Pure Fungal Cultures Research Articles

From Killer to Solution: Evaluating Bioremediation Strategies on Microbial Diversity in Crude Oil-Contaminated Soil over Three to Six Months in Port Harcourt, Nigeria

The study aimed to evaluate the efficacy of various bioremediation approaches on microbial diversity in crude oil-contaminated soil over three to six months in Port Harcourt, Nigeria. The objective was to assess the impact of different bioremediation strategies on microbial populations, particularly focusing on hydrocarbon-utilizing bacteria and fungi. Microbial populations were quantified using serial dilution and microbial count techniques. The vapor phase transfer mechanism was employed to estimate hydrocarbon-utilizing bacteria and fungi. Bacterial and fungal colonies were incubated for five days, followed by biochemical tests for isolate identification. Fungal pure cultures were observed under a microscope. The study observed a significant increase in microbial populations in soil free of crude oil pollution when bioremediators such as mushrooms and earthworms were introduced. Mushrooms exhibited a 50% increase in hydrocarbon-utilizing bacteria (HUB), while earthworms showed a 55% increase in HUB over the three to six-month period. The longer lifespan and nutrient absorption capabilities of earthworms facilitated faster growth. Furthermore, significant growth in the microbial population of hydrocarbon-utilizing bacteria and fungi was observed in crude oil-polluted soil after employing bioremediation, with the highest growth observed in soil treated with mushrooms at six months, followed by earthworms at six months. Conversely, the lowest microbial population was recorded in soil polluted with 10% crude oil and remediated with earthworms at three months. The results suggest that mushrooms and earthworms effectively increase microbial populations in crude oil-polluted soil. However, mushrooms demonstrated a higher microbial population increase compared to earthworms, especially in terms of promoting the growth of hydrocarbon-utilizing bacteria (HUB) and hydrocarbon-utilizing fungi (HUF). Based on the findings, it is recommended to prioritize using mushrooms as bioremediation agents in similar environmental restoration efforts due to their superior efficacy in increasing microbial populations, particularly HUB and HUF. This study underscores the potential of mushrooms and earthworms as effective bioremediation agents for restoring microbial diversity in crude oil-contaminated soil, offering insights for sustainable environmental restoration practices in oil-affected regions like Port Harcourt, Nigeria.

Exploring the biotechnological prospects of a recently discovered fungus isolated from marine mucilage

AbstractThis study aimed to determine the usability of the identified fungus isolated from the marine-mucilage formation in the biotechnological processes. For this purpose, the antimicrobial and antioxidant activities of the extract obtained from the fungus were examined, and the fatty acid methyl ester composition was determined. The molecular identification of the pure fungal culture was made using LSU regions. In addition, fungal extracts were prepared using different solvents, and the antimicrobial activity of these extracts was investigated by disk diffusion and minimum inhibition concentration methods. At the same time, the antioxidant properties of these extracts were analyzed using the DPPH and ABTS free radical removal methods. Also, FAME analysis was performed to determine the fatty acid content of the fungal extract. According to the study results, the new isolate was identified as the fungus Rhizopus stolonifer. Although fungal extracts have no significant antimicrobial activity, it has been determined that they performed successful DPPH and ABTS scavenging activity without needing additional reactions. FAME results indicate that the mucilage-originated fungus R. stolonifer is a valuable provider of fatty acids that, when purified at large bioreactors, can be good and cheap sources of next-generation biologicals for wide-ranging biotechnological applications.

Molecular Identification of Ascomycetes from American Cranberry (Vaccinium macrocarpon Aiton) Grown in Plantation in Poland

The American cranberry is a perennial North American fruit plant that is grown successfully on commercial plantations in Poland. The purpose of this study was to recognize filamentous fungi that colonize roots, leaves, and fruits without visible disease symptoms. Pure fungal cultures were isolated from disinfected plant fragments in agar media and identified by sequencing common taxonomic DNA markers such as the ITS region, the TEF-1α, or RPB2 genes. Of the 141 isolates studied, 59% were identified as closely related to soil saprotrophs. They were classified primarily as showing the greatest similarity to type strains of Trichoderma amoenum, Trichoderma dorothopsis, Paraphaeosphaeria sporulosa, and Penicillium murcianum. Additionally, isolates that are most similar to strains of Penicillium crustosum, Aspergillus flavus, and Aspergillus versicolor that produced mycotoxins were detected. The fungi identified as closest to Alternaria geophila, Alternaria senecionicola, Paraphoma radicina, Pestalotiopsis unicolor, Pestalotiopsis scoparia, and Neopestalotiopsis spp., whose hosts are plants other than American cranberry, represented 33.81% of the isolates tested. Only 7.2% of the isolates corresponded to the species of Physalospora vaccinia, Diaporthe vaccinii, and Diaporthe eres, known cranberry pathogens. The results of this study can be used to identify latent plant infection and potential disease risks.

Revisiting soil fungal biomarkers and conversion factors: Interspecific variability in phospholipid fatty acids, ergosterol and rDNA copy numbers

Refined conversion factors for soil fungal biomarkers are proposed.High interspecific variability is present in all fungal biomarkers.A modeling approach supports the validity of biomarker estimates in diverse soils.ITS1 copies vary strongly, but are fungal-specific with least phylogenetic bias.A combination of fungal biomarkers will reveal soil fungal physiology and activity.The abundances of fungi and bacteria in soil are used as simple predictors for carbon dynamics, and represent widely available microbial traits. Soil biomarkers serve as quantitative estimates of these microbial groups, though not quantifying microbial biomass per se. The accurate conversion to microbial carbon pools, and an understanding of its comparability among soils is therefore needed. We refined conversion factors for classical fungal biomarkers, and evaluated the application of quantitative PCR (qPCR, rDNA copies) as a biomarker for soil fungi. Based on biomarker contents in pure fungal cultures of 30 isolates tested here, combined with comparable published datasets, we propose average conversion factors of 95.3 g fungal C g−1 ergosterol, 32.0 mg fungal C µmol−1 PLFA 18:2ω6,9 and 0.264 pg fungal C ITS1 DNA copy−1. As expected, interspecific variability was most pronounced in rDNA copies, though qPCR results showed the least phylogenetic bias. A modeling approach based on exemplary agricultural soils further supported the hypothesis that high diversity in soil buffers against biomarker variability, whereas also phylogenetic biases impact the accuracy of comparisons in biomarker estimates. Our analyses suggest that qPCR results cover the fungal community in soil best, though with a variability only partly offset in highly diverse soils. PLFA 18:2ω6,9 and ergosterol represent accurate biomarkers to quantify Ascomycota and Basidiomycota. To conclude, the ecological interpretation and coverage of biomarker data prior to their application in global models is important, where the combination of different biomarkers may be most insightful.

Development of functional consortia for the pretreatment of compostable lignocellulosic waste: A simple and effective solution to a large-scale problem

Microorganisms drive the degradation of organic matter thanks to their enzymatic versatility. However, the structure of lignocellulose poses a great challenge for the microbiota inhabiting a compost pile. Our purpose was to increase the biodegradability of vegetable waste in the early stages of the composting process by applying a microbial consortium with lignocelllulolytic capacity. For this, a previous screening was performed among the culturable microbiota from different composting processes to find inoculants with ligninocellulolytic activity. Selected strains were applied as a pure culture and as a microbial consortium. The starting material was composed of tomato plant and pruning remains mixed in a ratio (50:50 v/v), whose humidity was adjusted to around 65%. To determine the ability of both treatments to activate the biodegradation of the mixtures, moisture, organic matter, ash, C/N ratio, 4-day cumulative respirometric index (AT4) and degradation rates of cellulose, hemicellulose and lignin were evaluated. Subsequently, a real composting process was developed in which the performance of the microbial consortium was compared with the composting process without inoculum (control). According to our tests, three microbial strains (Bacillus safensis, Bacillus licheniformis and Fusarium oxysporum) were selected. The results showed that the application of the bacteria strains at low doses (104 CFU g−1 on the complete residual material of the pile) resulted in higher rates of lignocelullose degradation after 10 days of treatment compared to that observed after application of the fungus in pure culture or untreated controls. The implementation of the strategy described in this work resulted in obtaining compost with better agronomic quality than the uninoculated controls. Therefore, the application of this consortium could be considered as an interesting tool for bioactivation of lignocellulosic waste prior to the composting process.

An Overview of Mycorrhiza in Pines: Research, Species, and Applications.

In the latest literature, climate models show that the conditions for pines, spruces, larches, and birches will deteriorate significantly. In Poland, as well as in other European countries, there are already signs of the decline of these species. This review article deals with the symbiotic relationships between fungi and plants, which can hardly be overestimated, using the example of pine trees. These are the oldest known symbiotic relationships, which are of great benefit to both components and can help plants, in particular, survive periods of severe drought and the attack of pathogens on the roots. This article describes symbioses and their causal conditions, as well as the mycorrhizal components of pine trees and their properties; characterizes ectomycorrhizal fungi and their mushroom-forming properties; and provides examples of the cultivation of pure fungal cultures, with particular attention to the specificity of the mycorrhizal structure and its effects on the growth and development of Pinus species. Finally, the role of mycorrhiza in plant protection and pathogen control is described.

The indigenous microbial diversity involved in the spontaneous fermentation of red dragon fruit (Hylocereus polyrhizus) identified by means of molecular tools

Red dragon fruit (RDF) is well-known for its high nutritional content, especially the red pigment betacyanins that possess high antioxidant activity. Natural fermentation is an ancient yet outstanding technique that relies on the autochthonous microbiota from fruits and vegetables surfaces to preserve and improve the nutritional values and quality of the food product. The present study was to evaluate and identify the indigenous microbial community (bacteria and fungi) that are involved in the natural fermentation of RDF. Results revealed a total of twenty bacterial pure cultures and nine fungal pure cultures were successfully isolated from fermented red dragon fruit drink (FRDFD). For the first time, the PCR amplification of 16S rRNA and ITS regions and sequence analysis suggested nine genera of bacteria and three genera of fungi (Aureobasidium pullulans, Clavispora opuntiae, and Talaromyces aurantiacus) present in the FRDFD. Four dominant (≥10 % isolates) bacteria species identified from FRDFD were Klebsiella pneumonia, Brevibacillus parabrevis, Bacillus tequilensis and Bacillus subtilis. The carbohydrate fermentation test showed that all the indigenous microbes identified were able to serve as useful starter culture by fermenting sucrose and glucose, thereby producing acid to lower the pH of FRDFD to around pH 4 for better betacyanins stability. The present study provides a more comprehensive understanding of the indigenous microbial community that serves as the starter culture in the fermentation of RDF. Besides, this study provides a useful guide for future research to be conducted on studying the rare bacterial strains (such as B. tequilensis) identified from the FRDFD for their potential bioactivities and applications in medical treatment and functional foods industries.

Mycobiota of Ips sexdentatus (Bö;rner, 1776) (Coleoptera, Curculionidae: Scolytinae) in Belarus

The paper presents the results of a study of the fungal component of the microbiome associated with six-toothed bark beetle on the territory of Belarus to clarify the role of this xylophage as a vector of dangerous phytopathogenic fungi. More than 360 pure cultures of fungi isolated from the surface and hemocoel of Ips sexdentatus were analyzed by cultural-morphological and molecular-genetic methods. 35 species of fungi from 25 genera of 17 families in 3 divisions were identi ed. Based on their impact on the stored wood, all species are conventionally classi ed into three groups: (1) fungi that cause discoloration of wood (mold, blue stain, etc.), (2) fungi that cause rot of growing forest and harvested wood, and (3) fungi whose . Five species of fungi from the family Ophiostomatacea e were identi ed: Leptographium Lagerb. & Melin sp., Ophiostoma ips (Rumbold) Nannf., O. minus (Hedgc.) Syd. & P. Syd., O. canum (Münch) Syd. & P. Syd., and O. piceae (Münch) Syd. & P. Syd. associated with blue wood. Heterobasidion annosum (Fr.) Bref. was found in 7.8% of the studied samples of the fungal ora. Fairly high frequency of occurrence con rms the participation of the six-toothed bark beetle in the formation of complex centres of pine desiccation in the territory of Belarus. In the I. sexdentatus population, a signi cant proportion of entomopathogenic fungi (11.4%) was recorded using the molecular genetic method, which may indicate natural processes of self-regulation, or the attenuation of the outbreak of bark beetles in Belarus.

First Report of Cytospora azerbaijanica Causing Cytospora Canker and Shoot Dieback on Peach (Prunus persica) in California, U.S.A.

Peaches (Prunus persica L.) are an important crop in the United States with California leading the nation in peach production, with approximately 505,000 tons valued at $378.3 million (USDA National Agricultural Statistics Service, 2021, https://www.nass.usda.gov/). From April to July 2022, symptoms of branch and scaffold canker as well as shoot dieback were observed in three peach (cvs. Loadel, Late Ross and Starn) orchards located in San Joaquin County, California. Samples were collected from about 12 trees for each cultivar. Fast-growing, white, flat colonies were consistently isolated from active cankers on acidified potato dextrose agar (APDA) following the method described by (Lawrence et al. 2017). Pure fungal cultures were obtained by transferring single hyphal tips onto new APDA Petri plates. A total of 22 isolates were obtained. Each fungal isolate was recovered from a single diseased branch (40 to 55% recovery). All isolates in this study shared similar morphological characteristics. Fungal colonies were fast-growing with relatively even but slightly dentate margin, flat with white to off-white mycelium that turned vinaceous buff to pale greyish sepia (Rayner 1970) with age. Black, globose, ostiolated pycnidia, 0.8-(1.3)-2.2 mm diameter, with brownish surface hyphae formed on peach wood embedded in PDA after approximately three weeks and exudated buff-colored mucilage. Pycnidia were both solitary and aggregated and had multiple internal locules sharing invaginated walls. Conidiogenous cells were hyaline, smooth-walled, septate, tapering towards the apex, 13-(18.2)-25.1 × 0.8-(1.3)-1.9 µm (n = 40). Conidia were hyaline, allantoid, smooth, aseptate, 5.5-(6.3)-7.1 × 1.4-(1.9)-2.3 µm (n = 40). Genomic DNA was extracted and sequences of the internal transcribed spacer region (ITS) using ITS5/ITS4 universal primers, translation elongation factor 1α gene (TEF) using primers EF1-728F/EF1-986R, second largest subunit of RNA polymerase II (RPB2) using primers RPB2-5F2/fRPB2-7cR, and actin gene region (ACT) using primers ACT-512F/ACT-783R were obtained and compared with sequences available in GenBank (Lawrence et al. 2018; Hanifeh et al. 2022). Isolates were identified as Cytospora azerbaijanica following DNA sequencing and morphological identification. Consensus sequences of the four genes of two representative isolates (SJC-66 and SJC-69) were deposited into GenBank database (ITS: OQ060581 and OQ060582; ACT: OQ082292, OQ082295; TEF: OQ082290 and OQ082293; RPB2: OQ082291 and OQ082294). The Basic Local Alignment Search Tool (BLAST) indicated that the sequenced RPB2 genes of isolates (SJC-66 and SJC-69) were at least 99% identical to that of Cytospora sp. strain shd47 (Accession: MW824360) covering at least 85% of the sequences. The actin genes from our isolates were at least 97.85% identical to that of Cytospora sp. strain shd47 (Accession: MZ014513), covering 100% of the sequences. The translation elongation factor gene from isolates (SJC-66 and SJC-69) was at least 96.4% identical to that of Cytospora sp. strain shd166 (Accession: OM372512), covering 100% of the query. Those top hit strains belong to C. azerbaijanica, recently reported by Hanifeh et al. (2022). Pathogenicity tests were performed by inoculating eight wounded, 2- to 3-year-old healthy branches on each of eight 7-year-old peach trees, cvs. Loadel, Late Ross and Starn, using 5-mm-diameter mycelium plugs collected from the margin of an actively growing fungal colony on APDA. Controls were mock-inoculated with sterile agar plugs. Inoculation sites were covered with petroleum jelly and wrapped with Parafilm to keep moisture. The experiment was performed twice. After four months, inoculation tests resulted in vascular discoloration (canker) above and below the inoculation sites (average necrosis length of 114.1 mm). Cytospora azerbaijanica was re-isolated from all infected branches (70 to 100% recovery) completing Koch's postulates. Controls remained symptomless and no fungi were isolated from the slightly discolored tissue. Cytospora species are destructive canker and dieback pathogens of numerous woody hosts worldwide. Recently, C. azerbaijanica was reported in causing canker disease of apple trees in Iran (Hanifeh et al. 2022). To our knowledge, this is the first report of C. azerbaijanica causing canker and shoot dieback of peach trees in the United States and worldwide. These findings will aid towards a better understanding of genetic diversity and host range of C. azerbaijanica.

Occurrence of Nigrospora osmanthi Causing Leaf Blight on Water Lettuce in China.

Water lettuce (Pistia stratiotes L.), is one of the emerging invasive weeds for inland water bodies in Asia and become a major problem for local water ecosystem. Biocontrol of water lettuce by mycobiota is being considered as a promising and sustainable method (Kongjornrak et al. 2019). During July 2021, a leaf blight of water lettuce was observed within about 1.5 ha in Shenxi stream (N25°66', E119°05') in Putian, Fujian, China. The disease severity was about 100% with 80% incidence, early symptoms appeared as small irregularly yellow or brown blight, severely infected leaves turned to be rot, then death and sink. Small pieces (5 × 5 mm) of symptomatic leaves were excised and surface disinfected with 75% ethanol and 0.1% HgCl2 solution, air dried and plated on potato dextrose agar (PDA). 3~5 days after incubation at 28°C, six fungal pure cultures showing similar morphology were obtained from the infected leaves. On PDA, colonies were flat, aerial mycelium grew sparsely, most of it grew inside the agar medium, it reverses white to grey to black with age. Hyphae were branched, septate, smooth and hyaline. Conidiophores mostly reduced to conidiogenous cells and setae were not observed. Conidiogenous cells were monoblastic, discrete and solitary, at first hyaline, subspherical, then turning to pale brown, ampulliform, 4.5-10 × 3.5-6 μm in size. Conidia were solitary, globose or ellipsoidal, black, smooth, some of it formed directly from the mycelia, aseptate, 8-12 μm diam (n=10). Genomic DNA was extracted from one of the representative isolate Z1. ITS1/ITS4 (Mills et al. 1992), Bt-2a/Bt-2b (Glass and Donaldson 1995) and EF1-728F/EF-2 (O'Donnell et al. 1998) primer pairs were used to amplify the isolate's internal transcribed spacer (ITS), the Beta-tubulin fragment (TUB) and the partial translation elongation factor (TEF1), respectively. The isolate's sequences were deposited in the GenBank with accession numbers of OM279539 (ITS), OM296034 (TUB) and OM296035 (TEF1). Phylogenetic analysis using maximum likelihood based on the ITS-TUB-TEF1 concatenated sequences from Nigrospora species revealed that isolate Z1 is closely clustered with N. osmanthi strain LC4487. The fungus was identified as N. osmanthi based on the morphological characteristics and molecular analyses (Hao et al. 2020; Wang et al. 2017). Pathogenicity test were performed using twenty inoculated and control plants, respectively. Conidial suspensions (107 CFU/ml) of Z1 isolate were spray-inoculated on the leaves of healthy water lettuce seedlings, while sterile distilled water was used as control. Inoculated and control plants were kept in the differential 50-liter plastic tanks and maintained in a greenhouse at room temperature (19 to 24°C) for one month. Symptoms appeared 7 days post inoculation, which was similar to what occurs in the field. No symptoms occurred on controls. Pathogen was reisolated and confirmed by morphology and molecular analysis. Koch's postulates were conducted twice. N. osmanthi is a pathogenic fungus of many crop plants, such as buckwheat (Shen et al 2021), Java tea (Ismail et al. 2022) or buffalograss (Mei et al. 2019) in Asia and particularly in China. However, to our knowledge, this is the first report of N. osmanthi causing leaf blight on water lettuce. Further studies on how to apply formulated N. osmanthi will be required so that the strain could be effectively used to control water lettuce, moreover, its environmental safety also need a rigorous experimental evaluation.

LaeA-Regulated Fungal Traits Mediate Bacterial Community Assembly.

Potent antimicrobial metabolites are produced by filamentous fungi in pure culture, but their ecological functions in nature are often unknown. Using an antibacterial Penicillium isolate and a cheese rind microbial community, we demonstrate that a fungal specialized metabolite can regulate the diversity of bacterial communities. Inactivation of the global regulator, LaeA, resulted in the loss of antibacterial activity in the Penicillium isolate. Cheese rind bacterial communities assembled with the laeA deletion strain had significantly higher bacterial abundances than the wild-type strain. RNA-sequencing and metabolite profiling demonstrated a striking reduction in the expression and production of the natural product pseurotin in the laeA deletion strain. Inactivation of a core gene in the pseurotin biosynthetic cluster restored bacterial community composition, confirming the role of pseurotins in mediating bacterial community assembly. Our discovery demonstrates how global regulators of fungal transcription can control the assembly of bacterial communities and highlights an ecological role for a widespread class of fungal specialized metabolites. IMPORTANCE Cheese rinds are economically important microbial communities where fungi can impact food quality and aesthetics. The specific mechanisms by which fungi can regulate bacterial community assembly in cheeses, other fermented foods, and microbiomes in general are largely unknown. Our study highlights how specialized metabolites secreted by a Penicillium species can mediate cheese rind development via differential inhibition of bacterial community members. Because LaeA regulates specialized metabolites and other ecologically relevant traits in a wide range of filamentous fungi, this global regulator may have similar impacts in other fungus-dominated microbiomes.

Transcriptomics Reveals the Effect of Strain Interactions on the Growth of A. Oryzae and Z. Rouxii

The microbial community structure in traditional fermented foods is quite complex, making the relationship between strains unclear. In this regard, the co-culture system can simulate microbial interactions during food fermentation and reveal the morphological changes, metabolic processes, and gene expression of microbial communities. The present study sought to investigate the effects of microbial interactions on the growth of Aspergillus oryzae and Zygosaccharomyces rouxii through omics. After co-cultivation, the pH value and dry weight were consistent with the pure culture of Z. rouxii. Additionally, the consumption of reducing sugar decreased, and the enzymatic activity increased compared with the pure culture of fungus. The analysis of volatile organic compounds (VOCs) and transcriptomics showed that co-culture significantly promoted the effect on Z. rouxii. A total of 6 different VOCs and 2202 differentially expressed genes were identified in the pure and co-culture of Z. rouxii. The differentially expressed genes were mainly related to the endonucleolytic cleavage of rRNA, ribosome biogenesis in eukaryotes, and RNA polymerase metabolic pathways. The study results will provide insights into the effect of microbial interactions on the growth of A. oryzae and Z. rouxii.

Antibacterial eremophilane sesquiterpenoids from Xylaria feejeensis, an endophytic fungi of the medicinal plant Geophila repens

Geophila repens (L.) I.M. Johnst (Rubiaceae) is a traditional medicinal plant used in Sri Lanka for the treatment of bacterial infections. Due to its rich endophytic fungi content, it was postulated that endophytically-produced specialized metabolites may be responsible for its purported antibacterial effects. To test this hypothesis, eight pure endophytic fungal cultures were isolated from G. repens then extracted and screened for antibacterial activity in a disc diffusion assay against Staphylococcus aureus, Bacillus cereus, Escherichia coli and Pseudomonas aeruginosa. Large scale culturing, extraction, and purification of the most active fungal extract, obtained from Xylaria feejeensis, led to the isolation of 6′,7′-didehydrointegric acid (1), 13-carboxyintegric acid (2), and four known compounds including integric acid (3). Compound 3 was isolated as the key antibacterial component (MIC = 16 μg/mL against Bacillus subtilis, 64 μg/mL against Methicillin-Resistant S. aureus). Compound 3 and its analogues were devoid of hemolytic activity up to the highest tested concentration of 45 μg/mL. This study demonstrates that specialized metabolites produced by endophytic fungi may contribute to the biological activity of some medicinal plants. Endophytic fungi should be evaluated as a potential source of antibiotics, especially from unexplored medicinal plants traditionally used for the treatment of bacterial infections.

Etiology, Clinical Spectrum, Epidemiology, New Developments in Diagnosis and Therapeutic Management of Onychomycosis: An Update

Fungal infections are recognized as an important cause of morbidity as well as mortality, especially in the immune-suppressed subjects worldwide. Onychomycosis is a sporadic fungal infection that can affect any part of the nail unit, including the matrix, bed, and plate. It is caused by a wide variety of fungi that include dermatophytes, yeasts, and non-dermatophyte moulds. Dermatophytes are the most common cause of onychomycosis among the numerous fungi. The disease is mainly sporadic and has been recorded in sexes, all age groups, in rural and urban settings, and developing and developed countries. Onychomycosis affects more than 5% of the world's population today. Distal lateral subungual onychomycosis (DLSO), white superficial onychomycosis (WSO), proximal subungual onychomycosis (PSO), endonyx onychomycosis (EO), and candidal onychomycosis are the five primary subtypes of onychomycosis. The condition can manifest itself in a variety of clinical forms, all of which impact the nails on the fingers and legs. Generalized infections involving both hands have been found on occasion. The infection in immunocompromised patient may cause a serious health problem. A plethora of factors, such as nail trauma, occlusive foot wear, and humidity may predispose to fungal nail infections. Clinical diagnosis should be supported with laboratory findings. Direct microscopic demonstration of the fungal agent in the afflicted nail using the potassium hydroxide procedure and isolation of fungi in pure culture on mycological media, such as Sabouraud dextrose agar, dermatophyte test medium, and Pal sunflower seed agar helps to confirm the diagnosis of onychomycosis. The precise morphology of fungal cultures taken from the clinical specimens of patients can be easily investigated in staining solutions, such a PHOL and Narayan. Direct microscopy in potassium hydroxide is still considered the simplest, quickest, and cheapest way to detect fungal agents in nail scrapings. Onychomycosis medications can be applied locally (amorolfine, ciclopirox, efinaconazole, tavaborole) or taken orally (fluconazole, itraconazole, terbinafine). The cure rate is increased when topical and systemic treatments are used together. Depending on the extent of nail involvement, therapy might last from 3 to 6 months or even longer. It is imperative to manage the risk factors. Hygiene is considered the best prevention strategy of onychomycosis.

First report of a new leaf blight disease of bletilla (Bletilla striata) caused by Curvularia reesii in China.

Bletilla striata is an important Chinese herbal plant grown widely in southwest China (Qian et al. 2021). Leaf blight was found on cultivated bletilla crops in Yunnan in 2021. The disease infected bletilla leaves and it was present in the field from April to November with the highest incidence (86% plants diseased) recorded in early September in Puer area. Foliar lesions were circular (Φ0.5-1.8 cm) or oval, with pale-gray center and narrow gray-brown outer area surrounded by a yellow halo. The lesions coalesced later to form large irregular spots or blighted areas on leaves. Symptomatic bletilla leaves were sampled from fields in Jiangcheng (E101.8672o, N22.5803o) and Simao (E109.7816o, N22.7891o) counties, Yunnan in July 2021. Seven fungal isolates were obtained from (BJ01-BJ04) and Simao samples (HBJ05-HBJ07) via lesion-tissue culture and hypha-tip purification on PDA medium. A pathogenicity test following Koch's Postulates (Grimms et al. 2006) was conducted using each isolate by inoculating 45-day old bletilla plant (n=30, Zihua cultivar) in a greenhouse through spraying hypha-spore suspension (3.25×104 CFU/mL) prepared with 14 d fresh DNA culture. Non-inoculated plants (n=30) were used as controls. The experiment was repeated once. The isolates BJ02 and HBJ06 (deposited in Yunnan Agric. Univ. Microbes Herbarium) were shown pathogenic to bletilla since similar lesions formed on seedlings 7 d post inoculation and pure fungal cultures with the same colony morphology as those of BJ02 and HBJ06 were re-isolated from leaf lesions 14 dpi. Isolates BJ02 and HBJ06 produced identical colony and conidium morphology after they were incubated at 25oC for 7 d on PDA. Colonies were circular, pale brown, Φ5.5-7.5cm, with villous surface and abundant aerial hyphae. Mycelia were septate, colorless, Φ3-4 µm and with acute-angled branches. Conidiophores developed from hyphae were erect, septate, pale-brown colored and 60-200 µm long. Conidia (produced scarcely and ripened slowly) were long-oval or petaloid, straight or slightly curved, brown, sized 28-45×10-14 µm. Most conidia were divided into 4 cells by 3 septa; the middle two were bigger than the basal and apex cells. Both BJ02 and HBJ06 were identified as Curvularia sp. based on their morphological characters (Tan et al. 2018). The rDNA-ITS, TEF1α and GAPDH genes (Tan et al. 2018) were amplified from these isolates with PCR (White et al. 1990) and sequenced. ITS sequences of the two isolates were both 574 bp (acc. no. OL587997 & OL336480) and 100% (574/574 bp) identical shown by blast comparison. Further blast analyses of ITS (574 bp, OL587997), TEF1α (532 bp, ON637120) and GAPDH (881 bp, ON637121) from isolate BJ02 showed that they were 99.27% (547/551 bp), 100% (842/842 bp) and 99.8% (507/508 bp) identical respectively with those of Curvularia reesii BRIP4358 (MH414907). The 3 genes of BJ02 were concatenated and phylogenic analysis (Tamura et al, 2013) of the concatenated sequence with those of Curvularia spp. showed that BJ02 was clustered with C. reesii BRIP4358 on the same end-branch of the tree with 100% confidence. Therefore, BJ02 and HBJ06 are the same species identified as Curvularia reesii and it is the pathogen causing bletilla leaf blight. C. reesii was first isolated from the air in Australia in 1963 and was named by Tan et al. in 2018. It has not been reported as a plant pathogen elsewhere. This is the first record of this fungus causing bletilla leaf blight in China. Keywords: Bletilla striata; leaf blight; Curvularia reesii; disease symptoms; pathogen morphology; multigene identification References (1) D.J. Grimes. Microbes, 1(5): 223-228, 2006. (2) L.H. Qian et al. Jiangshu Agric. Sci. 49(19): 64-71, 2021. (3) K. Tamura et al. Mol. Bio. & Evol. 30 (12): 2725- 2729, 2013. (4) Y. P. Tan et al. MycoKeys, 35: 1-25. 2018. (5) T.J. White et al. In: PCR Protocols: A Guide to Methods and Applications (eds. M.A. Innis et al.), Acad. Press, Inc. New York. 315-322, 1990.

Diversity and Heavy Metal Tolerance of Fungi Associated with Different Coal Overburden Strata of Tikak Colliery, Assam.

Coal mine overburdens are generally highly acidic and contaminated with toxic heavy metals. Here, we studied the culturable fungal diversity associated with different coal overburden strata (OBS) of Assam, India, and assessed their heavy metal tolerance ability against five different heavy metals viz., As3+, Cd2+, Cr6+, Cu2+, and Ni2+. Among 15 distinct coal OBS considered in this study which spans a depth of ~ 35m from the ground surface, the isolation of fungi was successful only from 11 OBS samples and the colony-forming unit (CFU) counts were highly variable among the samples. A total of 66 fungal pure cultures were isolated which belong to 18 genera (17 known and 1 unknown) under 15 families and two divisions i.e., Ascomycota (89.4%) and Basidiomycota (10.6%). Acidiella bohemica was found relatively the most abundant species followed by Rhodotorula toruloides. A good number of fungal isolates was found tolerant to the test heavy metals at concentrations ≥ 1mM. Findings of some multi-metallotolerant fungal isolates along with a tolerance up to 5mM concentration of As3+, and up to 10mM each of Cu2+, Cr6+, Ni2+ and Cd2+ were noteworthy in the present study that could be useful in the management of heavy metal pollution or stress. Cultivable fungal diversity of coal mine overburden strata of Tikak colliery, Margherita, Assam, India. It shows a photograph of the coal mining site as the background, front view of the fungal colonies in the upper section, and a graphical representation of heavy metal tolerance of the isolates at different concentrations of As, Cd, Cr, Cu, and Ni in the lower section.

Antifungal properties of rhizobacterial strains in relation to fungi of agricultural crops

On earth, pathogenic fungal infections are considered one of the most common crop problems, accounting for more than 80% of all plant diseases. The production of biologics and the fight against pathogens are highly relevant priorities. In this article, rhizosphere cultures isolated from wheat root tubers have been studied in detail for the development of pathogenic fungi. In the course of research, the inhibitory properties of rhizobacteria against common pathogenic fungi were studied. The main mechanism of antagonism is that bacteria directly affect phytopathogens or increase plant resistance to diseases by enhancing immunity. The antagonistic activity of rhizospheric microorganisms was studied and the level of their action was determined. It has been established that wheat rhizospheric bacteria Escherichia hermannii, Enterobacter cloacae and Rahnella aquatilis affect the development of pathogens that cause Alternaria and Fusarium wilt. In the experiments, 46 strains of rhizobacteria of the genus Enterobacter (strain E. cloacae CCIM1022), Rahnella (R. aquatilis CCIM1023), Pantoea, Pseudomonas, Bacillus, Escherichia were isolated and the effectiveness of their antifungal properties on the reproduction of pathogenic fungi was tested. Pathogenic fungi Alternaria, Fusarium, causing diseases of vegetable and melon crops, were isolated from host plants. It is noted that rhizobacteria have a negative impact and block the development of pure cultures of fungi isolated from tomatoes, zucchini, figs, melons, causing fungal diseases on cups. The 46 isolated and tested strains of rhizobacteria effectively inhibited the development of pathogenic fungi. Of these, Rahnella is the first bacterium studied for this purpose, and has been given great importance in experiments to block the development of Alternaria (A. alternata CCIM 1021). The causative agents of alternariosis are widely open. However, large-scale analysis of their presence and control of pathogens has always been difficult. In experiments to combat the pathogen in the laboratory, a new agent was used to spread nitrogen-fixing bacteria.

First report of Calonectria canadiana causing peach fruit rot in China.

Peach (Prunus persica (L.) Batsch) is one of the most popular fruits grown in Northern China. In July 2021, a fruit rot outbreak on the peach cultivar "Yonglian Sweet" occurred after unusual rains in Baoding, Hebei Province, China. Sixty peach trees from three orchards were assessed, and a 30% disease incidence was estimated. The disease initiated as a small concave spot on the fruit surface expanding circularly rotting the fruit (3-5 cm deep) with the appearance of grayish-white mycelia (Figure S1A). The infected fruit did not disintegrate but turned light brown. To identify the pathogen, 20 infected fruits were collected, and fruit tissues from lesion margins were inoculated on the potato dextrose agar (PDA) medium. A total of 15 fungal pure cultures with highly similar morphological characteristics were obtained by the hyphal-tipping method. The fungal culture formed smooth-edged colonies of extensive, dense, wooly aerial mycelium, with color changing from sienna to luteous, and to grayish-white along the radius of colonies (Figure S1B) Chlamydospores were extensive and developed micro-sclerotia after 20 d of growth. The conidiophore produced three branches in a "broom" shape, with the primary branch ranging 7.5-25.0 μm in length, the secondary branch 5.5-15.5 μm, and the tertiary branch 10-12.5 μm (N = 30). The top of the tertiary branch tapered and produced conidia. Conidia were colorless and culm-like, 40.0-57.5 μm long and 3.8-6.25 μm wide (N = 30). Hyphae occasionally produced spherical chlamydospores with a diameter of around 7.5 μm (N = 30). Conidia germinated after 12 h in moist conditions, and germ tubes originated from multiple points on the conidia. Based on these morphological features, the isolated fungus was identified as Calonectria spp. (Lombard et al. 2010). Six loci, including ITS, act, cmdA, his3, tef1, and tub2, were amplified and sequenced for molecular identification of an isolate F099 using primers listed in Table S1. The obtained ITS (528 bp, GenBank accession no. OL635556), act (263 bp, OL694221), cmdA (470 bp, OL694222), his3 (432 bp, OL694223), tef1 (487 bp, OL694224), and tub2 (535 bp, OL694225) sequences showed 100% similarity to the ex-type strain of Calonectria canadiana, CMW 23673 (accession nos. MT359667, MT334976, MT335206, MT335446, MT412737, and MT412958, respectively; Figure S1D) (Kang et al. 2001, Lechat et al. 2010, Liu et al. 2020). The isolate F099 of C. canadiana was further subjected to pathogenicity tests. Koch's postulates were performed by placing three mycelial disks (ten-day old, 5 mm) with conidia on the sterile needle-acupunctured surface of healthy fruits of the peach cultivar "Yonglian Sweet" (N= 10). Mock inoculations with sterile PDA disks were served as a control. All the inoculated fruits were kept in a moist chamber (25℃, 16-h light and 8-h dark period). The inoculation assay was repeated twice. Rotting symptoms developed on all the inoculated fruits about 5 days post-inoculation (dpi) and grayish-white mycelia appeared around ten days post inoculation while mock inoculated fruits did not show any rotting. The pathogen of interest was re-isolated from the inoculated fruits and validated as C. canadiana by ITS and tef1 sequences. All above evidence collectively indicates that the fungal pathogen causing the peach fruit rot is C. canadiana. The new host plant and new geographic distribution reported here will inform future management of this fungal species.

The Diversity of Fungi Involved in Damage to Japanese Quince.

In recent years, Japanese quince (Chaenomeles japonica) plantations in Latvia have increased. Interest in breeding Japanese quince is also known in other European countries and Russia. The occurrence and harmfulness of fungal diseases have become increasingly significant. However, there is a lack of overall information in the literature about the diversity of fungi afflicting C. japonica. In our study, we aimed to determine the diversity of fungi associated with C. japonica in Latvia, with the possibility of identifying the fungi that are most characteristically associated with certain parts of the plant. Our research was conducted from 2017 to 2019 in eight Japanese quince plantations in Latvia. Samples of plant parts with disease symptoms were collected. Pure cultures of fungi were obtained and identified using ITS region sequencing. We determined the relative density of identified genera of fungi, which were grouped using hierarchical cluster analysis depending on the plant part from which they were found. Various disease-like symptoms were observed and described. A total of 538 isolates of fungi were obtained that belong to 36 genera and represent different ecological niches. Fusarium, Alternaria, Botrytis, and Sarocladium were the genera most frequently found during our study. The number of identified cases of fungal genera differed depending on the part of the plant from which the fungi were obtained. However, it is not possible to relate a specific genus of fungus to only one certain part of a plant. Further research is needed to clarify the pathogenicity of detected fungi and the composition of species in the detected genera of fungi.

ANTAGONIST, ANTIBACTERIAL ACTIVITY OF Stapylocossus aureus AND ISOLATE OF ORAL BACTERIA FROM THE ENDOGENOUS FUNGUS Apis dorsata BINGHAMI NEST

Apis dorsata Binghami is a honey bee endemic to Indonesia, living naturally in the forests of Sulawesi. This study aimed to obtain isolates and characteristics of endogenous fungi antibiotic activity from Apis dorsata Binghami nest. The study consisted of isolation of fungi from beehives using potato dextro agar, pure culture of fungi, antagonist test and antibiotic test using disc diffusion method. Antibiotic test was performed on oral bacteria and Staphylococcus aureus. The results obtained six fungal isolates from Apis dorsata Binghami's nest, namely isolates FAB1, FAB2, FAB3, FAB4, FAB5 and FAB6. The results of the antagonist test showed that the isolates FAB1, FAB2, FAB3, FAB4 and FAB5 were antagonistic. The results of the antibacterial test showed that all isolates produced an average bacterial growth inhibition of 17-18 mm on Staphylococcus aureus and isolates of oral bacteria. The inhibition zone lasts up to 3 x 24 hours so that the activity of the bacteria is bactericidal. From the results of this study, it can be concluded that the endogenous fungus Apis dorsata Binghami is a potential source of antibacterial bioactives.