Surface Sterilization Research Articles

Identification of Bacterial Endophytes Isolated from Different Medicinal Plants

Plants have very potential compounds to live on earth as they supply 90% of human calorie intake, 80% of protein intake directly, and perhaps the most vital sources of medicine with a vast diversity of microorganisms. As such it’s important to know those microorganisms, their kinds, the features they possess, and the significant compounds/metabolites they can produce. So, this study is based on identifying such microorganisms. To achieve this aim, isolation of endophytes has been done to know their biochemical activities and properties. Various identification procedures have been followed to get pure endophytic strains without any contamination. Surface sterilization of the plant tissue is a must in this progress, various surface sterilization techniques have been tried and finally, for 4/5 plant tissues, sodium hypochlorite and ethanol were given the best result and for 1/5 with the addition of mercuric chloride were the standardized method for surface sterilization. About 30 different bacterial endophytes have been isolated from five kinds of medicinal plants. 4% sodium hypochlorite and 75% ethanol were found effective in sterilizing the surface of Psidium guajava, Cassia occidentalis, Calotropis procera, and Hibiscus rosa-sinensa. While Mangifera indica required an addition of 0.1% mercuric chloride. 19 strains isolated were Gram-positive, 11 Gram-negative (5 were Lactose fermenters and 6 were not), and most of which were bacilli. All isolates have shown different biochemical results, 25 showed a positive result for oxidase, and 28 gave a positive result for catalase. Most of the endophytes identified in this work are Bacillus spp. However, this study highlighted the significance of surface sterilisation and most importantly the presence of potential endophytes capable of producing novel bioactive compounds usable in pharmaceutical/medicinal application.

Karakterisasi dan Potensi Acremonium sp. Secara In Vitro Terhadap Cendawan Patogen Fusarium oxysporum

Various natural interactions exist between fungi and plants, including mutualism and pathogenesis. This study evaluated the interaction of fungi Acremonium sp. to plants and pathogenic fungi. Acremonium sp. was isolated from the root tissue of an oil palm plant using surface sterilization methods before isolation. The potential pathogenicity interaction of Acremonium sp. was assayed towards Chinese white cabbage (Brassica rapa L. var. pekinensis) in vitro. Based on the PDA medium assay, isolated Acremonium sp. exhibited pathogenic traits against Chinese white cabbage within 14 days post-inoculation. Interestingly, Acremonium sp. showed antifungal activity against Fusarium oxysporum IPBCC.19.1468, with an average inhibition rate of 69.11%. Our data suggest the pathogenicity interaction of Acremonium sp. toward cabbage plant and fungi F. oxysporum IPBCC.19.1468.

ANTIOXIDANT ACTIVITY OF ENDOPHYTIC BACTERIA ORIGIN FROM ENDOCARP OF COCOA POD HUSK

Cocoa (Theobroma cacao L.) is one of the leading commodities in Indonesia. Cocoa pod husk is part of the mesocarp or part of the cacao pod husk that wraps the cocoa beans. Endophytic bacteria are microbes that live in host plant tissues and do not interfere with host plant growth. This study aimed to determine characteristics of endophytic bacterial isolates contained in endocarp cocoa pod husk and to determine the antioxidant power of endophytic bacterial isolates contained in cocoa pod husk. Isolation of endophytic bacteria from the inner pod of cocoa which be expected to produce antioxidants has been carried out. Isolation of bacteria using surface sterilization method. Antioxidant test using DPPH method (1,1-diphenyl-2-picrylhydrazyl) with UV-Vis spectrophotometer at 600 nm. Two isolates out of four endophytic bacteria were obtained from the results of the preliminary test of phenolic tests, namely isolates of bacteria KK2 and KK3 which had different colony morphology. The results of the secondary metabolite test showed that the bacterial isolates KK2 and KK3 contained flavonoids and phenolics. Based on the test results, the value of IC₅₀ of bacterial isolate KK2 was 2104.58 ppm and KK3 bacterial isolate was 1123.04 ppm. The highest antioxidant activity was possessed by bacterial isolate KK3 and was classified as a very weak antioxidant.

Optimizing Green Globular Body Induction for Micropropagation of Microsorum pteropus ‘Windeløv’

Microsorum pteropus ‘Windeløv’ is a water fern from Asia commonly used in aquarium landscapes. This study aimed to develop a mass production method through the induction of green globular bodies (GGBs) from leaf explants of M. pteropus. Surface sterilization was performed on adventitious buds on the fronds of M. pteropus ‘Windeløv’ as explants. The results showed that the optimal explant disinfection condition was 1% sodium hypochlorite solution for 20 min, achieving a successful rate of 87.5 ± 9.6%. The most effective GGB induction was achieved with MS medium supplemented with 5.0 mg/L of NAA (1-Naphthaleneaceticacid) and 0.5 mg/L of 6-BA (6-benzylaminopurine), producing the highest number and size of GGBs, with a mean value of 4.0 ± 0.8 GGBs per explant and a diameter of 2.45 ± 0.61 mm after 8 weeks of culture. The regeneration of multiple sporophytes from the GGBs was only observed under hormone-free MS medium, with a mean value of 34.2 ± 7.3 sporophytes per GGB after 6 weeks of culture. Subsequently, rootless and separated sporophytes could be directly transplanted into the aquarium with a 100% survival rate.

Comparative analysis of red and green lettuce microgreens under different artificial LED lighting conditions

This study investigated how LED light exposure influences the growth and nutrient content of two lettuce microgreens over 10 days in a randomized setup. Lettuce seeds underwent surface sterilization, germination in prepared soil, and exposure to different LED light conditions. Morphological parameters and pigment analysis, including stem length, petiole length, leaf area, plant height, root length, fresh weight, dry matter percentage, were evaluated. Green lettuce outperformed red lettuce in the studied morphological parameters, including stem length (2.74±0.22 cm), plant height (4.54±0.21 cm), and fresh weight (3.79±0.32 g/100 plants) under different LED. White light promoted taller plants with higher fresh weight (4.45±0.43 g/100plants), dry matter (4.84±0.38%), and leaf area (0.76±0.06 cm2) in both lettuce species. In contrast, red light reduced overall growth and development, as evidenced by a 54% decrease in leaf area, despite a 23.36% increase in plant height. Chlorophyll levels varied significantly among LED treatments, with white LED yielding the highest levels in both red and green lettuce. Highest chlorophyll a (146.37±6.27 µg/g FW), chlorophyll b (86.74±2.44 µg/g FW), total chlorophyll (233.11±8.69 µg/g FW) and relative chlorophyll (215.84±8.05 µg/cm²) content was found in green lettuce under white light condition. Similarly, green lettuce grown under white LED had the highest total carotenoid, β-carotene, and lutein. The study concludes that optimizing white LED illumination has the potential to improve the nutritional value of lettuce microgreens by enhancing growth and pigment content, particularly in green varieties. These findings emphasize the crucial role of LED light color in optimizing the nutritional quality of microgreens.

The impact of atmospheric air plasma treatment on the polyfunctional end-use polyester fabric using new synthetic pyrazole dye

Before gluing, bonding, and painting, a variety of materials' surfaces can be modified using the widely used plasma treatment technique. These materials include plastics, glass, metals, and wood. Materials' physical and chemical properties are changed in an environmentally responsible way to enhance or confer particular qualities. The recently used technology known as low-temperature atmospheric pressure plasma (LTAPP) allows heat-sensitive materials to be surface modified in a simple, one-step process. Polymer-based materials are frequently surface modified using LTAPP treatment to improve adhesion, printability, and surface sterility. Polyester's superior mechanical and physical qualities have led to its widespread use as a technical textile and garment material in the form of fibers, films, and plastics. Its limited versatility in terms of end use has been caused by its poor surface properties as the hydrophobic nature of polyester surface, roughness, the crystallinity, and lack of dyeability which are the main drawback restricting its use in different textile applications especially during wet treatments unlike natural fibers. In order to increase the fabric's hydrophilicity and dyeability, the surface of a polyester fabric was altered in this study using atmospheric pressure plasma treatment with atmospheric plasma jet and dielectric barrier discharge (APJ-DBD) technology in air. The primary obstacles in making dielectric barrier discharge (DBD) plasma treatment suitable for industrial purposes are the extended duration of treatment and the utilization of elevated voltage levels. The combination between the atmospheric plasma jet and the dielectric barrier discharge reduces the optimal treatment time to 1 min and the applied voltage to 3.3 kV. After being exposed to oxygen plasma species for a brief period of time (between 30 and 300 s), and were analyzed using an X-ray diffraction machine, an X-ray photoelectron spectroscopy (XPS), static contact angle, and a scanning electron microscope (SEM) to investigate changes in the morphology and chemical nature of the surface, respectively. Fabric wettability increased as a result of plasma treatment, which also increased the fabric's surface roughness, as demonstrated by SEM and X-ray diffraction. The contact angle decreases by increasing the treatment time. The dyeability of untreated and plasma-treated samples was examined with respect to washing, rubbing, perspiration, sublimation, and light fastness. Additionally, color strength was examined. Adequately, compared to the untreated fabric, polyester treated with plasma showed improved dyeing performances. The technical reactivity of poly (ethylene-terephthalate) (PET) fabrics was found to be effectively increased by atmospheric air plasma treatment, creating new avenues for surface modification in light of the expanding environmental and energy-saving concerns.

Ultrafast Solid-Phase Oxidation of Aldehydes to Carboxylic Acids by Atmosphseric Plasma Treatment.

Although atmospheric plasma treatment is an industrially widespread, scalable, and environmentally friendly method, it has been generally used for surface modification, decontamination, or sterilization. In this paper, a novel, sustainable, green, and ultrafast oxidation method is described for aldehydes on a preparative thin-layer chromatographic plate as a solid support. The plasma treatment has proven to be suitable for producing the corresponding carboxylic acids by using only air as a reactant source under mild reaction conditions, while the isolation of the products is also directly integrated into the oxidation process. Extensibility to other reaction types is not explored yet, but we are sure that this novel synthesis conception carries a lot of possibilities.

Endophytic Bacteria Improves the Growth of Tomato Plants: An Initial Trial Towards Sustainable Agriculture

Abstract Microbe-based biofertilizer remains high demand toward sustainable agriculture. Previous study isolated three potential endophytic bacteria from the root of klutuk banana plants namely K2, K8, and K117. The potencies were indicated by IAA production ability. This experiment examined the influence of root inoculation using these endophytic bacteria on the growth of tomato plants. Isolate preparation was initiated by shaking the three isolates in the Nutrient Broth medium for six hours. The seeds of tomato plants were germinated by soaking in sterile water for an hour after surface sterilization. Then, the soaked seeds were incubated for 3 days on the moist paper towel in the petri dish to achieve a high humidity. The germinated tomato seeds were inoculated by soaking the root tip in the bacterial isolates for 10 minutes. Subsequently, the inoculated tomato plants were grown on sterile soils in the greenhouse condition. The height of the plants, leaf count, root length, and quantity of roots were assessed 28 days after inoculation (dai). The t-test analysis indicated two isolates, K2 and K117, significantly improved the height of the plants, leaf count and root lengths compared to the control. In addition, all isolates significantly increased the number of roots of tomato plants. This finding revealed the plant growth improvement caused by root inoculation by these potential endophytic bacteria. This preliminary study gives additional information about a novel biofertilizer for sustainable agriculture.

Season, thermotherapy and surface sterilization play important roles in microbial contamination of hazelnut in vitro cultures

The initiation of cultivars for hazelnut micropropagation from mature field-grown plants is challenging due to high contamination levels and low regeneration rates. This study aimed to develop an efficient protocol for establishing in vitro cultures, the crucial first step in hazelnut micropropagation. Three independent experiments were conducted to achieve this goal. In the first experiment, plant materials were collected directly from the field at different times of the year to initiate cultures, with July to August identified as the most suitable period. In the second experiment, one-year-old potted suckers underwent a sanitation program in the greenhouse and were subjected to thermotherapy in the growth chamber before being utilized as explant sources. Nodal explants obtained from these plants were then used to initiate cultures. In the third experiment, various doses and durations of sterilization agents were applied to optimize surface sterilization. The combination of 1% silver nitrate (AgNO3), 0.1% mercury chloride (HgCl2), and 3% hydrogen peroxide (H2O2) after treatments with 70% ethanol and 2% sodium hypochlorite (NaOCl) yielded the best results, with the highest percentage of viable shoots. Overall, thermotherapy treatment to the mother plant was the most effective method for initiating cultures from plants in highly contaminated environments. While cultures initiated with explants from field-grown plants exhibited 53.7% visually clean shoots, this rate increased to 97.4% in plants subjected to thermotherapy. This study, for the first time, evaluated the effectiveness of applying thermotherapy to mother plants to reduce the incidence of microbial contaminants in the establishment stage of hazelnut in vitro propagation.

Callus induction of red ginger (Zingiber officinale var. rubrum) with 2,4-D and Kinetin combination to enhance total phenolic and flavonoid content

Abstract Plant tissue culture perform as in vitro technique which use any parts of plants under environmental and nutritional control. Tissue culture provides a competitive systems for effective secondary metabolites production, including in red ginger which has many functional properties with high demand in health and pharmaceutical sectors. This study aims to develop an appropriate procedure to cultivate red ginger with in vitro technique to enhance its phenolic and flavonoid content. The rhizome buds were use as explants for callus induction by MS medium with some combination of 2,4-D and kinetin (1:0; 1:1; 1:5; and 5:1). The 70% ethanol, 5,25% sodium hypochlorite, and 10% povidone iodine were used for surface sterilization of bud explants. The result reveals that in 1:0 combination of 2,4-D and Kinetin, the compact callus were initiated on day-16. The other three combinations showed shorter callus forming time in day-14 and they were friable. The 1:1 combination showed the highest percentage (70%) of callus formation compared to other three combinations. In this combination also indicated no contamination occurs. From this in vitro tissue culture, the methanolic extract of callus obtained highest total phenolic content (204,793 µg/mL) than rhizome (130.77 µg/mL). Though, the total flavonoid content of callus extract (0,289 µg/mL) was lower than rhizome extract (6.74 µg/mL). This study showed that the in vitro tissue culture was quite appropriate to enhance total phenolic content in red ginger, although not at the flavonoid content.

Influence of growth regulators on microclonal propagation of <em>Scrophularia umbrosa</em> Dumort under <em>in vitro</em> conditions

The possibility of effective microclonal propagation of Scrophularia umbrosa Dumort for producing planting materials, aimed at preservation of the species has been shown. This will aid in the creation of artificial introduction populations of this endangered species in the Republic of Tatarstan, Russia. Optimal conditions for surface sterilization of seeds, development of microshoots, in vitro rooting and acclimatization have been developed. The most effective surface sterilization and germination was achieved with 10 % commercial bleach. Nodal explants were cultured in Murashige and Skoog’s (MS) medium with different concentrations of 6-benzylaminopurine (BAP) and indole-3-acetic acid (IAA). The maximum number of microshoots was developed on MS medium containing 1.0 mg/l of BAP and 1.0 mg/l of IAA. Full strength MS medium with only IAA or together with (indole-3-butyric acid) IBA is optimal for rapid rooting of microshoots. The combination of soil: perlite (2:1) (v/v) was the best for ex vitro acclimatization of plantlets.

First report of Diaporthe eres causing leaf spot on Rosa roxburghii Tratt in Guizhou Province, China.

Rosa roxburghii Tratt is a plant from the Rosaceae family whose fruits are rich in vitamins, dietary fiber, flavonoids, phenolic acids, and other active components (Jiang, et al. 2024). In July 2023, about R. roxburghii 500 plants were investigated in a field of 6000 m2 in Guiding County (107°14'E, 26°45'N), Guizhou province, China, and the results showed a leaf spot incidence of s 20 to 30%. . The affected leaves had irregular, black lesions with a clear blackish brown boundary and faint black conidiomata in a brown center. Fifteen symptomatic leaves were collected from 10 plants washed with sterile distilled water, and 5 × 5 mm pieces of the infected tissues were cut. After surface sterilization for30 s with 75% ethanol, 2 min with 3% NaOCl, three washes in sterilized distilled water, the leaf pieces were dried and placed on potato dextrose agar (PDA) and incubated at 25℃ for 5 days. Three isolates (H3-Y-1-1, H3-Y-1-2, H3-Y-1-3) with identical morphology were obtained, and the isolate H3-Y-1-1was selected for further study. The colonies on PDA exhibited irregular growth patterns, with white felty aerial mycelium on the upper surface, and white mycelium on the lower surface. Conidiomata were irregularly distributed over the agar surface. The isolate H3-Y-1-1 produced darkly pigmented pycnidia on PDA after 30 days and oozed milky mucilaginous drops. The fungus produced two types of conidia, α and β. Regular α conidia were 4.74 - 5.96 × 1.52 - 2.24 μm (n = 50), hyaline, elongated, biguttulate and non-septate. Beta conidia were 20.13 - 25.74 × 0.86 - 1.29 μm (n = 50), aseptate, hyaline, smooth, spindle shaped, slightly curved to bent. The morphological features were consistent with the description of Diaporthe eres (Pereira, et al. 2022). The pathogen was confirmed to be D. eres by amplification and sequencing of the internal transcribed spacer region (ITS), the partial β-tubulin (TUB), the partial translation elongation factor 1-alpha (TEF) genes using primers ITS1/ITS4, Bt-2a/Bt-2b, EF1-728F/EF1-986R, respectively. Sequences from PCR amplification were deposited in GenBank with accession numbers PP411998 (ITS), PP502153 (TUB), PP502156 (TEF). BLAST searches of the sequences revealed (96%) (500/523nt), 97% (479/494 nt) and 99% (334/338 nt) homology with those of D. eres CBS 138594 from GenBank (OM698848, OM752196 and OM752197), respectively. Phylogenetic analysis using maximum-likelihood and Bayesian methods placed the isolate H3-Y-1-1 in a well-supported cluster with D. eres CBS 101742. The pathogen was thus identified as D. eres based on the morphological characterization and molecular analyses (Feng, et al. 2013; Tao, et al. 2020). To assess its pathogenicity, healthy R. roxburghii potted plants were inoculated with H3-Y-1-1 spore suspensions. Symptomatic leaves mirroring field symptoms were observed after XX days of incubations at XX°C, while control plants exhibited no symptoms. Diaporthe eres was consistently reisolated from the infected leaves showing brown irregular or round lesions at the initial stage of the disease, expanding and become more irregular over time ultimately causing leaf curling and plant death. To our knowledge, this is the first report of leaf spot on R. roxburghii caused by D. eres in China. The disease may become a serious threat to fruit of R. roxburghii production in China. Therefore, detection of this pathogen is very important to ensure timely disease management.

(Invited) III-Nitride Ultraviolet LEDs and Lasers for Applications in Biology and Medicine

Solid-state ultraviolet (UV) light emitters are being developed for emerging biomedical applications such as surface sterilization, air/water purification, and medical treatments as a replacement for mercury vapor lamps. While these UV emitters offer many advantages over mercury lamps, such as more compact form factors, tunable emission wavelengths, and greatly increased lifetimes, they also suffer from a number of issues which hinder their implementation in many potential applications. UV emitters are based on AlGaN heterostructures which usually have very poor external quantum efficiencies (EQEs) of less than 10%, with EQEs decreasing with emission wavelength. This makes it very challenging to realize high efficiency UV emitters whose high energy photons are capable of inactivating viruses, bacteria, and other pathogens. This work discusses recent developments made by the Zhang Research Group at the Rochester Institute of Technology in improving the efficiencies of UV LEDs and lasers.

Evaluation of antioxidant potential of crude extract of metabolites from endophytic fungi isolated from <i>Annona senegalensis</i> Pers

Degenerative diseases resulting from free radicals can be prevented through the use of natural antioxidants. This study aimed to assess the free radical scavenging potential of the underexplored endophytic fungal metabolites from Annona senegalensis Pers (wild custard apple). Freshly collected root, midrib, and leaf tissues were planted on malt extract agar following surface sterilization with 70% ethanol for 3 minutes, washed twice with distilled water, immersed in a sodium hypochlorite solution (4%) for 5 minutes, and subsequently rinsed with sterile water. Seven endophytic fungi were isolated, and the antioxidant activity of their metabolites was evaluated using the 2,2- diphenyl-1-picrylhydrazyl free radical scavenging method, with ascorbic acid serving as a reference antioxidant. The crude extract from the seven endophytic fungal isolates demonstrated concentration- dependent antioxidant activities. LB2 exhibited a strong antioxidant activity with an IC50 value of 38.15 μg/ml. Extracts from RT1 and LB1 showed antioxidant activities, with the highest inhibition of 60% and 65%, respectively, observed at a concentration of 1000 μg/ml. This was compared favorably to ascorbic acid, which exhibited 94% inhibition. This indicates that the crude extract from endophytic fungi isolated from A. senegalensis possesses free radical scavenging properties. Further purification and elucidation of the crude extract will reveal the bioactive compounds responsible for the antioxidant activities.

First Report of Postharvest Fruit Rot Caused by Neofusicoccum parvum on Prunus salicina (Nai Plum) in China.

Nai plum (Prunus salicina var. cordata cv. Younai) is one of the most popular fruit crop in South China. In July 2023, a fruit rot of nai plum with about 5 % disease incidence was observed in a fruit market of Changsha city, Hunan Province, China. Initially, small, brown lesions appeared randomly on the fruit surface, with disease progression, the lesions gradually expanded and developed into soft rot. To isolate possible fungi from rotten fruits, small pieces (2 × 2 mm) from the periphery of 10 infected fruits were surface-sterilized using 70% ethanol for 10 s, rinsed three times in sterile distilled water, air dried, and then placed onto potato dextrose agar (PDA) plates and incubated at 28℃ for three days. Emerging colonies were subcultured by hyphal tiptransfer on fresh PDA. A total of ten isolates with similar morphology were obtained. Fungal colonies were initially white, gradually turning gray and eventually becoming black, and aerial hyphae were dense and fluffy. Conidia were hyaline, single celled, ellipsoidal to fusiform, and range from 12.7 to 20.0 μm long (avg. 16.9 ± 2.39 μm) × 5.3 to 7.3 μm wide (avg. 6.3 ± 0.82 μm). These morphological characteristics of these isolates matched those of Neofusicoccum parvum (Phillips et al. 2013). To future confirmation of the identify, the internal transcribed spacer (ITS) region, translation elongation factor 1-alpha (TEF1-a), and beta-tubulin TUB2) genes of two representative isolates (JXNP1 and JXNP2) were amplified and sequenced using primer sets ITS5/ITS4 (White et al. 1990), EF1-728F/EF1-986R (Carbone and Kohn 1999; Phillips et al. 2013), and BT2A/BT2B (Glass and Donaldson 1995), respectively. The sequences of both isolates were deposited in GenBank for the ITS (accession nos. OR899331 and OR899332), TEF1-a gene (accession nos. OR909890 and OR909891) and TUB2 gene (accession nos. OR909892 and OR909893). BLAST analysis showed 99-100% identity with the ex-type strain of N. parvum (CMW9081) for ITS, TEF1-a and TUB2. A maximum likelihood phylogenetic tree was constructed using IQtree web server based on combined ITS, TEF1-a and TUB2 data set. The phylogenetic tree revealed that two isolates clustered with N. parvum in a clade with 90% bootstrap support. Based on morphological and molecular data analysis, the isolates were identified as N. parvum. To confirm the pathogenicity, five healthy nai plum fruits were wounded by using a sterile needle after surface sterilization with 75% ethanol, then a 5-mm-diameter mycelial disc of isolate JXNP1 was taped to the wound, the control fruits were taped with sterile agar plugs. All fruits were incubated at 25 ℃ with 80% humidity. After five days, typical naturally occurring fruit rot symptoms appeared on the fruits which inoculated with N. parvum, whereas control fruits remained asymptomatic. To fulfill Koch's postulates, the pathogen was re-isolated from the inoculated fruits and comfirmed as N. parvum by morphological and molecular analysis. Previous studies reported that N. parvum caused fruit rot on various common fruits in China, including loquat, kiwifruit and citrus (Lei et al. 2013; Zhai et al. 2019; Zhou et al. 2013). To our knowledge, this is the first report of N. parvum causing postharvest fruit rot on nai plum in China. This finding provides critical insights for the management of the high-risk disease on plum in China.

Inactivation of microorganisms on surfaces of a refrigerator compartment with low-pressure mercury lamps

Household refrigerator compartments and cold chain systems are highly susceptible to microbial contamination. To minimize the surface-contact disease transmission, low-temperature surfaces must be inactivated. Currently, there is no environmentally friendly and convenient means for surface sterilization at low temperatures. This investigation proposed the use of low-pressure mercury lamps to sterilize the bottom surface of a refrigerator. The mercury lamps emitted UVC irradiation and were mounted horizontally on the sidewalls of the refrigerator compartment to inactivate Escherichia coli (E. coli) on the bottom surface. The measured irradiances and E. coli inactivation efficiencies were used to validate the surface-to-surface (S2S) model. The impacts of the refrigeration temperature (20 °C, 4 °C, and −18 °C, respectively) on the UVC output and E. coli sterilization efficiency were evaluated. And so did for the impacts of the relative humidity (ranging from 45 % to 85 % at 4 °C). The required time for operation of the UVC lamps to reach a 3-log reduction in E. coli was calculated for different lamp placements. The results revealed that the UVC emission of low-pressure mercury lamps decreases remarkably with the temperature. The necessary UVC irradiation dose to achieve the same inactivation efficiency increases as the temperature decreases. To extend the operating life of UVC lamps, the installed lamps should make the irradiance distribution on the target surface as uniform as possible.

Phytosanitary problems in elephant garlic (Allium ampeloprasum var. holmense) in the “Val di Chiana” area (Central Italy), and evaluation of potential control strategies

Allium ampeloprasum var. holmense (elephant garlic) is traditionally cultivated in “Val di Chiana”, an area between Umbria and Tuscany regions of Central Italy, under the name “Aglione della Valdichiana”. This product has recently increased in importance, becoming a key economic resource for local farmers. In 2019, phytosanitary problems of elephant garlic cloves ready for transplanting emerged in this cultivation area. Symptom/sign observations and fungal isolations were performed for cloves divided into four components (tunic, basal plate, reserve tissue and shoot) from six farms in the “Val di Chiana” area. Isolates obtained were identified, using partial β-tubulin (BenA) and calmodulin (CaM) or translation elongation factor 1α (tef1α) genes sequences, as belonging to Penicillium [P. allii (95%), P. citrinum (4%), P. brevicompactum (1%)] or Fusarium [F. oxysporum (81%), F. proliferatum (19%)]. Fusarium spp. were mainly associated with clove tunics and basal plates, while Penicillium spp. with basal plates, reserve tissues and shoots. Fungi often also developed from asymptomatic components, but a correlation was found between isolated pathogens and disease symptoms. Pathogenicity and virulence towards elephant garlic cloves were verified for a representative isolate of each identified species, and Penicillium allii was the most virulent. Strategies to control Fusarium and Penicillium spp. on cloves were assessed, including chemicals, a biocontrol agent, surface sterilization and heat treatment. Among these, treatments with Patriot Gold® (active ingredient [a.i.] Trichoderma asperellum TV1, approved in organic farming on crops similar to elephant garlic), or Signum® (a.i. boscalid + pyraclostrobin, approved for Integrated Pest Management systems on crops similar to elephant garlic), were effective in simultaneous reduction of Penicillium spp. and Fusarium spp. Transplanting of asymptomatic cloves combined with the use of the above treatments showed promising effects for pathogens control, and to assist elephant garlic crop establishment.

First Report of Colletotrichum scovillei Causing Anthracnose Fruit Rot on Pepper in Ontario, Canada.

Anthracnose fruit rot affecting field peppers (Capsicum annuum L.) has been reported in Ontario, Canada, leading to significant crop losses of up to 80% over the past three years. Ten symptomatic fruits per field, exhibiting one or more soft, sunken lesions covered with salmon-colored spore masses (Fig. S1), were collected from one and two Banana pepper fields in August 2022 and 2023, respectively, all located in southwestern Ontario. Small sections of diseased tissue (0.5 cm in length) from lesion edges underwent surface sterilization and plated on 2% potato dextrose agar (PDA, Difco) supplemented with kanamycin (50 mg liter-1), neomycin sulfate (12 mg liter-1) and streptomycin sulfate (100 mg liter-1), and incubated at 22°C for 7 days in the dark. Fifteen fungal colonies were isolated and purified using the hyphal tipping method. All fungal isolates showed a pale gray colony morphology with a faint salmon tint on PDA (Fig. S1). Conidia, produced on PDA after incubating the 15 isolates at 22°C for 17 days in the dark, were hyaline, aseptate, smooth-walled, cylindrical with obtuse ends (Fig. S1), and measured 9.4 to 15.0 × 2.7 to 4.8 µm (mean ± standard deviation of 145 conidia = 11.3 ± 1.2 μm × 3.7 ± 0.5 μm), the typical morphology of Colletotrichum species (Damm et al. 2012). Internal transcribed spacer (ITS), actin (ACT), chitin synthase 1 (CHS-1), glyceraldehyde-3-phosphate dehydrogenase (GAPDH), glutamine synthetase (GS), histone H3 (HIS3) and beta-tubulin 2 (TUB2) gene regions of all isolates were amplified and sequenced with primers ITS1/ITS4, ACT-512F/ACT-783R, CHS-79F/CHS-345R, GDF1/GDR1, GSF1/GSR1, CYLH3F/CYLH3R and Bt2a/Bt2b and deposited in GenBank (Accession Nos. ITS: PP060584 to PP060596; ACT, CHS-1, GAPDH, GS, HIS3 and TUB2: PP085919 to PP086005), respectively. The sequences were 100% identical to Colletotrichum scovillei strains from different hosts and countries (ITS: PP079643; ACT: MN718468; CHS-1: MN718466, GAPDH: MN718465.1, HIS3: MT592502, TUB2: MK462971). The maximum likelihood-based phylogenetic analysis of ITS, ACT, CHS-1, GAPDH, GS, HIS3, and TUB2 concatenated sequences was conducted using IQ-TREE 2.2.2.7 (Minh et al. 2020). All isolates from this study were grouped with high bootstrap support values with the holotype C. scovillei CBS 126529 (Fig. S2). Living cultures of these isolates were deposited in the Canadian Collection of Fungal Cultures (DAOMC 252833 to 252847). Pathogenicity was tested by inoculating 4 Banana (cv. Jumbo Stuff) and 4 Bell (cv. Archimedes) pepper fruits with 10 μl droplet of a 1 × 105 conidia ml-1 suspension of each isolate onto a wound made with a sterile pipette tip. Eight control fruits were mock-inoculated with sterilized water. Nine days post-inoculation, necrotic lesions measuring 24.7 ± 0.3 mm on Bell and 27.9 ± 0.2 mm on Banana peppers were observed. Colletotrichum scovillei was re-isolated from all symptomatic fruits, and its species identity was confirmed through morphology, fulfilling Koch's postulates. Control fruits remained symptom-free, and no fungi were isolated from them. This is the first report of C. scovillei in Canada. Previously identified as a pathogen causing anthracnose on peppers in eastern Asia, the United States, Brazil, and Kosovo (Farr and Rossman 2024; Xhemali et al. 2023), its emergence in Ontario raises significant concerns for pepper crops. Additional research is essential to better understand the epidemiology of the disease and develop effective phytosanitary strategies for control.

Optimization of Sterilization Parameters for Isolation of Endophytes from Allium sativum and Exploring its Antibacterial Activity

Endophytic bacteria reside within the plant cell and are beneficial to it in a number of ways like growth, protection to environmental conditions and sustainability in favor of the hosts. Throughout the ages, Garlic (Allium sativum L.) has legendary therapeutic importance. The important step in endophyte isolation is the removal of plant tissue surface microflora, called as surface sterilization. Endophytes from medicinal plants may mimic the compound produced by the host plant and also plays an important role in production of bioactive compound, so it is necessary to isolate endophytes, not the epiphytic microbes. The present investigation was undertaken with an aim to optimize the sterilization parameters viz: time and concentration of ethanol and sodium hypochlorite for isolation of endophytic bacteria as well as exploring their antibacterial activity from Allium sativum. The results revealed that concentration and exposure time of sterilizing agent caused prominent surface sterilization but have negative effect on isolation of endophytes. Experimental results revealed that 70% ethanol (6 min), 2% sodium hypochlorite (1 min) followed by 70% ethanol (30 sec) is effective for surface sterilization of leaf and 70% ethanol (6 min), 3% sodium hypochlorite (1 min) followed by ethanol (30 sec) for bulb of Allium sativum. A total of 86 bacterial endophytes were recovered and screened for antibacterial activity against Enterococcus faecalis, Bacillus cereus ATCC 11778, Staphylococcus aureus, Pseudomonas aeruginosa, Escherichia coli, Enterobacter, Klebsiella pneumoniae, Salmonella typhi and Proteus. Out of 86 bacterial endophytes, only (9%) endophytes were found to be inhibiting one or all test human pathogen. The findings of the present study suggest that use of optimized sterilization conditions are effective for removing surface bacterial strains without affecting endophytes as well as the Allium sativum plant of the Jaunpur district represents an excellent reservoir of endophytic bacteria and novel source of bioactive compounds. On the other hand different concentration and time of their exposure to different parts of plant is required for proper surface sterilization of that part and isolation of endophytes.

First Report of Nigrospora sphaerica causing Leaf Spot on Rhododendron simsii in China.

Rhododendron simsii (indoor azalea) is widely cultivated for its high ornamental value (Xu et al. 2021). In April to May 2023, a leaf spot disease occurred in a field study at the Baili Azalea Forest Area (27°12'N, 105°48'E), Guizhou Province, China. About 500 plants were investigated, and the results showed that the incidence of leaf spot was 20 ~ 30%. To study this disease, 10 plants showing severe symptoms were collected. Initially, the symptoms were round or irregularly shaped brown spots (1 to 10 mm). With time, the spots enlarged and merged. Symptomatic leaves were washed with sterile distilled water, and 5 × 5 mm pieces of the infected tissues were removed. After surface sterilization (30 s with 75% ethanol, 2 min with 3% NaOCl, then washed three times with sterilized distilled water), the leaf pieces were dried and placed on potato dextrose agar (PDA) and incubated at 25℃ for 5 days. Fungal colonies developed from leaf tissues, and the germinated spores were transferred onto PDA for further purification and morphological observation. Three isolates (GUBJ23, GUBJ24, and GUBJ12) with similar morphology were obtained from five affected leaves. The representative strain GUBJ23 was selected for further study. On PDA the mycelium was initially white but with sporulation turned gray and then black. Black, single-celled conidia, spherical to sub-spherical, from 11.80 to 21.39 × 13.38 to 21.83 μm (n = 50) in diameter were borne singly on hyaline vesicles at the tips of conidiophores. These morphological characteristics were similar to those of Nigrospora sphaerica (Wang et al. 2017). To confirm the identification, primer pairs for the internal transcribed spacer (ITS) region (ITS5/ITS4), β-tubulin (TUB2) (Bt-2a/Bt-2b), and the translation elongation factor 1-alpha (TEF1-α) (EF1-728F/EF1-986R), were used for PCR amplification of DNA from strain GUBJ23 (Carbone and Kohn 1999; Glass et al. 1995; White et al. 1990). The resulting sequences were deposited in GenBank with accession numbers OR818025 (ITS), OR835150 (TUB2), and OR835147 (TEF1-α). BLAST searches of the sequences revealed 99.80% identity (503/504 bp) of the ITS sequence, 100.00% identity (395/395 bp) of the TUB2 sequence, and 100.00% identity of the TEF1-α sequence (241/241 bp) with N. sphaerica LC7294 (accessions KX985932, KY019602, and KY019397, respectively.) Based on a combined dataset of ITS, TEF1-α, and TUB2 sequences, a phylogenetic tree was constructed using the maximum likelihood method and confirmed that isolates GUBJ23, GUBJ24, and GUBJ12 were N. sphaerica (Wang et al. 2017). Leaves of three healthy R. simsii plants were spray-inoculated with a spore suspension (105 conidia/mL), and an additional three plants were sprayed with sterile water. These plants were incubated at 25℃ in 75% relative humidity. After 5 to 7 days of inoculation, 0.5 to 1.8 mm spots appeared on the leaves. At 10 to 14 days after inoculation, grayish brown, semicircular or irregular lesions appeared on the leaves, usually with a diameter of 0.8 to 3 mm. The symptoms were like symptoms seen on naturally infected leaves, while the control leaves remained asymptomatic. The pathogen was re-isolated from diseased leaves and identified by morphological characterization and molecular analyses (ITS, TUB and TEF1-α), and the reisolated pathogen was identical to N. sphaerica. Thus completing Koch's postulates. According to previous research, N. sphaerica is a widely distributed phytopathogenic fungus that has a wide host range (Wang et al. 2017). This study is the first to identify N. sphaerica as the cause of leaf spot disease in R. simsii. Given the popularity of R. simsii as a pot plant and landscape shrub in Asia and othr regions, the occurrence of leaf spot disease seriously affects its ornamental and economic value. Therefore, it is crucial to establish and implement effective disease management practices to reduce impact of the disease.