Agar Block Research Articles

Drug release profile of phenytoin-loaded starch-based biomaterials incorporating hierarchical microparticles with photothermal effects

This study aimed to synthesize phenytoin (PHT)-loaded water chestnut starch-based biomaterials and evaluate their drug release kinetics for use in transdermal drug delivery systems for antiepileptic therapy. Hierarchical microparticles (HMPs) extracted from human hair were also used to improve the PHT release efficiency. The physicochemical characteristics of PHT, HMPs, and the prepared biomaterials were evaluated by physical properties, antimicrobial activities, FE-SEM, FT-IR, XRD, 1H NMR, and 13C CPMAS solid-state NMR. The photothermal effect and the PHT release profile were confirmed through 808 nm NIR laser irradiation. After 30 min of the laser exposure, the temperature of the HMP-added biomaterials increased by 1.50–1.59 times compared to that of without the HMPs. PHT release in buffers and artificial skin test under NIR laser irradiation enhanced by 1.20–1.85 times owing to the photothermal effect. The release kinetics in pH buffer and artificial skin were determined using the Fickian diffusion and Korsmeyer-Peppas models. Additionally, to verify the transdermal penetration of PHT, drug-release simulations were conducted using rhodamine B in agar blocks and pig ears. The results implied that the photothermal effect of the HMPs enhanced the penetration of the drug.

First Report of Leaf Spot on Cucumis melo L. Caused by Arcopilus aureus in China.

Cucumis melo L. is an important fruit with widespread consumption and commercial value. However, an undescribed disease affecting Hami melon (Cucumis melo L. var. Luhoutian) plants has consistently emerged in the Qihe region of Dezhou, Shandong Province of China since 2021. The disease can occur in both seedling and mature stages of Hami melon plants, and in severely diseased areas, the incidence rate was seen as 40 to 80%. During the seedling stage, the initial symptom is the appearance of water-soaked spots on the leaves. As the disease progresses, the leaves develop necrotic spots, and severely affected plants may exhibit stem rot and decay. In the mature stage, the disease primarily affects the leaves, causing necrotic spots and chlorosis. Under conditions of high humidity, black mold can be observed in the affected areas. Small pieces of symptomatic leaves from six different infected plants were collected and surface-sterilized with 5% NaClO for 3 min and 75% alcohol for 30 s for pathogen isolation (Wang et al., 2020). After rinsing with sterile water and blotted on sterile filter paper, the tissues were established on potato dextrose agar (PDA) media and incubated at 28℃ for 3-4 days. Pure isolates showed up at PDA were obtained through single-spore isolation. Colonies of all 16 isolates obtained by single-spore isolation had similar morphological characteristics on the PDA medium, the mycelium of the isolate appears dense and yellowish-brown on the PDA medium, and also secretes a brownish-red pigment on PDA. Under the opticalmicroscope, the perithecia from PDA media are subglobose spherical in shape, 80-100 μm in diameter, brownish by reflected light, wholly and densely hairy. Terminal hairs are very dense, greyish by reflected light, olive brown to reddish brown by transmitted light, thick-walled, arcuate, circinate, or spirally coiled at the apex. The ascospores within the perithecia are elliptical or droplet-shaped, initially colorless hyaline but later becoming subhyaline slightly gray, with dimensions of 7-9 μm × 4-5 μm. The morphological characteristics of the isolates were consistent with the description of Arcopilus aureus (Wang et.al. 2016). The internal transcribed spacer (ITS) region and β-tubulin genes of three randomly selected isolates were PCR amplified and sequenced using primers ITS4/ITS5 and Bt2a/Bt2b. The sequences of ITS and β-tubulin genes were submitted to NCBI with GenBank Accession No. OR539527 and OR640972, respectively. Based on morphological features and phylogenetic analysis, we concluded that the isolates belonged to A. aureus. Pathogenicity tests were conducted by placing agar plugs-containing fungal mycelia and agar blocks (control) on leaves of Hami melon seedlings (n=12) grown at 28°C with 60% humidity in a greenhouse, the assay was repeated three times. Symptoms appeared on the pathogen-inoculated leaves seven days after inoculation, whereas the control treatment remained symptomless. The pathogens were reisolated from diseased leaves and identified as A. aureus based on morphological, and molecular phylogenetic analysis, while Koch'sostulate was used to confirm its life mode. To the best of our knowledge, this is the first report of leaf spot caused by A. aureus on Cucumis melo L. in China.

First Report of Fusarium verticillioides causing Stem Blight of Schizonepeta tenuifolia in China.

Schizonepeta tenuifolia is an important medicinal plant in China. Over 10000 ha of S. tenuifolia is cultivated in the country annually. However, fungal diseases are a major limiting factor in S. tenuifolia production. In 2022, 50 ha in several S. tenuifolia fields in Hebei province were observed to be severely affected by a disease causing a yield loss of 30%. Results from field surveys suggested an epidemic during seedlings stages that affected S. tenuifolia stems, causing irregularly watery brown lesions. Lesions ranged from 1.5 to 2 × 2.5 to 3 cm. To isolate the causal agent, tissue was removed from the border of lesions and surface sterilized in 75% ethanol for 30 sec and 0.1% HgCl2 for 1 min, then rinsed three times with steriled distilled water(SDW), plated on potato dextrose agar(PDA) at 25℃, and incubated in the dark for 7 days. Five putative isolates of the genus Fusarium were hyphal-tipped on new PDA plates. Isolates were cultured on synthetic low-nutrient agar(SNA) with a ~ 1 × 2-cm strip of sterile filter paper on the agar surface(Nirenberg 1976). Cultures were incubated for 7 to 10 days at 20℃ in dark conditions. When sporulation was observed, agar blocks were mounted on a microscopic slide with a drop of lactophenol cotton blue and examined at 400×. Colonies grew rapidly with abundant pink to violet aerial hyphae. Sporodochia formed on the agar, and the aerial conidiophores branched sparsely, often alternately or oppositely, terminating with up to three verticillate phialides. Microconidia produced on polyphialides and aggregating in heads were unicellular, ovoidal or ellipsoidal, 4.4 to 17 × 1.5 to 4.5 μm. Macroconidia were abundant, falcate to straight, three to five septate, with a distinct foot cell, 27 to 73 × 3.1 to 5.6 μm. Based on morphological characteristics, isolates were tentatively identified as F. verticillioides(A1-Hatmi et al. 2016; Guarro 2013). Pathogenicity tests were performed by injection inoculation of 0.1 mL of conidial suspensions(1×106 conidia/mL) into three S. tenuifolia stems using a disposable needle and syringe. Distilled water was injected into three mock controls. Inoculated plants were placed in a greenhouse at 32 to 34℃ and 95% relative humidity. Typical lesions were observed 7 days after inoculation, except in the control samples. Each treatment was replicated three times. The suspected pathogen was consistently reisolated from diseased tissue according to Koch's postulates, and was found to be morphologically similar to F. verticillioides. Preliminary morphological identification of the pathogen was further confirmed by using genomic DNA extracted from the mycelia of a 7-day-old culture grown on PDA at 25℃. The translation elongation factor 1-α gene(TEF1) was amplified(O'Donnell et al. 1998) and the TEF region(Genbank Accession No. OR105502) was sequenced by Sangon Biotech Co., Ltd.(Shanghai, China) and displayed 100% nucleotide similarity with rDNA-TEF of F. verticillioides(JF740717) separately after a BLASTn search in Genbank. Based on the symptoms, fungal morphology, TEF sequence, and pathogenicity testing, this fungus was identified as F. verticillioides. to our knowledge, this is the first report of F. verticillioides infecting S. tenuifolia in China. This report will promote further research of F. verticillioides on this host and lead to better understanding of disease prevalence, extent of damage, and possible management options.

Study of the fungicidal effect of the chimeric form of interferon alpha-2b in relation to fungi-pathogens of plants and animals

The purpose of this study is to evaluate the fungicidal effect of a chimeric form of recombinant interferon alpha-2b, fused with human apolipoprotein A-I, obtained by biosynthesis in the yeast Pichia рastoris, against significant fungal pathogens – pathogens of human and plant diseases.Material and methods. The fungicidal activity of the chimeric cytokine was assessed using the agar block method against fungal pathogens: Alternaria alternata, Fusarium oxysporum, Aspergillus spp. and Penicillum spp. The fungicidal effect was assessed visually and by microscopy of a section of the fungal body stained with methylene blue, the germination of fungal spores – by subculture, the cytotoxicity of interferon preparations – on Vero cell culture.Results and discussion. The studied recombinant interferons suppress the growth and reproduction of pathogenic fungi. Chimeric IFN has the greatest effect against pathogenic fungi A. alternate, Penicillum at a concentration of 15 ng/ml; inhibition of sporulation of fungi F. oxysporum and Aspergillus up to 87 % is achieved at a concentration of 150 ng/ml. The cytotoxicity of the new chimeric cytokine is 5,8 μg/ml, which is significantly more than toxic concentrations for fungi.Conclusions. The chimeric form of interferon alpha-2b can be used as an immunotherapeutic agent with fungicidal activity.

Using bifidobacterium and propionibacterium strains in probiotic consortia to normalize the gastrointestinal tract.

The gastrointestinal microflora regulates the body's functions and plays an important role in its health. Dysbiosis leads to a number of chronic diseases such as diabetes, obesity, inflammation, atherosclerosis, etc. However, these diseases can be prevented by using probiotics - living microorganisms that benefit the microflora and, therefore, improve the host organism's health. The most common probiotics include lactic acid bacteria of the Bifidobacterium and Propionibacterium genera. We studied the probiotic properties of the following strains: Bifidobacterium adolescentis АС-1909, Bifidobacterium longum infantis АС-1912, Propionibacterium jensenii В-6085, Propionibacterium freudenreichii В-11921, Propionibacterium thoenii В-6082, and Propionibacterium acidipropionici В-5723. Antimicrobial activity was determined by the 'agar blocks' method against the following test cultures: Escherichia coli ATCC 25922, Salmonella enterica ATCC 14028, Staphylococcus aureus ATCC 25923, Pseudomonas aeruginosa B6643, Proteus vulgaris ATCC 63, and Listeria monocytogenes ATCC 7644. Moderate antimicrobial activity against all the test cultures was registered in Bifidobacterium adolescentis АС-1909, Propionibacterium jensenii В-6085, and Propionibacterium thoenii В-6082. Antioxidant activity was determined by the DPPH inhibition method in all the lactic acid strains. Our study indicated that some Propionibacterium and Bifidobacterium strains or, theoretically, their consortia could be used as probiotic cultures in dietary supplements or functional foods to prevent a number of chronic diseases.

The behavior of the Bacillus probiotic species under conditions of co-cultivation

Combining probiotic bacteria is a promising strategy to increase the effectiveness and avoid side effects of probiotic therapy. Bacteria that find themselves in a common environment are able to both change their properties and show new ones under the influence of each other. The change of colonization and antagonistic properties, which provide bacteria with competitive advantages in the development of new spaces, deserves special attention.
 The aim of this research was to study the features of the mutual influence of probiotic bacilli: B. clausii, B. coagulans and B. subtilis on growth, swimming and swarming motility when co-cultivated on agar media of different solidity.
 Materials and methods. The study used commercial strains of bacilli from three probiotic preparations: Enterogermina, Lactovit forte and Subalin. The ability of the studied species of bacilli to influence each other’s growth was investigated by the agar block method (using 1.5 % nutrient agar) and the spot-on-lawn assay (using 0.7 % nutrient agar). The study of the mutual influence on swimming and swarming activity was carried out when bacilli were cultivated on 0.25 % and 0.70 % tryptone agar, respectively. The diameters of swimming halos and migration swarms formed by monoand mixed cultures were measured, and the areas covered by them were compared. The phenotypes of the meeting of swarms formed by cultures spotted on the swarm plates at different locations were also investigated.
 Results. The studied probiotic species of the bacilli did not show a strong ability to inhibit each other’s growth. The B. clausii culture had no inhibitory effect, and the B. coagulans culture demonstrated a moderate inhibitory influence on the growth of the other two species of bacilli when using both diffusion methods. The B. subtilis culture showed moderate or weak inhibitory activity against the B. clausii culture and weak or no inhibitory activity against the B. coagulans culture using the agar block method or spot-on-lawn assay, respectively. The B. coagulans + B. subtilis, B. clausii + B. coagulans and B. clausii + B. subtilis mixed cultures showed moderate, weak and no inhibitory activity against third cultures, respectively. The studied species of bacilli showed different swimming ability and swarming potential as well as the ability to influence each other’s motility. Swimming halos formed by the B. clausii + B. subtilis and B. clausii + B. сoagulans + B. subtilis mixed cultures covered significantly larger plate areas than the swimming halos formed by each culture separately during the same cultivation time. The highest swarming potential was observed in B. coagulans culture and B. cla usii + B. coagulans + B. subtilis mixed culture. The studied bacilli did not show the ability to merge swarms, but, on the contrary, their swarms at the point of contact formed visible “boundary” or “intermediate” lines, demonstrating the ability to identify nonself.
 Conclusions. The obtained results indicate the ability of the probiotic species of bacilli: B. clausii, B. coagulans and B. subtilis to mutually influence growth, swimming and swarm motility. The highest indicators of swimming and swarming of the triple mixed culture indicate an increase in the colonization potential of the studied bacilli when they are combined. The hypothesis of mutual induction of inhibitory compounds production by bacilli, which may increase the overall antagonistic potential of the triple mixed culture, is subject to further confirmation.

Effect of the Extracts from Lichens and Lichenophilic Fungi on <i>in vitro</i> Growth of Clinically Significant Microorganisms

Abstract—Activity of the ethanol extracts from lichens (LE), of the cultures of lichenophilic (endobiotic) fungi (LFE), and of ethanol extracts from these cultures was tested using the following test organisms: Escherichia coli, Salmonella typhimurium, Pseudomonas fluorescens, Staphylococcus aureus, Micrococcus luteus, Enterococcus faecium, Paenibacillus sp., and Candida parapsilosis. Selective activity of LE and LFE against yeasts and gram-positive and gram-negative bacteria was revealed. The extracts from Cladonia rangiferina exhibited no activity against any of the tested cultures. The previously reported locus effect on activity of metabolites of one lichen species was confirmed. Agar block technique was used to detect selective activity of 23 out of 61 analyzed fungal strains against gram-positive and gram-negative bacteria, and of 8 strains, against yeasts. Ethanol extracts of six cultures were shown to suppress growth of St. aureus, M. luteus, Paenibacillus sp., and E. coli. Strain NM10F28209 from the lichen Stereocaulon paschale, which exhibited inhibitory activity against Ca. parapsilosis was selected for priority development of an antifungal preparation.

First Report of Nigrospora oryzae causing Leaf Spot on Yam in China.

Yam (Dioscorea spp.) is an important source of food and energy in the world, and is also widely cultivated in China (Frossard et al. 2017). Yam is rich in fiber and antioxidants, and can significantly reduce blood sugar. Therefore, it is also known as a medicinal crop with high medicinal value in China (Cao et al. 2021). In October 2021, leaf spots were observed on the seven month old yam leaves of a commercial yam field in Nanjing city, Jiangsu Province, China. The field had a disease severity of approximately 25% and an incidence of 30%, and the infected plants displayed poor development. Symptoms in the leaves included irregular yellow to brown spots of different sizes, and lesion spots later turned dark brown, resulting in necrotic leaves (Fig. S1A). Four symptomatic leaves collected from 3 yam plants were rinsed with water, cut into 2-mm small square leaf pieces and were surface-sterilized in 70% ethanol and 2% sodium hypochlorite for 2 min, then rinsed three times with sterile distilled water. The sterilized leaf tissues were plated onto potato dextrose agar (PDA) containing ampicillin and rifampicin, and then were incubated at 25°C in the dark for 3-5 days. Three isolates (NAU-1, NAU-2, and NAU-3) were obtained from a total of four leaf samples by purifying of single-spore culture. Nine agar plugs (6×6 mm2) were transferred into 8 mL of PDA in a 70 mm plate at 25°C to observe colony morphology. The three isolates had identical morphological features. For the representative isolate NAU-1, colonies were white and fluffy in appearance during initial 4 days, and became gray-brown with the onset of sporulation after 5 days (Fig. S1B). Microscopic observation showed that mycelia were smooth, branched, and septate (Fig. S1C) and conidia were single-celled, black, spherical to subspherical, measuring 10.7 ± 0.9 µm × 12.5 ± 2.6 µm in diameter (n=50) (Fig. S1D). Morphological features suggested that these isolates possessed the same characteristics. For accurate identification, the genomic DNA was extracted from each isolate using Fungi Genomic DNA Extraction Kit (D2300; Solarbio, Beijing, China). The internal transcribed spacer (ITS) sequence, β-tubulin (TUB2) gene, and translation elongation factor 1-alpha (Tef-1α) gene, were amplified using the primer pairs ITS1/ITS4 (White et al. 1990), Bt2a/Bt2b (Glass and Donaldson 1995), and EF1/EF2 (O'Donnell et al. 1998), respectively. These sequences were deposited in GenBank (ITS: ON394529, OP735574, and OP735562; TUB2: ON427830, OP756526, and OP756528; Tef-1α: ON427831, OP756525, and OP756527 for NAU-1, NAU-2, and NAU-3, respectively). The ITS, TUB2, and Tef-1α sequences of three isolates showed high similarity (>98.5%) to the corresponding sequences (MT732051.1, KY019554.1, and KY019413.1) of Nigrospora oryzae in GenBank. Further phylogenetic analysis showed that the three isolates clustered with N. oryzae (Fig. S2). Therefore, based on morphological and molecular evidence, the three isolates were identified as N. oryzae. N. oryzae has been reported to cause leaf spot on cotton and ginger in China (Zhang et al. 2012; Liu et al. 2022). Furthermore, the pathogenicity of isolated N. oryzae was tested on healthy, potted 1-year-old yam plants. The 6-mm agar plugs-containing mycelia from 4-day-old PDA cultures and agar blocks (control) were used to inoculate sixteen wounded yam leaves, followed by maintaining in a growth chamber under 16 h day at 25°C and 8 h night at 20°C. After six days post inoculation, all the inoculated yam leaves exhibited similar symptoms observed in the field, whereas the uninoculated leaves remained symptomless (Fig. S1E). The experiments were repeated three times with similar results. N. oryzae was reisolated from the infected plants and confirmed to be the same pathogen by using morphological and molecular methods. To our knowledge, this is the first report of N. oryzae causing leaf spot disease of yams in China. Identification of this pathogen would assist in developing strategies to efficiently control the spread of the disease on Chinese yams.

Phylogenetic analysis of Streptomyces producing antimicrobial agent isolated from Kukup Beach Sand, Yogyakarta, Indonesia

Abstract. Ambarwati A, Santoso B, Sofyan A. 2023. Phylogenetic analysis of Streptomyces producing antimicrobial agent isolated from Kukup Beach Sand, Yogyakarta, Indonesia. Biodiversitas 24: 2374-2383. Microorganisms such as bacteria, fungi, and Streptomyces are sources of bioactive compounds. Streptomyces is known as the largest antibiotic-producing genus. This study aimed to determine the antimicrobial activity of Streptomyces isolates and analyze the relationship between Streptomyces isolates based on the 16S rRNA gene sequence. The Streptomyces isolates were screened for their antimicrobial activity based on their ability to inhibit test bacteria growth using the agar block method. The selected isolates that showed antimicrobial activity were molecularly characterized using 16S rRNA gene sequence analysis. The result showed 32 Streptomyces isolates successfully isolated on SCA and Raffinosa Histidine Agar media. Among 32 isolates, it was observed that five isolates demonstrated the ability to inhibit the test bacteria growth on 9 to 25 mm of inhibition zone diameters. The BRI-18 isolate showed the highest antimicrobial activity inhibiting Bacillus subtilis G. FNCC 0060 on 25 mm of inhibition zone diameter (strong inhibition category). Based on 16S rRNA sequences, it was known that five isolates belonged to Streptomyces. BLAST analysis on the ARA-5 isolate revealed that it was closely related to Streptomyces griseoincarnatus (Preobrazhenskaya et al. 1957) Pridham et al. 1958 strain JCM 4381 with 99.62% sequence similarities. The AR3-29 isolate was a sister clade identical to Streptomyces rochei Berger et al. 1953 strain NRRL B-1559 with a 99.35% similarity level. The BRI-18 and BRI-19 isolates were the most similar to Streptomyces fradiae (Waksman & Curtis) Waksman & Henrici 1948 strain NBRC 12773 on 96.87% and 95.15% of index similarity, respectively, whereas the BRI-35 isolate demonstrated the highest similarity to Streptomyces zhihengii Huang et al. 2017 strain YIM T102 (97.02%). The research demonstrated that Streptomyces isolates from Kukup beach sand showed the potential as an antimicrobial agent.

IMPACT OF ACTINOBACTERIA ON THE GROWTH AND ACCLIMATIZATION OF MICROPROPAGATED RUBUS FRUTICOSUS L. PLANTS TO EX VITRO CONDITIONS

Microcloning is an effective method of plant reproduction that is actively developing in Ukraine for the mass propagation of valuable food crops such as blackberries. However, the problem of losing a large number of microclones often arises during the ex vitro acclimatization stage. Inoculation of the rhizosphere with potentially useful microorganisms could have a positive impact on the survival rate and biometric characteristics of acclimatized seedlings. The aim of this study was to establish the impact of 23 actinobacteria isolates on blackberry microclones (Rubus fruticosus L.) during acclimatization to ex vitro conditions, and to determine the protective potential and plant growth-promoting effects of these bacteria. Methods. The antagonistic properties of the experimental microorganisms were determined using the agar block method. Bacteria were inoculated into the rhizosphere of blackberry microclones before planting in the soil. Results. The antagonistic activity of actinobacteria against phytopathogenic fungi P. expansum, P. variotii, A. niger, C. cladosporioides, F. oxysporum, A. alternata, R. cerealis, and A. tenuissima was established. The positive effect of bacteria on micropropagated blackberry plants during acclimatization to ex vitro conditions resulted in an increase in the survival rate of microclones in the soil by 34.8%, average height of experimental plants by 2.0 cm, node number by 3.4 nodes, and leaf area by 0.4 cm2. Conclusion. It was established that isolates of mycelial actinobacteria Lim4, Myt7ch, Conc32, Conc4 were promising inoculants for ex vitro acclimatization of micropropagated plants and could be recommended for the subsequent research in order to establish the interaction mechanisms between these microorganisms and plants.

First Report of Fusarium verticillioides Causing Fusarium Ear Rot of Corn in Jordan.

Sweet corn (Zea mays L.) is one of the most popular crops grown in Jordan. Fusarium verticillioides (Sacc.) is a major pathogen of corn and a producer of mycotoxin fumonisins (Blacutt et al. 2018). During September and October 2019, ear rot symptoms were observed on ̴30% of the sweet corn variety Mis Dolce grown in the Jordan Valley. The disease caused substantial losses, including damage to greater than 50% of the kernels within 15-20 days after harvesting. A total of 350-corn kernels were randomly taken from 70 plants distributed in five fields with a total area of 2 ha. About 35% of the samples showed typical symptoms of the disease. Discolored corn kernels were surface sterilized with 5% NaOCl solution for 1 min, then rinsed three times with sterilized distilled water (SDW), plated on potato dextrose agar (PDA) at 25°C, and incubated in the dark for 7 days. Twelve putative isolates of the genus Fusarium were hyphal-tipped on new PDA plates. Isolates were cultured on synthetic low-nutrient agar (SNA) with a ca. 1 × 2-cm strip of sterile filter paper on the agar surface (Nirenberg 1976). Cultures were incubated for 10 to 14 days at 20°C in dark conditions. When sporulation was observed, agar blocks were mounted on a microscopic slide with a drop of lactophenol cotton blue and examined under the microscope at 400x. Colonies grew rapidly with abundant pink to violet aerial hyphae. Sporodochia formed on the agar, and the aerial conidiophores branched sparsely, often alternately or oppositely, terminating with up to three verticillate phialides. Macroconidia were abundant, falcate to straight, three- to five-septate, with a distinct foot cell, 27 to 73 × 3.1 to 5.6 μm. Microconidia produced on polyphialides and aggregating in heads, were unicellular, ovoidal, or ellipsoidal, 4.4 to 17 × 1.5 to 4.5 μm (Fig. 1A, B, C, D, E, and F). Based on morphological characteristics, isolates were tentatively identified as F. verticillioides (Al-Hatmi et al. 2016; Guarro 2013). Two representative isolates were DNA extracted and the translation elongation factor 1-α gene (TEF1) was amplified (O'Donnell et al. 1998), and sequenced from both directions at Macrogen Inc, South Korea. The consensus sequences of the two isolates Fvcorn2021JO-03 (OK040159) and Fvcorn2021JO-04 (OK040160) were used as BLASTn query on the NCBI website and were 100% and 99% similar with F. verticilloides JF740717 and JF740737 accessions, respectively. Similarly, the two isolates were 100% and 99.85% similar with F. verticilloides reference sequences MH582332 and MH582327 on the Fusarium MLST database, respectively. The pathogenicity of the two isolates was tested on 15 cobs by injecting 2 ml of a 2.5 × 105 conidia/ml suspension into the silk channel and into kernel wounds of the primary ear (three replicates) for each treatment (Reid and Hamilton 1996). Inoculated kernels were incubated at 25°C for 2 weeks in plastic boxes. The healthy kernels were injected with 2 ml of SDW as a negative control. Grains started to rot after 2 weeks, in the form of a thick, cottony, crimson-looking growth between the ear and its covers, with only some grains or a group of a adjacent grains rotting, and then white lines appear on the outer skin of the grain, yielding symptoms similar to those in the field (Fig. 2A, B, C, and D). The fungus was re-isolated from the inoculated kernels and was morphologically identified as F. verticilloides thus fulfilling Koch's postulates. The fumonisins-producing potential of the isolated F. verticillioides was confirmed using the AgroQuant Total Fumonisins Assay (Romer Labs, Singapore). To our knowledge, this is the first report of F. verticillioides causing Fusarium ear rot on corn in Jordan. Further investigation is needed to gain a better understanding of the spatio-temporal dynamics of this novel pathogen.

Variations in decay resistance of Cryptomeria fortunei

Cryptomeria fortunei has been widely planted in many cities in southern China. Eventually some of this material may be utilized for timber, but there are relatively few studies of durability of this resource. There is also some question as to whether Cryptomeria fortunei is a synonym for Cryptomeria japonica or Japanese cedar (Sugi). Evaluating the durability of the Chinese resource will help ensure that the decay resistance of this urban plantation resource is properly categorized. The decay resistance of Cryptomeria fortunei wood was assessed in soil block and agar block tests against Trametes versicolor, Gloeophyllum trabeum and Rhodonia placenta. Hot water and ethanol extractive contents of the heartwood were determined on sections from various distances above ground and then FTIR spectroscopy was used to characterize the wood before and after fungal exposure. Weight losses in sapwood were consistent with the minimal decay resistance of this portion of the wood. Inner and outer heartwood weight losses were more variable suggesting that the heartwood of this species would be considered to be only moderately durable. Extractives were weakly correlated with decay resistance. FTIR results were more variable, although they suggested heavier attack of lignin components by the brown rot fungi. The results suggest that Cryptomeria fortunei would need to be protected from the weather unless supplemental preservative treatments were applied.

Development and validation of an advanced ex vivo brain slice invasion assay to model glioblastoma cell invasion into the complex brain microenvironment.

Organotypic cultures of murine brain slices are well-established tools in neuroscience research, including electrophysiology studies, modeling neurodegeneration, and cancer research. Here, we present an optimized ex vivo brain slice invasion assay that models glioblastoma multiforme (GBM) cell invasion into organotypic brain slices. Using this model, human GBM spheroids can be implanted with precision onto murine brain slices and cultured ex vivo to allow tumour cell invasion into the brain tissue. Traditional top-down confocal microscopy allows for imaging of GBM cell migration along the top of the brain slice, but there is limited resolution of tumour cell invasion into the slice. Our novel imaging and quantification technique involves embedding stained brain slices into an agar block, re-sectioning the slice in the Z-direction onto slides, and then using confocal microscopy to image cellular invasion into the brain tissue. This imaging technique allows for the visualization of invasive structures beneath the spheroid that would otherwise go undetected using traditional microscopy approaches. Our ImageJ macro (BraInZ) allows for the quantification of GBM brain slice invasion in the Z-direction. Importantly, we note striking differences in the modes of motility observed when GBM cells invade into Matrigel in vitro versus into brain tissue ex vivo highlighting the importance of incorporating the brain microenvironment when studying GBM invasion. In summary, our version of the ex vivo brain slice invasion assay improves upon previously published models by more clearly differentiating between migration along the top of the brain slice versus invasion into the slice.

Sensitivity of the North Caucasian and Belarusian populations of Microdochium nivale (Fr.) Samuels & hallet to fungicides

Pink snow rot (pathogen Microdochium nivale) is the most common low-temperature pathogen worldwide. Sensitivity of two geographically distant populations of the pink snow rot pathogen (southern Russia and the Republic of Belarus) to nine modern fungicides was studied. The fungicides included in the State Catalogue of pesticides and agrochemicals permitted for use in the Russian Federation and recommended for treatment against snow rot were selected for the study. The material for the study was a pure culture of the fungus M. nivale. The agar block method was used in this study. The fungicide solutions were introduced into the nutrient medium using two standard methods: by interfering with the medium and by rubbing the preparation on the medium surface with a spatula. The preparations with 100% fungicidal effect against both studied populations were identified: Polaris, OE, Quinto Duo, SC and Bariton Super, SC. Oplot Trio, WS, Vybrance Trio, FC, Maxim Forte, SC showed 100% efficacy only against the Belarusian population of the pathogen. It was determined that the use of two methods of introducing the preparation into the nutrient medium (intervention and rubbing on the agar surface) has a high correlation coefficient (for the Belarusian population - rxy = 1.0, for the North Caucasian population - rxy = 0.99). However, intervention is less likely to inhibit colony growth and is therefore preferable in drug sensitivity studies of pure culture of the fungus M. nivale. A statistically significant difference was found between the sensitivity to fungicides of populations from geographically distant regions (Ft 5.32 < Ff 23.2 for the intervention method, Ft 5.32 < Ff 37.7 for the rubbing method). The data indicate the heterogeneity of the snow rot pathogen in terms of sensitivity to the modern assortment of seed dressing agents.

Impact of Enterococcus italicus ONU547 on the growth and acclimatization of micropropagated Rubus fruticosus L. and Paulownia tomentosa Steud. plants to ex vitro conditions.

Clonal micropropagation is an effective method for plant reproduction, applicable in both scientific and industrial domains. However, a significant number of microclones are lost during the ex vitro acclimatization process. To address this, the introduction of beneficial microorganisms into the rhizosphere of micropropagated plants could have a positive effect on the survival rates and external characteristics of acclimatized plantlets. The aim of this study was to determine the protective and growth-promoting potential of Enterococcus italicus ONU547 and its effect on micropropagated plants during acclimatization. The antagonistic activity of the bacteria was determined using the agar block method. Lepidium sativum L. seeds were inoculated with bacterial suspensions at concentrations of 106, 107, and 108 CFU/ml. Subsequently, the roots of the microclones were treated with suspensions of 106 and 107 CFU/ml, and biometric characteristics were measured. The results demonstrated antagonistic properties against various phytopathogenic fungi, including Aspergillus niger, Cladosporium cladosporioides, Alternaria alternata, Alternaria tenuissima, Rhizoctonia cerealis, Penicillium expansum, and Paecilomyces variotii. Inoculation of L. sativum L. seeds resulted in improved germination rates, increased root numbers, and enhanced root and shoot lengths. Similarly, the effects of the studied bacteria on Rubus fruticosus L. and Paulownia tomentosa Steud. during the acclimatization stage led to higher survival rates, increased shoot lengths, greater node numbers, and larger leaf areas. A concentration of 107 CFU/ml was identified as optimal for inoculating the microclones. The findings indicate that E. italicus ONU547 holds promise for the inoculation of micropropagated plants during the acclimatization process. Further research is recommended to establish the specific interaction mechanisms between these bacteria and plants.

Antagonistic properties of a probiotic preparation containing bacteria of the genera Bacillus and Enterococcus

Complex probiotic preparations with spore-forming microorganisms of the genus Bacillus and other types of bacteria in their composition have a high biological activity and are of scientific and practical interest. In our laboratory experiments, we determined the level of antagonistic activity of the newly developed probiotic preparation containing strains of bacteria B. subtilis, B. licheniformis, B. coagulans in a complex with Enterococcus faecium, products of their own fermentation, other natural substances and a sorbent made of natural aluminosilicates. The antagonistic properties of the probiotic preparation were examined by diffusion methods of delayed antagonism and agar blocks. In order to identify the antagonistic action of the experimental probiotic preparation, we used Gram-negative and Gram-positive test cultures of bacteria from the Museum of Strains of Microorganism Cultures of the Research Bacteriology Department. The results of laboratory studies indicated the antagonistic effectiveness of the experimental probiotic preparation in its interaction with test microorganisms. This was confirmed by the indicators of the diameters of the growth inhibition zones of the tested bacteria, ranging 27–36 and more and corresponding to high and very high levels of antagonistic activity of the experimental probiotic. The use of the developed probiotic preparation in poultry farming would ensure the correct formation of microbiocenosis in gthe astrointestinal tract of poultry from hatching, increase local immunity, support a stable epizootic situation by preventing bacterial infections among poultry through biological means, and therefore contribute to production of ecologically high-quality and safe products of poultry farming.

Secondary metabolites from endophytic fungi of moringa oleifera: antimicrobial and antioxidant properties

The exponential increase in antimicrobial resistance among several microbes, now more than ever, poses a tremendous challenge to the health system. This has created an urgency to look outside the “norm” for antimicrobial agents that would be useful in tackling the various disease-causing microorganisms. Endophytes are microorganisms that not only have a niche within the plant tissues but also produce bioactive compounds which could be a significant breakthrough in the pharmaceutical industry, especially with the manufacture of novel drugs having an impactful effect on these disease-causing microbes. The study examined the secondary metabolites produced by endophytic fungi associated with Moringa oleifera as well as the antioxidant and antimicrobial activities of the crude extracts of these secondary metabolites against microbes of medical importance. To achieve this, the fresh leaves of moringa (Moringa oleifera) were collected from Ngwo in Enugu’s north local government area, Enugu State. Isolation of endophytic fungi was done using the protocol described by.13 The fungus was cultured by placing agar blocks of actively growing pure culture (3 mm in diameter) in a 500 ml Erlenmeyer flask containing 100 g of rice medium. Incubation was done at room temperature for 3 weeks. Afterward, the fermentation was stopped by introducing 500 ml of ethyl acetate into the flask. Whatman No. 1 filter paper was used to separate the fermentation mixture. Cultures of the bacteria and fungi were maintained on nutrient and Sabouraud dextrose agar respectively at 4 ºC. After the extraction had been done, antimicrobial activity was determined by agar well diffusion assay by Kirby-Bauer’s method. The result of the DPPH antioxidant assay of the endophytic fungal extract revealed that at a concentration of 100 µg/ml, Two Endophytic fungi, MA and MB showed antioxidant activity with % Inhibition of 55.1 and 48.3% respectively. The result of HPLC analysis of the fungal endophytic fungal metabolites revealed the presence of active compounds in the extracts. It was concluded that Moringa oleifera leaves harbor endophytic fungi. These endophytes could be exploited in medicine as novel metabolites.

First Report of Leaf Spot Disease caused by Nigrospora oryzae on Nicotiana tabacum in China.

Tobacco (Nicotiana tabacum) is an important economic crop and widely cultivated in rural areas in south of China. A previously uncharacterized disease was observed on field-grown tobacco during 2020 and 2021 around Tongren city, Guizhou province of China (27°59'25.73" N, 108°7'2.43" E). The disease mainly occurred from fast growing period (about 13-16 leaves) to leaf maturity stage. In severely diseased areas, the incidence rate was between 20%-100%. Symptoms first began as yellow-brown necrotic spots on leaves, then merged into larger irregular necrotic spots surrounded by chlorotic halos. Similar lesions were also found on the stems. Ten symptomatic leaf and stem samples were collected from the different infected plants for pathogen isolation. The small pieces of discolored tissues were surface-disinfected with 2% sodium hypochlorite for 3 min and 75% ethanol for 30 s, rinsed three times with sterile water, and blotted on sterile filter paper, placed on potato dextrose agar thenincubated at 28°C in the dark for 3-4 days. The obtained isolates were purified through single-spore culture. Colonies were initially white and fluffy in appearance, later turning gray. Hyphae were smooth, branched, septa, transparent or light brown. Spores were solitary, oblate or nearly spherical, dark brown to black, smooth, 14.3 to 16.1μm × 11.8 to 15.2 μm in diameter. DNA of fungal isolates were extracted using Fungi Genomic DNA Extraction Kit (Solarbio, Beijing, China), the internal transcribed spacer (ITS) of the ribosomal DNA, β-tubulin (TUB2) gene and translation elongation factor 1-alpha (TEF1-α) were amplified with primers ITS1/ITS4, βt2a/βt2b and EF1-1728F/EF1-986R, respectively. The resulting ITS, TUB2 and TEF1-α sequences were deposited at GenBank, NCBI under accessions MZ882151, MZ927749, MZ927747, respectively. The sequence identity of ITS, TUB2 and TEF1-α with those of Nigrospora oryzae strains HBN (KU254608), HGUP191068 (MZ724102) and LC7307 (KY019409) were 99.64%, 99.29% and 99.65%, respectively. Based on morphological features and phylogenetic analysis, the pathogen was identified as N. oryzae (Wang et al. 2017). Pathogenicity tests were conducted by placing agar plugs-containing fungal mycelia and agar blocks (control) on leaves of tobacco plants grown at 28°C with 60% humidity in greenhouse. Symptoms appeared on the pathogen inoculated leaves seven days after inoculation, whereas the control treatment remained symptomless. The pathogens were reisolated from diseased leaves and identified as N. oryzae based on morphological, molecular and phylogenetic analysis, which were fulfilling Koch's postulates. This pathogen was recently identified from watermelon and kiwifruit in the Guizhou (Far and Rossman, 2021). To our knowledge, this is the first report of leaf spot caused by N. oryzae on Nicotiana tabacum in China.

Antibacterial Activity Screening Of Bacterial Isolates Associated With Seaweed Eucheuma cottonii From Coastal Area In Buleleng, Bali

Seaweeds like other marine organisms develop a strong association with prokaryotic communities, especially bacteria. Many of these associated bacteria play an important role in synthesizing secondary metabolites which are beneficial for their host, especially against predation or infection. In Indonesia, Eucheuma cottonii is one of the seaweed species that has been cultivated widely. However, only limited study has been reported on bacterial isolates and their antibacterial activity from E. cottonii. Bacterial isolates from E. cottonii were cultivated in six agar media namely Zobell Marine Agar, Nutrient Agar, Plate Count Agar, ISP-1, ISP-2, and Starch-M protein agar. Cultivation of bacterial isolates yielded 23 isolates with nine bacterial isolates were classified as Gram-positive bacteria and 14 isolates were grouped among Gram-negative bacteria. All the bacterial isolates were screened for their antibacterial activity against six bacterial indicator strains namely Staphylococcus aureus ATCC 25923, Streptococcus mutans FNCC 0405, Escherichia coli ATCC 25922, and Klebsiella pneumoniae ATCC 700603 using perpendicular streak and agar block method. Six out of 23 bacterial isolates displayed antibacterial activity against at least one of the bacterial indicator strains. The bacterial isolate ISP1RL4 showed the highest antibacterial activity with average inhibition of > 20 mm against all bacterial indicators. Overall, our result indicated the potential of bacterial isolates associated with E. cottonii as an antibacterial producer.

Preservation of Streptococcus pneumoniae quality control strains using chocolate agar blocks in glycerol at minus 70 degrees celsius

We present a method for storage of the quality control strain of S. pneumoniae that could be used in low resource settings. Small blocks of chocolate agar with growth were placed in glycerol (60%V/V) and stored at minus 70 °C. Viability and cultural characteristics were maintained for the testing period of 10 months. The wide availability of both chocolate agar and glycerol make this a suitable alternative to other specialized formulations usually used to maintain stocks of S. pneumoniae ATCC 49619 strain required for quality control of susceptibility testing.