Protoplast fusion between Blakeslea trispora 14,271 (+) and 14,272 (-) enhanced the yield of lycopene and ß-carotene.

Blakeslea trispora, a heterothallic Zygomycota with two mating types (termed "plus" and "minus"), is an ideal source of lycopene and ß-carotene. The lycopene and ß-carotene yields when the two type strains are used for fermentation separately are lower than those when they are joint together. To enhance the yield of lycopene and ß-carotene in B. trispora, protoplast fusion technology was carried out between ATCC 14,271 (+) and ATCC 14,272 (-). After protoplast preparation, protoplast fusion, fusion sorting, fusion regeneration, and high-throughput screening, two fusions (Fu-1and Fu-2) with high lycopene and ß-carotene yields were obtained. The lycopene yields of Fu-1 and Fu-2 were increased to 0.60 mg/gDW and 0.90 mg/gDW, which were respectively 3.62- and 5.44-fold those of 14,271 and 1.76- and 2.64-fold those of 14,272. The ß-carotene yields of Fu-1 and Fu-2 were increased to 22.07 mg/gDW and 36.93 mg/gDW, which were respectively 1.72- and 2.89-fold those of 14,271 and 1.23- and 2.06-fold those of 14,272. In this study, the protoplast fusion technique was successfully used in Blakeslea trispora, providing new ideas for improving lycopene and ß-carotene production.

Comparative analysis of nonvolatile and volatile metabolites in Lichtheimia ramosa cultivated in different growth media.

Lichtheimia ramosa is one of the predominant filamentous fungi in Korean traditional nuruk. The nonvolatile and volatile metabolites of L. ramosa cultivated in three growth media: complete medium (CM), potato dextrose broth (PDB), and sabouraud dextrose broth (SDB), were investigated and compared. Among nonvolatile metabolites, serine, lysine, and ornithine increased in CM and PDB cultivated with L. ramosa during the exponential phase. In addition, glucose level increased in CM whereas decreased in PDB and SDB. The major volatile metabolites in the extract samples were acetic acid, ethanol, 3-methyl-2-buten-1-ol, 2-phenylethanol, ethylacetate, 2-furaldehyde, 5-(hydroxymethyl)-2-furaldehyde, 2,3-dihydro-3,5,-dihydroxy-6-methyl-4H-pyran-4-one, and α-humulene. In particular, the levels of volatile metabolites related to makgeolli (e.g., acetic acid, ethanol, and ethyl acetate) were highest in extracts cultivated in CM. On the other hand, the level of 2-phenylethanol was relatively higher in PDB and SDB, possibly due to there being more phenylalanine present in the biomass sample in media.

Changing Epidemiology of Mucoralean Fungi: Chronic Cutaneous Infection Caused by Mucor irregularis.

INTRODUCTION: The fungi pertaining to order Mucorales usually cause an acute form of clinical disease called mucormycosis. A primary chronic presentation in an immunocompetent patient is a rare form of mucormycosis. Mucor irregularis is known for causing chronic cutaneous infections geographically confined to Asia, mainly in China. We describe a case of primary chronic cutaneous mucormycosis caused by M. irregularis from a new geographical niche in India, highlighting changing aspects of its epidemiology. CASE PRESENTATION: The patient was a farmer with a history of skin lesions over the lower limb for the past 6 years. The biopsy taken from the lesions showed pauci-septate hyphae with right-angle branching on KOH wet mount as well as special fungal stains. On fungal culture, greyish-white cottony mycelial growth of Mucormycetes was obtained. The strain was finally identified as M. irregularis on macro- and microscopic features on 2 % MEA and DNA sequencing. The antifungal susceptibility was done using EUCAST broth microdilution method and was found to be susceptible to commonly used antifungal agents. The patient was started on oral itraconazole and saturated solution of potassium iodide (SSKI). While undergoing treatment for 2 months, he was lost to follow-up, however, after a year when he recently visited the hospital; the disease got completely healed with no new crops of skin lesions. CONCLUSION: Mucoralean fungi should also be suspected in cases with chronic presentation, in immunocompetent host, as there is emergence of such fungi in new endemic areas, particularly located in Asia. The role of other antifungal agents apart from amphotericin B for the treatment of chronic mucormycosis needs to be explored.

Invasive Apophysomyces variabilis infection in a burn patient.

Apophysomyces variabilis is an emerging fungal pathogen that can cause significant infections in immunocompetent patients. We report a case of A. variabilis invasive wound infection in a 21-year-old male after a self-inflicted burn injury.

Molecular phylogeny and proposal of two new species of the emerging pathogenic fungus Saksenaea.

Saksenaea is a monotypic genus belonging to the order Mucorales and capable of producing severe human infections. Through a polyphasic study based on analysis of the sequences of the internal transcribed spacer (ITS) region, domains D1 and D2 of the 28S rRNA gene, and the elongation factor 1α (EF-1α) gene, as well as by evaluation of relevant morphological and physiological characteristics of a set of clinical and environmental strains, we have demonstrated that Saksenaea vasiformis is a complex of species. We propose as new species Saksenaea oblongispora, characterized by oblong sporangiospores and unable to grow at 42°C, and Saksenaea erythrospora, characterized by large sporangiophores and sporangia and by ellipsoid sporangiospores, biconcave in the lateral view. Itraconazole, posaconazole, and terbinafine were active against all isolates included in the study, while amphotericin B, voriconazole, and the echinocandins showed low activity.

Taxonomic study of Endogonaceae in the Japanese islands: New species of Endogone, Jimgerdemannia, and Vinositunica, gen. nov.

Species of Endogonaceae (Endogonales, Mucoromycotina) are characterized by the formation of relatively large sporocarps and zygosporangia. Numerous species in this family remain undescribed or have unclear phylogenetic positions. In Asia specifically, the species diversity of this family is almost completely unknown. However, many mycobionts of bryophytes belonging to several novel clades in Endogonaceae have recently been identified phylogenetically. Therefore, establishing a robust taxonomic system for this family is essential. We obtained numerous sporocarps of undescribed Endogonaceae-like species from the Japanese islands. Morphological observation and multilocus phylogenetic analysis of nuc 18S rDNA (18S), nuc 28S rDNA (28S), and portions of two nuclear protein-coding regions-translation elongation factor 1-alpha (tef1) and RNA polymerase II large subunit (rpb1)-from these species resulted in the description of one new species each of Endogone and Jimgerdemannia and two new species of Vinositunica, gen. nov. Because Vinositunica is characterized by purplish sporocarps and red-wine-colored chlamydospores up to 700 µm in diameter, we emended the definition of Endogonaceae.

A comparison of histomorphologic diagnosis with culture- and immunohistochemistry-based diagnosis of invasive aspergillosis and mucormycosis.

Background: Due to the low sensitivity of mould culture, clinicians usually depend on the histomorphologic diagnosis of invasive mould infection for empirical antifungal therapy. However, definite diagnosis is not always possible based on the mould morphology. We thus compared the histomorphologic diagnosis with immunohistochemistry (IHC)- and culture-based diagnosis.Methods: All adult patients who underwent tissue biopsy and in whom the histomorphologic diagnosis revealed invasive mould infection were enrolled at a tertiary hospital, Seoul, South Korea, between 1992 and 2014 (retrospectively) and 2015 and 2019 (prospectively). Their histomorphologic diagnoses were classified as two categories: (1) acute-angled branching, septate hyphae with parallel walls and a uniform width ('morphologic aspergillosis') and (2) right-angled branching pauciseptate, broader and ribbon-like hyphae with nonparallel walls ('morphologic mucormycosis').Results: A total of 113 patients were finally analysed and their histomorphologic diagnoses were classified as follows: 51 (45%) with morphologic aspergillosis, 62 (55%) with morphologic mucormycosis. Of the 51 patients with morphologic aspergillosis, 46 (90%) received the same diagnosis based on culture and/or IHC, and the remaining five (10%) gave positive IHC result for mucormycosis. Of the 62 patients with morphologic mucormycosis, 60 (97%) had the same diagnosis based on culture and/or IHC, and the remaining two (3%) yielded a positive aspergillus culture or a positive IHC result for aspergillosis, respectively.Conclusions: The majority of histomorphologic diagnoses appear to be consistent with definitive diagnoses based on sterile culture and IHC tests. However, about 10% of 'morphologic aspergillosis' diagnoses were mucormycosis cases.

Fatal Actinomucor elegans var. kuwaitiensis infection following combat trauma.

We report the first case of invasive mucormycosis secondary to Actinomucor elegans infection. A severely injured soldier with a fatal A. elegans var. kuwaitiensis infection is described. The identification of this fungus was performed by classical and molecular methods, and this report documents the pathogenicity of the recently described variety Actinomucor elegans var. kuwaitiensis.

Mucormycotic pseudoaneurysm of the common carotid artery with tracheal involvement.

Mucormycosis is an emerging and fatal fungal infection. A high index of suspicion and the knowledge of its potential manifestations are essential for early diagnosis. We describe a patient with acute lymphoblastic leukaemia (L2 subtype) who developed a neck mass following a course of induction chemotherapy. Doppler ultrasonography and angiography of the neck revealed a pseudoaneurysm of the right common carotid artery. The patient then developed haemoptysis. Surgical exploration revealed a necrotic right common carotid artery with anteromedial pseudoaneurysm and adjacent tracheal wall perforation. Local debridement and tracheal repair were performed. Nonseptate hypheal invasion (mucormycosis) was found on the microscopic examination of the excised arterial wall. A subsequent recurrence of pseudoaneurysm was treated with local surgical debridement and intravenous amphotericin B (Fungizone) administration. Although rare, clinicians should be aware of these possible presenting features of mucormycosis as early diagnosis and treatment may potentially improve the survival.

Decomposition of myceliar matrix and extraction of chitosan from Gongronella butleri USDB 0201 and Absidia coerulea ATCC 14076.

Free chitosan, 2 g/100g mycelia from Gongronella butleri and 6.5 g/100g mycelia from Absidia coerulea were isolated by 1M NaOH at 45 degrees C for 13 h and 0.35 M acetic acid at 95 degrees C for 5 h. Both myceliar matrixes did not break down under these conditions. However, myceliar matrix could be decomposed by treatment with 11 M NaOH at 45 degrees C for 13 h and 0.35 M acetic acid at 95 degrees C for 5 h and then extracted the total chitosan, 8-9 g/100g mycelia from both fungi. According to these results, G. butleri has higher amount of complexed chitosan and A. coerulea has higher amount in free chitosan.

Siepmannia, a new genus in the Mucoraceae.

A new genus Siepmannia of the Mucoraceae family is described. Siepmannia pineti sp. nov. and S. lariceti sp. nov. were isolated from forest soils in Poland. The species were differentiated on the basis of morphology and ITS1/2 rDNA sequencing. Siepmannia pineti and S. lariceti are identical in some aspects. Both show distinct morphological dimorphism in culture depending on conditions of growth and exhibit distinct mycotrophism manifested by sporulation in the presence of other fungi. Their morphological elements are minute. The phylogenetic grouping of Siepmannia with Absidia parricia and A. zychae leads to the creation of two new combinations, S. parricida and S. zychae.

Diversity of mucoralean fungi in soils of papaya (Carica papaya L.) producing regions in Mexico.

Mexico is the fifth largest producer of papaya worldwide and has recently reported problems with mucoralean fungi in this crop. These fungi are considered saprophytes in the soil and are ubiquitous in nature. In this work, they were isolated from soil in regions of intensive papaya cultivation in Mexico. Collections were made in the states of Colima, Oaxaca and Veracruz in Apr 2016. A total of 72 mucorales fungal isolates was obtained and morphologically characterized and then molecular characterization (28S ribosomal region) of 25 representative isolates was carried out. Phylogenetic analysis of the sequences confirmed the presence of the species Gilbertella persicaria, Rhizopus oryzae, Rhizopus stolonifer, Mucor circinelloides and Mucor hiemalis, which cause soft rot in papaya fruits, therefore, spores of these fungi found in the orchard soils can be considered as a constant source of contamination that affects healthy fruits. Additionally, Choanephora cucurbitarum, Mucor ellipsoideus, Rhizopus homothallicus, Rhizopus microsporus, Rhizopus schipperae, Lichteimia ramosa, Gongronella butleri, Cunninghamella bertholletiae and Cunninghamella blakesleeana were identified which are considered to have agricultural, biotechnological and medical importance.

A remodeling system for the oligosaccharide chains on glycoproteins with microbial endo-beta-N-acetylglucosaminidases.

Endo-M, endo-beta-N-acetylglucosaminidase from Mucor hiemalis, transferred the complex type oligosaccharide of sialoglycopeptide to partially deglycosylated proteins (N-acetylglucosamine-attached proteins), which were prepared by excluding high-mannose type oligosaccharides from glycoproteins with Endo-H, endo-beta-N-acetylglucosaminidase from Streptomyces plicatus. This finding indicated that the high-mannose type oligosaccharides on glycoproteins can be changed to complex type ones by the transglycosylation activity of Endo-M. This is the first report of the establishment of a remodeling system for the different types of oligosaccharides on glycoproteins with microbial endo-beta-N-acetylglucosaminidases having different substrate specificities. Endo-M is a powerful tool for the in vitro synthesis of glycoproteins containing complex type oligosaccharides from glycoproteins produced by yeast.

Selection of reference genes for quantitative real-time RT-PCR assays in different morphological forms of dimorphic zygomycetous fungus Benjaminiella poitrasii.

Benjaminiella poitrasii, a dimorphic non-pathogenic zygomycetous fungus, exhibits a morphological yeast (Y) to hypha (H) reversible transition in the vegetative phase, sporangiospores (S) in the asexual phase and zygospores (Z) in the sexual phase. To study the gene expression across these diverse morphological forms, suitable reference genes are required. In the present study, 13 genes viz. ACT, 18S rRNA, eEF1α, eEF-Tu,eIF-1A, Tub-α, Tub-b, Ubc, GAPDH, Try, WS-21, NADGDH and NADPGDH were evaluated for their potential as a reference, particularly for studying gene expression during the Y-H reversible transition and also for other asexual and sexual life stages of B. poitrasii. Analysis of RT-qPCR data using geNorm, normFinder and BestKeeper software revealed that genes such as Ubc, 18S rRNA and WS-21 were expressed at constant levels in each given subset of RNA samples from all the morphological phases of B. poitrasii. Therefore, these reference genes can be used to elucidate the role of morpho-genes in B. poitrasii. Further, use of the two most stably expressed genes (Ubc and WS-21) to normalize the expression of the ornithine decarboxylase gene (Bpodc) in different morphological forms of B. poitrasii, generated more reliable results, indicating that our selection of reference genes was appropriate.

Mitosis in the fungus Zygorhynchus moelleri: evidence for stage specific enhancement of microtubule preservation by freeze substitution.

Interphase and mitotic nuclei of Zygorhynchus moelleri were examined by serial sections of cells fixed with a conventional procedure incorporating an enzymatic cell wall degrading pretreatment (CF) and with a freeze substitution process (FS). CF is superior for analysis of nucleus-associated organelles (NAO) and the nuclear envelope. FS is best for microtubules because, during interphase, it alone retains a population of about 10 short intranuclear microtubules and also preserves more NAO-associated cytoplasmic microtubules. However, during mitosis, while FS gives clearer images of spindles, both procedures show the same numbers and types of spindle and cytoplasmic microtubules, thus supporting the use of enzyme pretreatment schedules for studies of fungal spindles. Mitosis characteristically utilizes about 6 presumptive kinetochore microtubules which remain very short (0.04-0.18 mum) and clustered around the spindle poles throughout mitosis. The rest of the spindle initially contains both interdigitating and continuous microtubules which elongate, reduce in numbers, and probably slide apart as the spindle elongates. The variously oriented, NAO-associated cytoplasmic microtubules do not appear to aid in spindle elongation. Chromatin remains dispersed throughout the nuclear cycle. The presence of presumptive kinetochore microtubules clarifies earlier reports on this group of fungi, but their unusual behaviour supports previous suggestions about the primitive nature of the spindle.

Histologic features of zygomycosis: emphasis on perineural invasion and fungal morphology.

OBJECTIVE: Invasive zygomycosis is rapidly progressive and is associated with angioinvasion and infarction. Invasive disease requires emergent surgical and medical intervention. Because it is important for surgical pathologists to recognize these fungi and their preferential sites of growth, the objective of this article is to describe the fungal morphology and histopathologic findings in biopsies from patients with zygomycotic disease, with emphasis on preferential sites of fungal growth. DESIGN: Medical record and histologic review identified 20 patients with zygomycosis. Inclusion criteria included the presence of typical ribbonlike hyphae and positive culture, a clinical history of invasive zygomycosis, or both. The histologic features of disease and the fungal morphology were assessed. RESULTS: Fungus ball (15%), rhinocerebral (55%), and pulmonary (30%) disease were the types of disease represented. The inflammatory responses were predominantly neutrophilic (50%), predominantly granulomatous (5%), pyogranulomatous (25%), or absent (20%). Invasive disease was characterized by prominent infarcts (94%), angioinvasion (100%), and, surprisingly, prominent perineural invasion (90%) in biopsies that contained nerves for evaluation. At least rare hyphal septa were always seen (100%), and most branches (95%) varied from 45 degrees to 90 degrees. CONCLUSIONS: As known to mycologists, zygomycetes are pauciseptate, rather than aseptate, molds. Therefore, the presence of an occasional septum is expected. Perineural invasion is a common finding in invasive zygomycosis, as are angioinvasion and infarcts. Therefore, prior to excluding the presence of these fungi in biopsies suspected to contain zygomycetes, the perineural space should be carefully examined.

Production of beta-carotene from beet molasses by Blakeslea trispora in stirred-tank and bubble column reactors: development of a mathematical modeling.

The effect of aeration rate and agitation speed on beta-carotene production from molasses by Blakeslea trispora in a stirred-tank fermentor and optimization of the production of the pigment in a bubble column reactor were investigated. In addition, a central composite design was employed to determine the maximum beta-carotene concentration at optimum values for the process variables (aeration rate, sugar concentration, linoleic acid, kerosene). By image analysis of the morphology of the fungus, a quantitative characterization of the hyphae and zygospores formed was obtained. The hyphae were differentiated to intact hyphae, vacuolated hyphae, evacuated cells and degenerated hyphae. An increased proportion of zygospores was correlated to high beta-carotene production. In the stirred-tank fermentor, the highest concentration of the carotenoid pigment (92.0 mg/L) was obtained at an aeration rate of 1.5 vvm and agitation speed of 60 rpm. In the bubble column reactor, the aeration rate and concentration of sugars, linoleic acid, kerosene, and antioxidant significantly affected the production of beta-carotene. In all cases, the fit of the model was found to be good. Aeration rate, sugar concentration, linoleic acid, and kerosene had a strong positive linear effect on beta-carotene concentration. Moreover, the concentration of the pigment was significantly influenced by the negative quadratic effects of the given variables and by their positive or negative interactions. Maximum beta-carotene concentration (360.2 mg/L) was obtained in culture grown in molasses solution containing 5% (w/v) sugar supplemented with linoleic acid (37.59 g/L), kerosene (39.11 g/L), and antioxidant (1.0 g/L).

Water soluble exo-polysaccharide from Syncephalastrum racemosum, a strong inducer of plant defence reactions.

This study examines the production, characterization and bioactivity on plant cell cultured in vitro of exopolysaccharides (EPS) from Syncephalastrum racemosum CBS 443.59. Firstly, the influence of the fungus culture condition in shake flasks (pH, temperature and different carbon and nitrogen sources) on EPS and biomass production was evaluated. In order to enhance EPS production, a new protocol based on two-stage pH fermentation in a 3 L stirred fermentor was developed. Under this condition, EPS production increased by 3.55 times, compared to a constant pH process, reaching a maximal EPS concentration of 2.62 g/L. Structurally, the EPS contains a polyglucuronic acid backbone, linked essentially with mannose and fucose units and some galactose and glucose units. The bioactivity of EPS as inducer of defence reactions in plant suspension-cultured cells was also studied. Our results show, for first time, that EPS from S. racemosum CBS 443.59 induces, depending on the concentration, PAL activation and H2O2 synthesis in Arabidopsis thaliana cell suspensions.

Isolation of mutants and construction of intersexual heterokaryons of Blakeslea trispora.

The Mucoral fungus Blakeslea trispora is used for the industrial production of ß-carotene and lycopene. Two genetic techniques have been used to increase carotene accumulation: the isolation of mutants and the formation and segregation of heterokaryons. Because all life stages are multinucleated, recessive mutants are isolated after exposure to N-methyl-N'-nitro-N-nitrosoguanidine, a strong mutagen and inactivator of nuclei. Intersexual heterokaryons are obtained easily, because they are formed spontaneously during sexual interaction. Here are the pertaining methods, based on those previously developed for Phycomyces blakesleeanus, a related and better-known fungus.