ITS, LSU, TUB2 and RPB2 using MEGA 11.0 showed the strains TYJ-SP1 and TYJ-SP2 formed a clade with S. pogostemonis (Fig. 2). Thus, the strains were identified as S. pogostemonis (Dong et al. 2021). To test pathogenicity, the strain TYJ-SP1 was inoculated onto 30-day-old S. rebaudiana seedlings which were surface sterilized with 70% alcohol and washed 3 times with water and air dried prior to inoculation. Ten seedlings were sprayed with a conidial suspension (105 conidia/mL) and ten seedlings were sprayed with sterile water to serve as the negative control. All seedlings were maintained in a growth chamber (25°C, 90% relative humidity) with a 16 h photoperiod. Brown spots were first observed on inoculated leaves 48 h after inoculation; typical symptoms appeared by 7 days post inoculation. All inoculated plants developed symptoms similar to naturally infected plants in the nursery beds, and the disease incidence reached 100% while control plants remained symptom free (Fig. 1 e-f). The same Stagonosporopsis isolates were reisolated from the inoculated plants and identified based on morphological and phylogenetic analyses. S. pogostemonis has been reported to cause leaf spot in Pogostemon cablin and Brassica oleracea var. botrytis (Dong et al. 2021; Habib et al. 2024). To our knowledge, this is the first report of S. pogostemonis causing leaf spot on S. rebaudiana in China. As a medicinal and economic plant, S. rebaudiana is widely planted in China and other Asian countries. The occurrence of this leaf spot disease seriously affects its medicinal and economic value. Therefore, it is crucial to establish and implement effective disease management practices to reduce the impact of the disease.