Extensive Fungal Colonization Research Articles

Endophytic fungal diversity and bioactive potentials: investigating antimicrobial and antioxidant activities.

This study investigates the colonization of endophytic fungi in nettle leaf tissues and evaluates their antibacterial and antioxidant activities. Using an inverted optical microscope, extensive fungal colonization was observed in all leaf parts, with hyphae prevalent in epidermal cells, parenchyma cells, and vascular tissues. 144 endophytic fungal isolates were isolated from 800 leaf fragments, indicating an 18% retention rate. ANOVA analysis revealed significant differences (p < 0. 001) in colonization frequencies among 20 subjects, with subject 3 showing the highest frequency (40%) and subject 11 the lowest (2. 5%). Ethyl acetate extracts of the three most abundant endophytic fungi demonstrated notable antibacterial activity against both Gram-positive and Gram-negative bacteria, including methicillin-resistant Staphylococcus aureus (MRSA). Inhibition zones ranged from 9. 5 to 15. 16 mm, with minimum inhibitory concentrations (MICs) between 0. 19 to 25 mg/mL. Alternaria sp. exhibited the highest antimicrobial activity against MRSA. Antioxidant activity was assessed using the DPPH radical scavenging test and FRAP method. All extracts showed substantial free radical scavenging properties, with IC50 values close to those of standards like BHT. Alternaria sp. had the highest antioxidant activity, followed by Epicocum sp. and Ulocladium sp. The FRAP method confirmed high reducing potential, with Alternaria sp. again exhibiting the highest activity. These findings highlight the potential of endophytic fungi in nettle leaves as sources of antimicrobial and antioxidant agents, with significant implications for pharmaceutical and biotechnological applications.

Characterization of fungal species involved in white haze disorder on apples in Northern Italy and description of Golubevia mali sp. nov. and Entyloma mali sp. nov.

White haze, caused by an extensive fungal colonization of the apple surface, is an emerging postharvest issue in several European apple production areas. It results in compromised quality and decreased marketability of the fruits, leading to economic losses. In this study, the occurrence and the diversity of white haze-related fungi associated with apples was investigated in Northern Italy. Fungal strains were isolated from apple tissues and the species diversity was assessed using molecular and phylogenetic tools. Moreover, the ability of the isolated species to reproduce symptoms on healthy apples artificially inoculated was tested. Seventy-two fungal isolates were collected. Six species belonging to different basidiomycetous genera (Entyloma, Golubevia, Tilletiopsis) were identified, showing high diversity of fungi involved in white haze development in Northern Italy. The strains were identified as E. belangeri, E. randwijkense, G. pallescens, T. washingtonensis. Moreover, two new species, E. mali sp. nov. and G. mali sp. nov. were described. The most frequently isolated strains were inoculated on healthy apple fruit, showing to be able to reproduce symptoms on red-skin apples, fulfilling Koch’s postulates. This work provides new insights to increase knowledge about the causal agents of white haze on apple. In addition, the names Golubevia, Golubeviaceae and Golubeviales have been nomenclaturally validated.

Protoplast-mediated transformation in Sporisorium scitamineum facilitates visualization of in planta developmental stages in sugarcane.

Sporisorium scitamineum is the causative agent of smut disease in sugarcane. The tricky life cycle of S. scitamineum consists of three distinct growth stages: diploid teliospores, haploid sporidia and dikaryotic mycelia. Compatible haploid sporidia representing opposite mating types (MAT-1 and MAT-2) of the fungus fuse to form infective dikaryotic mycelia in the host tissues, leading to the development of a characteristic whip shaped sorus. In this study, the transition of distinct stages of in vitro life cycle and in planta developmental stages of S. scitamineum are presented by generating stable GFP transformants of S. scitamineum. Haploid sporidia were isolated from the teliospores of Ss97009, and the opposite mating types (MAT-1 and MAT-2) were identified by random mating assay and mating type-specific PCR. Both haploid sporidia were individually transformed with pNIIST plasmid, harboring an enhanced green fluorescent protein (eGFP) gene and hygromycin gene by a modified protoplast-based PEG-mediated transformation method. Thereafter, the distinct in vitro developmental stages including fusion of haploid sporidia and formation of dikaryotic mycelia expressing GFP were demonstrated. To visualize in planta colonization, transformed haploids (MAT-1gfp and MAT-2gfp) were fused and inoculated onto the smut susceptible sugarcane cultivar, Co 97009 and examined microscopically at different stages of colonization. GFP fluorescence-based analysis presented an extensive fungal colonization of the bud surface as well as inter- and intracellular colonization of the transformed S. scitamineum in sugarcane tissues during initial stages of disease development. Noticeably, the GFP-tagged S. scitamineum led to the emergence of smut whips, which established their pathogenicity, and demonstrated initial colonization, active sporogenesis and teliospore maturation stages. Overall, for the first time, an efficient protoplast-based transformation method was employed to depict clear-cut developmental stages in vitro and in planta using GFP-tagged strains for better understanding of S. scitamineum life cycle development.

Wheat root transcriptional responses against Gaeumannomyces graminis var. tritici

Wheat root rot caused by Gaeumannomyces graminis var. tritici (Ggt) results in severe yield losses in wheat production worldwide. However, little is known about the molecular mechanism that regulates systemic symptom development in infected wheat. Fluorescent microscopy observation of the stained wheat roots infected by Ggt showed that lesions were visible when the fungus could be detected in the endodermis, pericycle and phloem at 5 days post inoculation (dpi), and rust symptoms were visible when there was extensive fungal colonization in the root cortex at 6 dpi. Transcriptome sequencing of Ggt-inoculated wheat roots and healthy control root samples was performed at 5 dpi to identify Ggt-induced gene expression changes in wheat roots at the time of lesion formation. A total of 3973 differentially expressed genes (DEGs) were identified, of which 1004 (25.27%) were up-regulated and 2969 (74.73%) were down-regulated in Ggt-inoculated wheat roots compared with those in control roots. GO annotation and KEGG pathway analysis of these DEGs revealed that many of them were associated with pathogen resistance, such as those involved in oxidation-reduction process, tryptophan biosynthesis process, and phenylpropanoid biosynthesis process. Analysis of DEGs revealed that 15 DEGs were involved in cellular regulation, 57 DEGs in signal transduction pathways, and 75 DEGs in cell wall reorganization, and 23 DEGs are pathogenesis-related proteins. Reverse transcription quantitative PCR (RT-qPCR) of 13 of those DEGs showed that these genes may play roles in wheat resistance against Ggt. Overall, this study represents the first transcriptional profiling of wheat roots in response to Ggt infection and further characterization of DEGs identified in this study may lead to better understanding of resistance against take-all in wheat.

Fusarium oxysporumcolonizes the stem of resistant tomato plants, the extent varying with the R-gene present

The plant immune system employs resistance (R) genes to detect the presence of pathogenic microbes by the avirulence (Avr) factors they produce. Whereas some R-genes confer extreme resistance, completely blocking pathogen proliferation, others act later during infection and allow initial microbial multiplication in the host. We hypothesized that transmembrane R-proteins – as opposed to intracellular R-proteins - may recognize and arrest pathogen entry earlier in the infection process. In tomato, two transmembrane R-proteins (I and I-3) and one intracellular R-protein (I-2) have been identified conferring resistance against the same pathogen: the soil borne fungus Fusarium oxysporum f.sp. lycopersici (Fol). We found that in all root inoculations with Fol on resistant tomato plants, the fungus was able to reach the vasculature of the stem. However, the extent of host vasculature colonization was less than that in susceptible plants. This indicates that a complete blockade of fungal ingress does not occur in any of the incompatible interactions. However, resistance mediated by the intracellular R-protein I-2 allowed more extensive fungal colonization than resistance mediated by the transmembrane R-proteins I or I-3. The first phases of invasion – penetration of the root epidermis, cortex colonization and early stage xylem colonization – are unaffected by R-gene mediated resistance of the host. We suggest that all 3 R-proteins only limit Fol colonization after the fungus has reached the xylem tissues, and that I and I-3 act prior to and/or are more effective than I-2 in reducing pathogen proliferation and spread.

Fungal Communities Associated with the Biodegradation of Polyester Polyurethane Buried under Compost at Different Temperatures

Plastics play an essential role in the modern world due to their low cost and durability. However, accumulation of plastic waste in the environment causes wide-scale pollution with long-lasting effects, making plastic waste management expensive and problematic. Polyurethanes (PUs) are heteropolymers that made up ca. 7% of the total plastic production in Europe in 2011. Polyester PUs in particular have been extensively reported as susceptible to microbial biodegradation in the environment, particularly by fungi. In this study, we investigated the impact of composting on PUs, as composting is a microbially rich process that is increasingly being used for the processing of green waste and food waste as an economically viable alternative to landfill disposal. PU coupons were incubated for 12 weeks in fresh compost at 25°C, 45°C, and 50°C to emulate the thermophilic and maturation stages of the composting process. Incubation at all temperatures caused significant physical deterioration of the polyester PU coupons and was associated with extensive fungal colonization. Terminal restriction fragment length polymorphism (TRFLP) analysis and pyrosequencing of the fungal communities on the PU surface and in the surrounding compost revealed that the population on the surface of PU was different from the surrounding compost community, suggesting enrichment and selection. The most dominant fungi identified from the surfaces of PU coupons by pyrosequencing was Fusarium solani at 25°C, while at both 45°C and 50°C, Candida ethanolica was the dominant species. The results of this preliminary study suggest that the composting process has the potential to biodegrade PU waste if optimized further in the future.

Fungal growth necessary but not sufficient for effective biopulping of wood for lignocellulosic ethanol applications

A lignocellulosic biomass treatment method utilizing wood rot fungi to improve cellulosic sugar solubilization for ethanol production was modified to provide larger samples for chemical analyses. Liriodendron tulipifera wood chips were treated in a novel application of aerobic polypropylene “spawn bags” as experimental bioreactors for biopulping. Treatments were inoculated with culture suspensions of white rot ( Ceriporiopsis subvermispora) and brown rot ( Postia placenta) fungi in 40 day incubations. Fungal growth occurred in all treatments, with extensive hyphal coverage of the biomass (ca. >80% of wood chip surfaces). After treatment, physical biodegradation, holocellulose, α-cellulose, and lignin contents of the fungal-biopulped wood chips were comparable to previous results. However, unlike in previous studies, the biopulped wood did not exhibit significant increases in soluble sugars after enzymatic hydrolysis. These results demonstrate that extensive fungal colonization of biomass is not necessarily sufficient for effective biopulping to increase production of free soluble sugars from wood. Environmental controls on hydrolytic activity by wood rot fungi are poorly known, but we hypothesize that treatment inoculation using nitrogen rich medium may have inhibited lignin-specific fungal hydrolysis of the wood. Future development of effective, standardized biopulping techniques for improving soluble sugar production from lignocellulosic biomass will require optimization of methods including characterization of inoculation nutrients and container effects on fungal metabolic activity.

Editorial: What is in a Name? A Proposal to Use Geomycosis Instead of White Nose Syndrome (WNS) to Describe Bat Infection Caused by Geomyces Destructans

In 2006, a mysterious condition was first observed in hibernating bats near Albany, NY, US [1]. The affected animals and carcasses displayed a prominent cotton-like growth around the nostrils, which came to be colloquially termed White Nose Syndrome (WNS). Subsequent studies indicated that the cottony appearance was due to fungal growth. The extensive fungal colonization of bat skin and hair, characteristic histopathological appearance, and exclusive presence of a specific fungal DNA in the animals’ tissues led to the discovery of the causal agent—a newly described species called Geomyces destructans [2–6]. Subsequent studies revealed that this condition has caused the deaths of nearly a million bats in the Northeast and it has rapidly spread to other parts of the country [7]. Unfortunately, the prognosis for bats remains poor at present. The US Fish and Wildlife Service has decided to impose extreme measures, such as barring human access to the caves and the mines where bats live, in an attempt to stop further spread of this disease (http:// www.fws.gov/whitenosesyndrome/pdf/NWRS_WNS_ Guidance_Final1.pdf). Recently, G. destructans growth was reported on bats from many European countries, but the associated morbidity or mortality seen in the US bats was absent from the European animals [8, 9]. Thus, G. destructans causes both colonization and invasive infections in Chiroptera. The underlying mechanisms causing such a wide spectrum of host– pathogen interactions still remain unknown. Bats afflicted with WNS have many symptoms, such as, emaciation, epidermal erosion and ulcers, and extensive wing damage [4, 10]. Accordingly, this disease can be confirmed by the morphological appearance of the fungus on any part of the body, histopathology of affected tissues with demonstration of hyphae–conidia, or demonstration of G. destructans by PCR–nucleotide sequencing [3, 4, 6, 11]. Thus, it is reasonable to conclude that WNS is neither an exclusive presentation nor an all-encompassing description of G. destructans infections in bats. The continued use of this terminology to describe bat disease carries the risk of undue focus on one symptom of what is likely to be a complex host– pathogen interaction. In the fungal kingdom, Geomyces destructans has the closest affinity with Geomyces pannorum, which is widely distributed from the Arctic and the Antarctic to temperate climate zones in cave soil and other substrates [12–15]. The two species can be distinguished by their characteristic conidia and subtle differences in the nucleotide sequences of the ribosomal DNA [5, 16]. Geomyces pannorum is a rare pathogen of humans and animals. It has been V. Chaturvedi (&) S. Chaturvedi Mycology Laboratory, Wadsworth Center, New York State Department of Health and Department of Biomedical Sciences, University at Albany School of Public Health, 120 New Scotland Ave., Albany, NY 12208, USA e-mail: vishnu@wadsworth.org

A Prospective Study on the Clinical Performance of Polysiloxane Soft Liners: One-year Results

The aim of the present study was to evaluate the clinical performance of four denture soft liners up to 12 months. Thirty-three edentulous patients who experienced difficulties when using hard denture bases because of changes in denture-supporting tissues were accepted for the study and randomly received Molloplast B, GC Reline Soft, Silagum Comfort, or Mollosil Plus relines. Performance of the materials was evaluated using nine criteria at 3, 6, and 12 months: physical integrity, surface detail, adhesion, color, odor, plaque accumulation, resilience, hygiene, and mucosal condition. A four-point categorized scale (1=poor, 2=fair, 3=good, 4=excellent) was used. Unscheduled maintenance events and the presence of fungal colonization were also recorded. The percentage of patients available at 3, 6, and 12 months were 91%, 91%, and 66%. Main reasons for dropouts and discontinuation were fractured dentures and patient dissatisfaction. At 6 months, 96% of the performance scores were good or excellent and the largest changes were observed for physical integrity, surface detail, color, and fungal colonization. Fungal colonization was the most commonly observed problem and was the only reason of failure at 12 months. The clinical performance of all soft liners was slightly impaired over the 12-month observation. Except for cases showing extensive fungal colonization, the observed changes in clinical performance did not necessitate remaking of the dentures. Mollosil Plus showed a performance comparable to that of Molloplast B, and the other materials had slightly lower performance especially in terms of fungal colonization.

Host–Pathogen Interactions in Douglas-Fir Seedlings Infected by Phellinus sulphurascens

ABSTRACT Several aspects of the host-pathogen interaction between Douglas-fir (Pseudotsuga menziesii) and the fungal pathogen Phellinus sulphurascens were investigated in an in vitro inoculation system using young seedlings and fungal mycelia. Light microscopy confirmed that P. sulphurascens mycelia can successfully penetrate host epidermal cells within 3 days postinoculation (dpi). Extensive fungal colonization and cortical cell decay occurred within 14 dpi. Western immunoblot studies showed significant upregulation (five to sixfold) of four specific pathogenesis-related (PR) proteins in infected roots. These proteins were a Douglas-fir thaumatin-like protein (PmTLP), an endochitinase protein (ECP), a Douglas-fir PR10 (DF-PR10) protein (PsemI), and a 10.6-kDa antimicrobial peptide (PmAMP1). The highest accumulation of PmTLP and PmAMP1 occurred at 12 dpi, whereas accumulations of the ECP and DF-PR10 proteins peaked at 7 dpi. For both inoculated and control Douglas-fir seedlings, only one of the four PR proteins, PmAMP1, was clearly detectable in needles. Immunolocalization experiments using fluorescein isothiocyanate-conjugated secondary antibodies confirmed accumulation of all four PR proteins mainly in and around cell walls of root cortical tissues. Overall, the highest immunofluorescence was observed in infected roots at 12 dpi, whereas labeling in control roots was negligible at all sample times. The ECP produced the highest fluorescence; the DF-PR10 the lowest. Upregulation and localization of these PR proteins in cortical tissues of inoculated roots suggest that they play a defensive role in response to infection by P. sulphurascens. This in vitro inoculation system will facilitate further proteomic and genomic studies of this important pathosystem.

A potato carboxypeptidase inhibitor gene provides pathogen resistance in transgenic rice

A defensive role against insect attack has been traditionally attributed to plant protease inhibitors. Here, evidence is described of the potential of a plant protease inhibitor, the potato carboxypeptidase inhibitor (PCI), to provide resistance to fungal pathogens when expressed in rice as a heterologous protein. It is shown that rice plants constitutively expressing the pci gene exhibit resistance against the economically important pathogens Magnaporthe oryzae and Fusarium verticillioides. A M. oryzae carboxypeptidase was purified by affinity chromatography and further characterized by mass spectrometry. This fungal carboxypeptidase was found to be a novel carboxypeptidase B which was fully inhibited by PCI. Overall, the results indicate that PCI exerts its antifungal activity through the inhibition of this particular fungal carboxypeptidase B. Although pci confers protection against fungal pathogens in transgenic rice, a significant cost in insect resistance is observed. Thus, the weight gain of larvae of the specialist insect Chilo suppressalis (striped stem borer) and the polyphagous insect Spodoptera littoralis (Egyptian cotton worm) fed on pci rice is significantly larger than that of insects fed on wild-type plants. Homology-based modelling revealed structural similarities between the predicted structure of the M. oryzae carboxypeptidase B and the crystal structure of insect carboxypeptidases, indicating that PCI may function not only as an inhibitor of fungal carboxypeptidases, but also as an inhibitor of insect carboxypeptidases. The potential impact of the pci gene in terms of protection against fungal and insect diseases is discussed.

Evidence that two independent host genes influence the severity of tissue damage and susceptibility to acute pyelonephritis in murine systemic candidiasis

Tissue damage in the kidney and brain after systemic infection withCandida albicanswas examined in recombinant inbred strains (AKXL) derived from AKR and C57/L progenitors. Nine of the 15 strains showed mild (C57/L-like) tissue damage. Of the remainder, two strains developed lesions comparable to the AKR parental strain, whereas four exhibited a much more severe pattern of tissue damage. This was characterized by pronounced mycelial growth in the brain, and gross oedema of the kidney, with extensive fungal colonization and marked tissue destruction. The presence of the null allele of the haemolytic complement gene (Hc) may be necessary, but not sufficient, for the expression of the very severe lesions. The results were interpreted as reflecting the actions of two independent genes, which have been designatedCarg1andCarg2(Candida albicansresistance genes 1 and 2).

Quantitative Localization of the Phytoalexin Glyceollin I in Relation to Fungal Hyphae in Soybean Roots Infected with Phytophthora megasperma f. sp. glycinea

A radioimmunoassay specific for glyceollin I was used to quantitate this phytoalexin in roots of soybean (Glycine max [L.] Merr. cv Harosoy 63) after infection with zoospores of either race 1 (incompatible) or race 3 (compatible) of Phytophthora megasperma Drechs. f. sp. glycinea Kuan and Erwin. The sensitivity of the radioimmunoassay and an inmmunofluorescent stain for hyphae permitted quantitation of phytoalexin and localization of the fungus in alternate serial cryotome sections from the same root. The incompatible interaction was characterized by extensive fungal colonization of the root cortex which was limited to the immediate vicinity of the inoculation site. Glyceollin I was first detected in extracts of whole roots 2 hours after infection, and phytoalexin content rose rapidly thereafter. Significant concentrations of glyceollin I were present at the infection site in cross-sections (42 micrometers thick) of such roots by 5 hours, and exceeded 0.6 micromoles per milliliter (EC(90)in vitro for glyceollin I) by 8 hours after infection. Longitudinal sectioning (14 micrometers thick) showed that glyceollin I accumulated particularly in the epidermal cell layers, but also was present in the root cortex at inhibitory concentrations. No hyphae were observed in advance of detectable levels of the phytoalexin and, in most roots, glyceollin I concentrations dropped sharply at the leading edge of the infection. In contrast, the compatible interaction was characterized by extensive unchecked fungal colonization of the root stele, with lesser growth in the rest of the root. Only small amounts of glyceollin I were detected in whole root extracts during the first 14 hours after infection. Measurable amounts of glyceollin I were detected only in occasional cross-sections of such roots 11 and 14 hours after infection. The phytoalexin was present at inhibitory concentrations in the epidermal cell layers, but the inhibitory zone did not extend appreciably into the cortex. Altogether, these data support the hypothesis that the accumulation of glyceollin I is an important early response of soybean roots to infection by P. megasperma, but may not be solely responsible for inhibition of fungal growth in the resistant response.

Lipid fraction in forest litter: Early stages of decomposition

The lipid fractions of leaf or needle litters of ash, beech, birch, larch, oak, pine and spruce generally disappear more rapidly than non-lipid fractions, though the trend may be obscured by extensive fungal colonization as observed in 3-month old oak and ash samples. While the behaviour of most lipid subfractions is approximately species-independent, changes in concentration of material soluble in hot heptane but only sparingly soluble in cold ether are inversely related to bulk disappearance rate. The exceptional behaviour of this fraction in birch litter is perhaps a result of grazing by soil animals being the dominant removal process for birch leaves. Hot acid hydrolysis, presumably of ester linkages to lignin or polysaccharide, liberates a bound lipid fraction composed principally of fatty acids and accounting for up to 32% (in larch) of total lipid. Data for all species reveal rapid liberation and subsequent disappearance of free fatty acids by biochemical processes in the field.

Cyanogenic potential of Trifolium repens L. in relation to pepper spot caused by Stemphylium sarciniforme

White clover (Trifolium repens L.) contains two cyanogenic β-glucosides (G), linamarin and lotaustralin. Upon injury to the tissues a β-glucosidase (E) effects the release of cyanide from the β-glucosides. Four selections of white clover differing in cyanogenic properties (G+E+, G+E−, G−E+, G−E−) were examined for differences in susceptibility to Stemphylium sarciniforme. Symptom development was determined at 24, 48, 72, 96, and 120 h after the leaves were inoculated. Symptoms for all four selections consisted initially of small brown flecks (1 mm in diameter), then dark spots (1–2 mm in diameter) with definite borders, and finally senescence and wilt of the interlesion tissue. Plants containing both β-glucosidase (EC 3.2.1.21)and cyanogenic glucosides (G+E+) and those with only glucosides (G+E−) developed symptoms 24 h earlier than did the G−E+ and G−E− selections. The earliest symptoms were observed at 48 h; by 96 h all four selections showed comparable degrees of senescence and wilt. Pathogen development in infected leaves prepared as whole mounts was examined microscopically. Through 72 h after inoculation, development of S. sarciniforme in the G+E+ and G+E− selections was slower than in the G−E+ selections. By 96 h, however, fungal development in all four selections was comparable; commencement of leaf senescence coincided with extensive fungal colonization of necrotic tissue. Accelerated rates of symptom expression and retarded pathogen development apparently were positively correlated with cyanogenic potential of each white clover selection. Necrosis of suscept tissue was not correlated with cyanogenic potential of each selection.

Histochemistry and identification of disease-induced terpenoid aldehydes in Verticillium-wilt-resistant and -susceptible cottons

Hemigossypol (HG) and 6-methoxyhemigossypol (MHG) were the major terpenoid aldehydes isolated from stem stele tissue of 6-week-old verticillium-wilt-susceptible Rowden and verticillium-wilt-resistant Seabrook Sea Island 12B2 (SBSI) cotton plants infected for 14 days with Verticillium dahliae. HG constituted 90 and 45 mol % and MHG 6 and 41 mol % of the induced terpenoid in the steles of Rowden and SBSI, respectively. No terpenoids were detected in extracts of noninfected steles.The cellular localization of terpenoids in infected and noninfected stems was studied histochemically with a SbCl3 reagent. Terpenoids first occurred in both SBSI and Rowden in scattered, usually solitary, paratracheal parenchyma cells appressed to infected xylem vessels. After extensive fungal colonization, diffusion of induced terpenoids obscured the initial, discrete sites of localization.