Fungal Hyphae Research Articles

First report of Powdery Mildew Caused by Podosphaera xanthii on Siraitia grosvenorii in Guizhou, China.

Swingle (Siraitia grosvenorii), a member of the Cucurbitaceae family, stands out as a distinctive plant with both economic and medicinal significance. In October 2023, severe powdery mildew were observed on S. grosvenorii in Guiyang City (26.50°N; 106.66°E), Guizhou Province, China. About 80 % of the plants in the greenhouse showed powdery mildew symptoms. Three infected plant samples were selected for morphological and molecular analysis (GZAAS 23-0801, GZAAS 23-0802 and GZAAS 23-0803). The voucher specimens are deposited in the Key Laboratory of Agricultural Biotechnology of Guizhou Province. The symptoms initially manifested as irregular to nearly circular, small yellow spots, with distinct depressions as well as surfaces covered in white mycelium. Over time, these spots gradually expanded and merged patches. In the final stages, the entire leaves turned into yellow and withered. Microscopic observations showed that fungal hyphae were septate, branched, and flexuous to straight and 5 to 9 µm wide, and appressoria were indistinct to slightly nipple-shaped. Conidia were hyaline and ellipsoid to oval with fibrosin bodies and measured 31 to 43 × 18 to 24 μm (n = 50) with a length/width ratio of 1.3 to 2.3. Conidiophores were unbranched, straight, 120 to 268 × 14 to 22 µm (n = 30), producing two to five immature conidia in chains. Foot cells of conidiophores were cylindrical, 39 to 84 × 8 to 14 μm (n = 30), followed by one to three short cells. Short cells were cylindrical, 12 to 32 × 8 to 15 μm (n = 50). The morphological characteristics were identical with the previous description of Podosphaera xanthii (Braun and Cook, 2012). Total DNA was extracted from conidia and mycelia by the Chelex method (Walsh et al., 1991). The ribosomal DNA internal transcribed spacer (ITS) and nuclear ribosomal large subunit (LSU) were amplified by using the primers ITS1/ITS4 (White et al., 1990) and LSU1/LSU2 (Scholin et al., 1994), respectively. The ITS (OR825802, OR825803 and OR825804, respectively) and LSU (OR825805, OR825806 and OR825807, respectively) sequences of three isolates, were deposited in GenBank. The BLAST results revealed that both the ITS and LSU region sequence were 100% identical to those of P. xanthii (ITS: MF043939, MG754404 and KJ698669; LSU: OQ061319, AB936277and OP218411). Phylogenetic analyses of ITS and LSU sequences showed that our three isolates were clustered with P. xanthii (KX842351, LC270782 and LC270779) with high statistical support (ML/MP/BI: 100%/97%/1.00). Combined with their morphological characteristics, these three isolates were identified as P. xanthii. Pathogenicity tests were performed by gently brushing conidia onto the leaves of five healthy S. grosvenorii plants. Five non-inoculated plants were used as the control. All plants were maintained in a greenhouse at 25 ± 2°C. One week after inoculation, similar symptoms were observed in the inoculated plants, whereas no symptoms occurred on the control plants. By microscopic observation, the fungus on the inoculated plants was morphologically identical to those on originally diseased plants. Powdery mildew caused by P. xanthii has been reported on Vernonia cinerea (Wu et al., 2023), Vigna unguiculata (Zhang et al., 2023), Cucumis melo (Meesam et al., 2023). To our knowledge, this is the first report of powdery mildew caused by P. xanthii on S. grosvenorii in Guizhou, China. The occurrence of powdery mildew on S. grosvenorii may pose a potential threat to its large-scale cultivation. The pathogen could become a threat to other Cucurbitaceae members in the future.

Clinicopathological study of pigmented fungal lesions with special reference to subcutaneous phaeohyphomycosis: A 3-year study in a tertiary care hospital of Eastern India

Background: Fungal infections are predominantly opportunistic infections in immunocompromised patients and in immunocompetent adults with a history of trauma. Recently, there is an increase in trends of fungal infections in immunocompetent individuals also without any history of trauma. Pigmented fungi are a group of fungi that have pigment in their cell wall and also known as dematiaceous fungi. These pigmented fungi include phaeohyphomycosis with a differential diagnosis of chromoblastomycosis, cryptococcosis, mycetoma, and aspergillus. Phaeohyphomycosis is a subcutaneous or systemic infection characterized histologically by dark-colored fungal hyphae and yeast forms (black yeast). Aims and Objectives: The aim of the present study is to analyze the clinicopathological features of pigmented fungal infections in biopsy specimen based on their histomorphology. Materials and Methods: This was a prospective study in which we have analyzed the dematiaceous fungal species by their histomorphological characteristics in biopsy specimens. Results: A total of 21 cases of biopsy specimens (with the clinical presentation of cutaneous/subcutaneous lesions) were studied histologically which showed 13 cases of phaeohyp homycosis, seven cases of eumycotic mycetoma, and one case of chromoblastomycosis. There was male predominance (17, 80.95%) and the majority of the patients were >60 years (16, 76.19%). Most common site of infection was foot with predominant clinical presentation as subcutaneous mass lesion. Conclusion: All the cases of dematiaceous fungal infections were diagnosed by histopathological examination. Melanin in the cell wall of dematiaceous fungi is the pathogenic factor. Phaeohyphomycosis is no longer considered as a rare infection rather there is an increasingly incidence of recognized infection. Although culture and histopathological study remain important tools for final diagnosis, various molecular studies might be helpful for accurate diagnosis.

Plant Growth Promotion and Plant Disease Suppression Induced by Bacillus amyloliquefaciens Strain GD4a.

Botrytis cinerea, the causative agent of gray mold disease (GMD), invades plants to obtain nutrients and disseminates through airborne conidia in nature. Bacillus amyloliquefaciens strain GD4a, a beneficial bacterium isolated from switchgrass, shows great potential in managing GMD in plants. However, the precise mechanism by which GD4a confers benefits to plants remains elusive. In this study, an A. thaliana-B. cinerea-B. amyloliquefaciens multiple-scale interaction model was used to explore how beneficial bacteria play essential roles in plant growth promotion, plant pathogen suppression, and plant immunity boosting. Arabidopsis Col-0 wild-type plants served as the testing ground to assess GD4a's efficacy. Additionally, bacterial enzyme activity and targeted metabolite tests were conducted to validate GD4a's potential for enhancing plant growth and suppressing plant pathogens and diseases. GD4a was subjected to co-incubation with various bacterial, fungal, and oomycete pathogens to evaluate its antagonistic effectiveness in vitro. In vivo pathogen inoculation assays were also carried out to investigate GD4a's role in regulating host plant immunity. Bacterial extracellular exudate (BEE) was extracted, purified, and subjected to untargeted metabolomics analysis. Benzocaine (BEN) from the untargeted metabolomics analysis was selected for further study of its function and related mechanisms in enhancing plant immunity through plant mutant analysis and qRT-PCR analysis. Finally, a comprehensive model was formulated to summarize the potential benefits of applying GD4a in agricultural systems. Our study demonstrates the efficacy of GD4a, isolated from switchgrass, in enhancing plant growth, suppressing plant pathogens and diseases, and bolstering host plant immunity. Importantly, GD4a produces a functional bacterial extracellular exudate (BEE) that significantly disrupts the pathogenicity of B. cinerea by inhibiting fungal conidium germination and hypha formation. Additionally, our study identifies benzocaine (BEN) as a novel small molecule that triggers basal defense, ISR, and SAR responses in Arabidopsis plants. Bacillus amyloliquefaciens strain GD4a can effectively promote plant growth, suppress plant disease, and boost plant immunity through functional BEE production and diverse gene expression.

A Bacillus velezensis strain shows antimicrobial activity against soilborne and foliar fungi and oomycetes.

Biological control uses naturally occurring antagonists such as bacteria or fungi for environmentally friendly control of plant pathogens. Bacillus spp. have been used for biocontrol of numerous plant and insect pests and are well-known to synthesize a variety of bioactive secondary metabolites. We hypothesized that bacteria isolated from agricultural soil would be effective antagonists of soilborne fungal pathogens. Here, we show that the Delaware soil isolate Bacillus velezensis strain S4 has in vitro activity against soilborne and foliar plant pathogenic fungi, including two with a large host range, and one oomycete. Further, this strain shows putative protease and cellulase activity, consistent with our prior finding that the genome of this organism is highly enriched in antifungal and antimicrobial biosynthetic gene clusters. We demonstrate that this bacterium causes changes to the fungal and oomycete hyphae at the inhibition zone, with some of the hyphae forming bubble-like structures and irregular branching. We tested strain S4 against Magnaporthe oryzae spores, which typically form germ tubes and penetration structures called appressoria, on the surface of the leaf. Our results suggest that after 12 hours of incubation with the bacterium, fungal spores form germ tubes, but instead of producing appressoria, they appear to form rounded, bubble-like structures. Future work will investigate whether a single antifungal molecule induces all these effects, or if they are the result of a combination of bacterially produced antimicrobials.

Recent advances in heavy metal/metalloid ion treatment from wastewater using nanocomposites and bionanocomposites

Wastewater remediation is of paramount importance for the water-stressed modern civilization from the perspective of sustainable development and the management of limited freshwater resources. Contaminants such as heavy metal and metalloid ions, toxic dyes, and pathogenic microbes present in the aquatic ecosystem have adverse effects not only on the environment but also on human health. Traditional wastewater remediation processes are costly, energy-consuming, and have less efficiency in removing pollutants from wastewater. Multi-phasic nanomaterials or nanocomposites have enormous potential for removing contaminants from water bodies. These materials have an enormous tunable surface area for interacting with the target toxicants and are extremely stable in the aquatic system, but they readily agglomerate. Many research groups have exploited biosorption, bioleaching, and bioprecipitation capacities of fungal hyphae for wastewater remediation. Recently, researchers have attempted to explore the synergistic effect of the bionanocomposite of fungal hyphae and nanomaterials for removing pollutants from wastewater. This bionanocomposite has already shown promise as a superior material for wastewater treatment and can easily be separated from the polluted water for reuse over several cycles. The bionanocomposite-mediated decontamination of water can lead to a total paradigm shift in wastewater management strategies.

Cultural techniques capture diverse phosphate-solubilizing bacteria in rock phosphate-enriched habitats.

Phosphorus (P) deficiency is a common problem in croplands where phosphate-based fertilizers are regularly used to maintain bioavailable P for plants. However, due to their limited mobility in the soil, there has been an increased interest in microorganisms that can convert insoluble P into a bioavailable form, and their use to develop phosphate-solubilizing bioinoculants as an alternative to the conventional use of P fertilizers. In this study, we proposed two independent experiments and explored two entirely different habitats to trap phosphate-solubilizing bacteria (PSBs). In the first experiment, PSBs were isolated from the rhizoplane of native plant species grown in a rock-phosphate (RP) mining area. A subset of 24 bacterial isolates from 210 rhizoplane morphotypes was selected for the inorganic phosphate solubilizing activities using tricalcium phosphate (TCP) as the sole P source. In the second experiment, we proposed an innovative experimental setup to select mycohyphospheric bacteria associated to arbuscular mycorrhizal fungal hyphae, indigenous of soils where agronomic plant have been grown and trapped in membrane bag filled with RP. A subset of 25 bacterial isolates from 44 mycohyphospheric morphotypes was tested for P solubilizing activities. These two bacterial subsets were then screened for additional plant growth-promoting (PGP) traits, and 16S rDNA sequencing was performed for their identification. Overall, the two isolation experiments resulted in diverse phylogenetic affiliations of the PSB collection, showing only 4 genera (24%) and 5 species (17%) shared between the two communities, thus underlining the value of the two protocols, including the innovative mycohyphospheric isolate selection method, for selecting a greater biodiversity of cultivable PSB. All the rhizoplane and mycohyphospheric PSB were positive for ammonia production. Indol-3-acetic acid (IAA) production was observed for 13 and 20 isolates, respectively among rhizoplane and mycohyphospheric PSB, ranging, respectively, from 32.52 to 330.27 μg mL-1 and from 41.4 to 963.9 μg mL-1. Only five rhizoplane and 12 mycohyphospheric isolates were positively screened for N2 fixation. Four rhizoplane PSB were identified as siderophore producers, while none of the mycohyphospheric isolates were. The phenotype of one PSB rhizoplane isolate, assigned to Pseudomonas, showed four additive PGP activities. Some bacterial strains belonging to the dominant genera Bacillus and Pseudomonas could be considered potential candidates for further formulation of biofertilizer in order to develop bioinoculant consortia that promote plant P nutrition and growth in RP-enriched soils.

Retrieved from the jaws of death – A successfully managed case of rhino- cerebral mucormycosis despite healthcare constraints due to economic turmoil

Rhino- orbital-cerebral mucormycosis is a rapidly progressive life-threatening fungal infection which primarily affects the nose, paranasal sinuses and orbit. It is an opportunistic fungal infection caused by fungi of order Mucorales. They are a group of ubiquitous fungi which grow on decaying vegetation and a wide variety of organic compounds, and they pose a huge threat to life by direct invasion of the vital structures in the body. Early identification and treatment of this disease is therefore of paramount importance for a better outcome. We report a case of a 65-year-old man with a background history of poorly controlled type 2 diabetes mellitus, hypertension and chronic kidney disease, who presented with a diffuse headache of 5 days along with left sided facial oedema, redness of left eye and visual disturbance. Imaging studies revealed sinusitis involving multiple sinuses and the nasal mucosal biopsy showed fungal hyphae. Considering the clinical presentation and the overall findings from the investigations a diagnosis of rhino-cerebral mucormycosis was made. Medical management was done with administration of intravenous Amphotericin B and IV itraconazole along with other supportive medication. Patient had a successful recovery after a lengthy hospital stay which included surgical debridement of affected tissues and the medical treatment even with challenges faced due to unavailability of drugs due to economic constraints on health.

Elucidating fungal decomposition of organic matter at sub-micrometer spatial scales using optical photothermal infrared (O-PTIR) microspectroscopy.

In microbiological studies, a common goal is to link environmental factors to microbial activities. Both environmental factors and microbial activities are typically derived from bulk samples. It is becoming increasingly clear that such bulk environmental parameters poorly represent the microscale environments microorganisms experience. Using infrared (IR) microspectroscopy, the spatial distribution of chemical compound classes can be visualized, making it a useful tool for studying the interactions between microbial cells and their microenvironments. The spatial resolution of conventional IR microspectroscopy has been limited by the diffraction limit of IR light. The recent development of optical photothermal infrared (O-PTIR) microspectroscopy has pushed the spatial resolution of IR microspectroscopy beyond this diffraction limit, allowing the distribution of chemical compound classes to be visualized at sub-micrometer spatial scales. To examine the potential and limitations of O-PTIR microspectroscopy to probe the interactions between fungal cells and their immediate environments, we imaged the decomposition of cellulose films by cells of the ectomycorrhizal fungus Paxillus involutus and compared O-PTIR results using conventional IR microspectroscopy. Whereas the data collected with conventional IR microspectroscopy indicated that P. involutus has only a very limited ability to decompose cellulose films, O-PTIR data suggested that the ability of P. involutus to decompose cellulose was substantial. Moreover, the O-PTIR method enabled the identification of a zone located outside the fungal hyphae where the cellulose was decomposed by oxidation. We conclude that O-PTIR can provide valuable new insights into the abilities and mechanisms by which microorganisms interact with their surrounding environments.IMPORTANCEInfrared (IR) microspectroscopy allows the spatial distribution of chemical compound classes to be visualized. The use of conventional IR microspectroscopy in microbiological studies has been restricted by limited spatial resolution. Recent developments in laser technology have enabled a new class of IR microspectroscopy instruments to be developed, pushing the spatial resolution beyond the diffraction limit of IR light to approximately 500 nm. This improved spatial resolution now allows microscopic observations of changes in chemical compounds to be made, making IR microspectroscopy a useful tool to investigate microscale changes in chemistry that are caused by microbial activity. We show these new possibilities using optical photothermal infrared microspectroscopy to visualize the changes in cellulose substrates caused by oxidation by the ectomycorrhizal fungus Paxillus involutus at the interface between individual fungal hyphae and cellulose substrates.

Is the amount of water transported by arbuscular mycorrhizal fungal hyphae negligible? Insights from a compartmentalized experimental study

Is the amount of water transported by arbuscular mycorrhizal fungal hyphae negligible? Insights from a compartmentalized experimental study

Antifungal Activity of Cedrol from Cunninghamia lanceolate var. konishii against Phellinus noxius and Its Mechanism.

Phellinus noxius is a highly destructive fungus that causes brown root disease in trees, leading to decay and death. In Taiwan, five prized woods-Taiwania cryptomerioides, Calocedrus macrolepis var. formosana, Cunninghamia lanceolata var. konishii, Chamaecyparis formosensis, and Chamaecyparis obtusa var. formosana-are known for their fragrance and durability. This study aims to explore the anti-brown-root-rot-fungus activity of Cunninghamia lanceolata var. konishii (CL) essential oil (CLOL) and its primary components, while also delving into their mechanisms of action and inhibition pathways. The essential oil (CLOL) from CL wood demonstrated significant efficacy against P. noxius, with an inhibitory concentration (IC50) of 37.5 µg/mL. Cedrol, the major component (78.48%) in CLOL, emerged as a potent antifungal agent, surpassing the reference drug triflumizole. Further assays with cedrol revealed a stronger anti-brown-root-disease activity (IC50 = 15.7 µg/mL) than triflumizole (IC50 = 32.1 µg/mL). Scanning electron microscopy showed deformation and rupture of fungal hyphae treated with CLOL and cedrol, indicating damage to the fungal cell membrane. Cedrol-induced oxidative stress in P. noxius was evidenced by increased reactive oxygen species (ROS) levels, leading to DNA fragmentation, mitochondrial membrane potential reduction, and fungal apoptosis through the mitochondrial pathway. Gel electrophoresis confirmed cedrol-induced DNA fragmentation, whereas TUNEL staining demonstrated increased apoptosis with rising cedrol concentrations. Moreover, protein expression analysis revealed cedrol-triggered release of cytochrome c, activation of caspase-9, and subsequent caspase-3 activation, initiating a caspase cascade reaction. This groundbreaking study establishes cedrol as the first compound to induce apoptosis in P. noxius while inhibiting its growth through oxidative stress, an increase in mitochondrial membrane permeability, and activation of the mitochondrial pathway. The findings offer compelling evidence for cedrol's potential as an effective antifungal agent against the destructive brown root disease caused by P. noxius.

Antagonistic activity of secondary metabolites from rhizofunctional bacteria extracts against <i>Fusarium</i>species

Background: Fusarium species remain important fugal pathogens that produce several mycotoxins with adverse effects on both plant and animals. This work aimed to identify biocontrol agent from rhizofunctional bacteria and assess its antagonistic activity against Fusarium sp. using dual culture technique.
 Methodology: Briefly a circular disc of the Fusarium sp. was inoculated at the center of Potato Dextrose Agar (PDA) plate and incubated for three days. The bacterial isolates were then inoculated about 2cm from the Fusarium hyphal tips and incubated for three days, and zone of inhibition was examined. Isolates that showed antagonistic activities against the fungi were subculture in nutrient broth for three days and the metabolites were extracted using ethyl acetate. The metabolic extracts were tested against the fungi using the agar disc diffusion method.
 Results: Of the 20 rhizofunctional bacterial isolates screened for antagonistic activities against Fusarium sp., 5 showed active antagonism against the fungi with observed clear zone of inhibition in the dual culture, and microscopic examination of the fungal hyphae showed excessive and diffused hyphal branching with hyphal swelling. Ethyl acetate extracts from nutrient broth cultures did not show any zone of inhibition in dual culture against the Fusarium sp. All the 5 bacterial isolates were Gram positive strains but only 2 isolates (2a and 3K) were lipase positive, which may indicate that the mechanisms of antagonism could be due to the production of enzymes that have the ability to hydrolyze the cell wall and membrane lipids of the fungi.
 Conclusion: The rhizoplane and rhizosphere of plants could be great sources of biocontrol agents and that bacterial isolates 2a and 3K have the potential to be used as antifungal agents against Fusarium sp. Molecular identification of 2a and 3K bacterial isolates to the species level is recommended.

Biological Control Activities of Rhizosphere Fungus Trichoderma virens T1-02 in Suppressing Flower Blight of Flamingo Flower (Anthurium andraeanum Lind.).

Flower blight caused by Neopestalotiopsis clavispora is an emerging disease of flamingo flower (Anthurium andraeanum Lind.) that negatively impacts flower production. The use of rhizosphere fungi as biocontrol agents is an alternative way to control this disease instead of using synthetic fungicides. This research aimed to screen the potential of rhizosphere fungi, Trichoderma spp., with diverse antifungal abilities to control N. clavispora and to reduce flower blight in flamingo flowers. A total of ten isolates were tested against N. clavispora by dual culture assay, and T1-02 was found to be the most effective isolate against N. clavispora, with inhibition of 78.21%. Morphology and molecular phylogeny of multiple DNA sequences of the genes, the internal transcribed spacer (ITS), translation elongation factor 1-α (tef1-α), and RNA polymerase 2 (rpb2) identified isolate T1-02 as Trichoderma virens. Sealed plate method revealed T. virens T1-02 produced volatile antifungal compounds (VOCs) against N. clavispora, with inhibition of 51.28%. Solid-phase microextraction (SPME) was applied to trap volatiles, and GC/MS profiling showed VOCs emitted from T. virens T1-02 contained a sesquiterpene antifungal compound-germacrene D. The pre-colonized plate method showed that T. virens T1-02 aggressively colonized in tested plates with inhibition of 100% against N. clavispora, and microscopy revealed direct parasitism onto fungal hyphae. Furthermore, the application of T. virens T1-02 spore suspension reduced the disease severity index (DSI) of flower blight in flamingo flowers. Based on the results from this study, T. virens T1-02 displays multiple antagonistic mechanisms and has the potential ability to control flower blight of flamingo flowers caused by N. clavispora.

EVALUATION OF CABIN-SEQUESTERING METHOD TO ELIMINATE BACTERIAL CONTAMINATION FROM AQUATIC FUNGAL CULTURES

Bacterial contamination is a major problem that needs to be addressed in fungal culture. Therefore, a novel technique, cabin sequestering (CS), eliminates bacterial contamination of aquatic fungal cultures. Two treatment groups were formed, which were Group A (CS method) and Group B (subculture on antibiotic-free media) to evaluate the effectiveness of the CS method. A sterile inoculation needle was used to pierce a square hole approximately 3-5 mm in diameter, referred to as a "cabin". Then a coverslip was placed over the inoculated site at 20-30̊ C for 7-10 days. Fungal hyphae were selected and re-inoculated onto new PDA culture plates when they had grown beyond the edge of the coverslip. The result showed no bacterial growth in group A. Meanwhile, in group B, bacteria were present, but no fungal growth was detected. The CS method is advantageous because it eliminates the risk of contamination, takes less time, and requires only simple procedures using standard and easily accessible equipment.

Control of phytopathogen organisms using bioactive compounds contained in orange wax

AbstractOrange wax (OW) is a waxy waste generated in the essential oil purification process (winterization). It has no commercial value. It is discarded or burnt, polluting the environment. The present study demonstrates the possibility of applying it to control phytopathogens. The ethanol‐soluble bioactive compounds of OW were identified and quantified. The effect of orange wax extracts on the growth inhibition of phytopathogenic fungi (Fusarium oxysporum and Colletotrichum gloeosporioides) and Spodoptera frugiperda larvae was evaluated. High‐performance liquid chromatography–mass spectrometry (HPLC‐MS) and gas chromatography–mass spectrometry (GC–MS) quantified 22 bioactive compounds representing 23% (w/w) of OW. Terpineol, limonene, and eremophilene were the most abundant bioactive substances in this material. The distortion of the radial growth of the fungal hyphae was measured in a potato dextrose agar (PDA), and all tested concentrations (6.3, 12.5, 25, 50, and 100 mg/mL) inhibited the radial expansion of C. gloeosporioides. In contrast, only the OW extracts at 50 and 100 mg mL−1 inhibited F. oxysporum. Spray treatments were performed using a Potter spray tower on third‐instar Spodoptera frugiperda larvae. Spraying OW extract at 50 mg mL−1 for 10 s caused the highest larval mortality. However, third‐instar larvae were attracted to consume corn leaves moistened with extract at lower concentrations. Up to 93% of the first‐instar larvae died after eating corn leaves moistened with the extract at 50 mg mL−1. As far as the authors are aware, this is the first scientific report about the use of bioactive compounds of orange wax to control fungi. Spodoptera frugiperda could form the basis for the manufacture of new biopesticides.

The mutual value of histopathology and ITS sequencing in the diagnosis of mucormycosis.

Mucormycosis is a fast-progressing disease with a high mortality rate. The most important factor determining survival of patients is early and accurate diagnosis. Although histopathology often recognises invasive mould infections at first, histomorphology alone is insufficient in providing an accurate diagnosis. Unbiased molecular methods to detect and identify fungi are promising, yet their role in complementing routine histopathological workflows has not been studied sufficiently. We performed a retrospective single-centre study examining the clinical value of complementing histopathology with internal transcribed spacer (ITS) sequencing of fungal DNA in the routine diagnosis of mucormycosis. At our academic centre, we identified 14 consecutive mucormycosis cases diagnosed by histopathology and subsequent ITS sequencing. Using histomorphological examination, fungal hyphae could be detected in all cases; however, morphological features were unreliable regarding specifying the taxa. Subsequent ITS sequencing identified a remarkable phylogenetic diversity among Mucorales: the most common species was Rhizopus microsporus (six of 14; 42.9%), followed by Lichtheimia corymbifera (three of 14, 21.4%) and single detections of Rhizopus oryzae, Actinomucor elegans, Mucor circinelloides, Rhizomucor pusillus and Rhizomucor miehei (one of 14; 7.1%, respectively). In one case, we additionally detected Pneumocystis jirovecii in the same lung tissue specimen, suggesting a clinically relevant co-infection. Fungal culture was performed in 10 cases but yielded positive results in only two of 10 (20%), revealing its limited value in the diagnosis of mucormycosis. Our study demonstrates that a combination of histopathology and ITS sequencing is a practically feasible approach that outperforms fungal culture in detecting Mucorales in tissue-associated infections. Therefore, pathologists might adapt diagnostic workflows accordingly when mucormycosis is suspected.

LysM protein BdLM1 of Botryosphaeria dothidea plays an important role in full virulence and inhibits plant immunity by binding chitin and protecting hyphae from hydrolysis.

Botryosphaeria dothidea infects hundreds of woody plants and causes a severe economic loss to apple production. In this study, we characterized BdLM1, a protein from B. dothidea that contains one LysM domain. BdLM1 expression was dramatically induced at 6 h post-inoculation in wounded apple fruit, strongly increased at 7 d post-inoculation (dpi), and peaked at 20 dpi in intact shoots. The knockout mutants of BdLM1 had significantly reduced virulence on intact apple shoots (20%), wounded apple shoots (40%), and wounded apple fruit (40%). BdLM1 suppressed programmed cell death caused by the mouse protein BAX through Agrobacterium-mediated transient expression in Nicotiana benthamiana, reduced H2O2 accumulation and callose deposition, downregulated resistance gene expression, and promoted Phytophthora nicotianae infection in N. benthamiana. Moreover, BdLM1 inhibited the active oxygen burst induced by chitin and flg22, bound chitin, and protected fungal hyphae against degradation by hydrolytic enzymes. Taken together, our results indicate that BdLM1 is an essential LysM effector required for the full virulence of B. dothidea and that it inhibits plant immunity. Moreover, BdLM1 could inhibit chitin-triggered plant immunity through a dual role, i.e., binding chitin and protecting fungal hyphae against chitinase hydrolysis.

Ophidiomycosis in Wild Dice Snakes (Natrix tessellata) in Germany

Abstract Ophidiomycosis (formerly snake fungal disease) is an emerging disease in snakes caused by the fungal pathogen Ophidiomyces ophidiicola (Oo). The disease is characterized by skin lesions that can range from mild to severe with associated fatalities documented. Subclinical infections have also been described presenting a wide variability of the impact Oo has on the host. In wild snakes, infections have most often been documented in North America, but increasing numbers of reports are becoming available from other parts of the world, including Europe. While monitoring dice snake (Natrix tessellata) populations in 2018 at Schleuse Hollerich/Lahn, a nature reserve in Germany, three dice snakes were observed with cutaneous granulomas. One year later, snakes with granulomas were also observed, and two dice snakes were captured and examined. Fungal hyphae were found in skin biopsies of the lesions and Oo was detected by PCR in the shed skin from one of the snakes. After shedding, the snakes appeared clinically healthy and were released back to the location at which they had been found. This is the first report of histologically confirmed ophidiomycosis in dice snakes and the first confirmed report of ophidiomycosis in a wild snake in Germany.

Does electrical activity in fungi function as a language?

All cells generate electrical energy derived from the movements of ions across membranes. In animal neurons, action potentials play an essential role in the central nervous system. Plants utilize a variety of electrical signals to regulate a wide range of physiological processes, including wound responses, mimosa leaf movements, and cell turgor changes, such as those involved in stomatal movements. Although fungal hyphae exhibit electrical fluctuations, their regulatory role(s), if any, is still unknown. In his paper “Language of fungi derived from their electrical spiking activity”, Andrew Adamatzky, based on a quantitative analysis of voltage fluctuations in fungal mycelia, concludes that the patterns of electrical fluctuations he detects can be grouped into “words” analogous to those found in human languages. He goes on to speculate that this “fungal language” is used “to communicate and process information” between different parts of the mycelium. Here we argue on methodological grounds that the presumption of a fungal language is premature and unsupported by the evidence presented, that the voltage fluctuations he detects are likely to originate as nonbiological noise and experimental artifacts, and that the measured electrical patterns show no similarity to any properties of human language.

Clinicopathological Study of Mucormycosis in COVID-19: A Cross-sectional Study from a Tertiary Care Centre in North Kerala, India

Introduction: Mucormycosis is an angioinvasive disease associated with high morbidity and mortality caused by fungi of the order Mucorales. The rise in the incidence of mucormycosis, along with the surge of Coronavirus Disease- 2019 (COVID-19), justifies reassessment of associated risk factors and the susceptible population. The present study aims to highlight the specific clinicopathological features and risk factors of mucormycosis in COVID-19 patients. Aim: To investigate the histopathological features, key clinical findings in mucormycosis, and the predisposing factors associated with mucormycosis in post-COVID-19 patients. Materials and Methods: This descriptive cross-sectional study was conducted at the Department of Pathology, Government Medical College, Manjeri, Malappuram, Kerala, India, in the Department of Pathology. It included all histopathologically proven Mucormycosis specimens from confirmed COVID-19 positive and post-COVID-19 cases in this Medical College from January 1, 2021, to December 31, 2021, amounting to a total of 30 cases. The type of tissue reaction, fungal hyphae, angioinvasion, necrosis, and fungal co-infections were assessed. Histopathologic analysis was performed using routine Haematoxylin and Eosin (H&E) slides, followed by special stains such as Periodic Acid Schiff (PAS) and Grocott Methenamine Silver (GMS). Data was collected using a proforma, and the clinical details were obtained from the request form received along with the biopsy specimen and case sheets. Age, gender, site of involvement, and risk factors were assessed and subjected to descriptive tabulation and analysis. Results: The mean age was 49.5 years with a male-to-female ratio of 1.30:1. The major risk factors observed in the present study were Diabetes Mellitus (DM) and steroid therapy, 30 all cases (100%), along with others such as chronic kidney disease 2 cases (6.67%) immune disorders, and renal transplantation 1 case each (3.33%). The most commonly involved site was sinuses 27 cases (90.00%). Multiple sinuses were affected in 22 cases (73.33%), with the ethmoid sinus being the most common site 24 cases (80.00%), followed by the maxillary sinus 21 cases (70.00%). The most common reaction pattern observed was acute inflammation 15 cases (50.00%). Angioinvasion was noticed in 19 cases (63.33%) and necrosis was present in 28 cases (93.33%). Co-infection with fungal pathogens was observed in 8 cases (26.67%), with aspergillus being the most common co-infection in all except one of the eight cases. Fungal hyphae were visible on H&E slides in 28 cases (93.33%), while the remaining cases were identified only through special stains (PAS and GMS). Conclusion: Mucormycosis infection is strongly associated with DM and steroid therapy. A more even distribution between males and females was noted, deviating from the male predominance described in most studies. The histopathological appearance of angioinvasion was less frequent compared to most studies, consistent with previous studies on the Indian population. Higher rates of co-infection and the presence of granulomatous inflammation were observed. Further studies are needed to explore the relationship between host immunity, morphological patterns, and their influence on prognosis.

Disseminated Gastrointestinal Basidiobolomycosis: A Case Report with Review of Diagnostic Clues

Introduction. Basidiobolomycosis is a rare fungal infection caused by an environmental saprophyte, Basidiobolus ranarum. It usually presents as a chronic subcutaneous infection; however, few cases of gastrointestinal involvement have been reported. The exact transmission route of gastrointestinal cases is not clear, and diagnosis always requires a high index of suspicion because it tends to mimic other inflammatory and neoplastic conditions. Case Report. A 31‐year‐old immunocompetent woman presented with abdominal pain and an advanced colon mass. She was completely well until about 1.5 years ago, when she underwent bariatric surgery. One year after surgery, chronic abdominal pain developed. A colonoscopy showed an ulcerative lesion in the descending colon, and the biopsy was in favor of ulcerative colitis. Despite immunosuppressive treatment, there was no improvement, and with worsening symptoms, more investigations revealed advanced colon mass with entrapment of the stomach and pancreas. Colonic mucosa biopsy and trucut biopsy of the mass showed just necrosis and acute inflammation; thus, she underwent exploratory laparotomy with colectomy, partial gastrectomy, distal pancreatectomy, and left nephrectomy. On pathologic examination, there was granulomatous inflammation plus the Splendore–Hoeppli phenomenon around fungal hyphae, which was diagnostic for gastrointestinal basidiobolomycosis. Previous pathology slides were reviewed and revealed a tiny focus of basidiobolomycosis. After 6 months of treatment with itraconazole, she is relatively well without any clinical or radiologic abnormalities. Conclusion. Our case highlights the significance of suspicion for basidiobolomycosis in ulcerative and necrotic lesions with increased eosinophils, especially in the presence of abdominal mass and systemic eosinophilia.