Biocontrol potential and growth-promoting effect of endophytic fungus Talaromyces muroii SD1-4 against potato leaf spot disease caused by Alternaria alternata.

BACKGROUND: Alternaria alternata is the primary pathogen of potato leaf spot disease, resulting in significant potato yield losses globally. Endophytic microorganism-based biological control, especially using microorganisms from host plants, has emerged as a promising and eco-friendly approach for managing plant diseases. Therefore, this study aimed to isolate, identify and characterize the endophytic fungi from healthy potato leaves which had great antifungal activity to the potato leaf spot pathogen of A. alternata in vitro and in vivo. RESULTS: An endophytic fungal strain SD1-4 was isolated from healthy potato leaves and was identified as Talaromyces muroii through morphological and sequencing analysis. The strain SD1-4 exhibited potent antifungal activity against the potato leaf spot pathogen A. alternata Lill, with a hyphal inhibition rate of 69.19%. Microscopic and scanning electron microscope observations revealed that the strain SD1-4 grew parallel to, coiled around, shrunk and deformed the mycelia of A. alternata Lill. Additionally, the enzyme activities of chitinase and ß-1, 3-glucanase significantly increased in the hyphae of A. alternata Lill when co-cultured with the strain SD1-4, indicating severe impairment of the cell wall function of A. alternata Lill. Furthermore, the mycelial growth and conidial germination of A. alternata Lill were significantly suppressed by the aseptic filtrate of the strain SD1-4, with inhibition rates of 79.00% and 80.67%, respectively. Decrease of leaf spot disease index from 78.36 to 37.03 was also observed in potato plants treated with the strain SD1-4, along with the significantly increased plant growth characters including plant height, root length, fresh weight, dry weight, chlorophyll content and photosynthetic rate of potato seedlings. CONCLUSION: The endophyte fungus of T. muroii SD1-4 isolated from healthy potato leaves in the present study showed high biocontrol potential against potato leaf spot disease caused by A. alternata via direct parasitism or antifungal metabolites, and had positive roles in promoting potato plant growth.

Violet colonies of Talaromyces marneffei produce on CHROMagar candida medium.

In the present report, we describe an unusual case of mixed infection of Candida albicans and Talaromyces marneffei in the oral cavity and oropharynx with cutaneous involvement. On the CHROMagar Candida plate, green colonies (identified as C. albicans) and tiny violet colonies (identified as T. marneffei) grew from the throat swab after incubation for 96 hours. 10 clinical isolates of T. marneffei were used to verify their color production on CHROMagar Candida. All colonies were violet on the fourth, seventh and ninth day incubated at 37 °C. T. marneffei appears violet on the CHROMagar Candida plate, but it may be easily ignored because of its slow growth and small colony size, especially after incubation for 48 hours. Therefore, when using CHROMagar Candida plate to detect specimens in AIDS patients, special attention must be paid to detect non-yeasts such as T. marneffei for up to 96 hours.

Transcription in fungal conidia before dormancy produces phenotypically variable conidia that maximize survival in different environments.

Fungi produce millions of clonal asexual conidia (spores) that remain dormant until favourable conditions occur. Conidia contain abundant stable messenger RNAs but the mechanisms underlying the production of these transcripts and their composition and functions are unknown. Here, we report that the conidia of three filamentous fungal species (Aspergillus nidulans, Aspergillus fumigatus, Talaromyces marneffei) are transcriptionally active and can synthesize mRNAs. We find that transcription in fully developed conidia is modulated in response to changes in the environment until conidia leave the developmental structure. Environment-specific transcriptional responses can alter conidial content (mRNAs, proteins and secondary metabolites) and change gene expression when dormancy is broken. Conidial transcription affects the fitness and capabilities of fungal cells after germination, including stress and antifungal drug (azole) resistance, mycotoxin and secondary metabolite production and virulence. The transcriptional variation that we characterize in fungal conidia explains how genetically identical conidia mature into phenotypically variable conidia. We find that fungal conidia prepare for the future by synthesizing and storing transcripts according to environmental conditions present before dormancy.

OVAT Analysis and Response Surface Methodology Based on Nutrient Sources for Optimization of Pigment Production in the Marine-Derived Fungus Talaromyces albobiverticillius 30548 Submerged Fermentation.

Pigment production from filamentous fungi is gaining interest due to the diversity of fungal species, the variety of compounds synthesized, and the possibility of controlled massive productions. The Talaromyces species produce a large panel of metabolites, including Monascus-like azaphilone pigments, with potential use as natural colorants in industrial applications. Optimizing pigment production from fungal strains grown on different carbon and nitrogen sources, using statistical methods, is widespread nowadays. The present work is the first in an attempt to optimize pigments production in a culture of the marine-derived T. albobiverticillius 30548, under the influence of several nutrients sources. Nutrient combinations were screened through the one-variable-at-a-time (OVAT) analysis. Sucrose combined with yeast extract provided a maximum yield of orange pigments (OPY) and red pigments (RPY) (respectively, 1.39 g/L quinizarin equivalent and 2.44 g/L Red Yeast pigment equivalent), as well as higher dry biomass (DBW) (6.60 g/L). Significant medium components (yeast extract, K2HPO4 and MgSO4·7H2O) were also identified from one-variable-at-a-time (OVAT) analysis for pigment and biomass production. A five-level central composite design (CCD) and a response surface methodology (RSM) were applied to evaluate the optimal concentrations and interactive effects between selected nutrients. The experimental results were well fitted with the chosen statistical model. The predicted maximum response for OPY (1.43 g/L), RPY (2.59 g/L), and DBW (15.98 g/L) were obtained at 3 g/L yeast extract, 1 g/L K2HPO4, and 0.2 g/L MgSO4·7H2O. Such optimization is of great significance for the selection of key nutrients and their concentrations in order to increase the pigment production at a pilot or industrial scale.

CRISPR-Cas9-mediated seb1 disruption in Talaromyces pinophilus EMU for its enhanced cellulase production.

Filamentous fungi are working horses for industrial enzyme production. Combinatory approaches, such as random mutagenesis and rational genetic engineering, were adopted to improve their enzyme productivity. The filamentous fungus Talaromyces pinophilus EMU is a hyper cellulase-producing filamentous fungus obtained through random mutagenesis. This study further enhanced its cellulase production through the disruption of seb1 gene, which encodes Seb1, a transcription factor that binds to the stress response element (STRE) and regulates a variety of cellular processes. Gene seb1 was cloned from strain T. pinophilus EMU and disrupted using CRISPR-Cas9 technology. The seb1-disruptants (TpΔseb1 strains) showed distinct morphology from its parent strain. They presented a hyphal branching phenotype with decreased transcription levels of rhoA and ras1 genes involved in hyphal branching. Furthermore, TpΔseb1 strains displayed lower cell biomass, higher specific protein content, and 20%-40% enhancement in filter paper cellulase (FPase) activity, however, insignificant changes in the transcription levels of cbh1 and bgl1 genes involved in cellulase production. Through this study, we confirmed that seb1 gene disruption in T. pinophilus EMU caused more hyphal branching, reduced cell growth, increased protein secretion, and enhanced cellulase production. In addition, we successfully established the CRISPR-Cas9 genome-editing platform in T. pinophilus EMU.

Comparing thermal inactivation to a combined process of moderate heat and high pressure: Effect on ascospores in strawberry puree.

High pressure processing is a mild preservation process that inactivates pathogenic and spoilage micro-organisms in food products, but preserves the fresh characteristics of a product. Compared to untreated product, an enhanced shelf life is obtained during refrigerated storage. Knowledge on the use of high pressure pasteurisation aimed for ambient storage is limited. The aim of this research was to investigate if a combination of high pressure and moderate heat could be used to produce a shelf-stable high-acid fruit product. Ascospores of the heat resistant fungi Talaromyces macrosporus and Aspergillus fischeri were added to fresh strawberry puree that served as a model system. The effect of the processing steps and storage at ambient temperature for 2 weeks was studied on viability of the ascospores. A preheating step at 69 °C/2 min resulted in full or partial activation of A. fischeri and T. macrosporus spores, respectively. The pressure build-up by the process without any holding time resulted in additional activation of spores. A combination of moderate heat (maximum 85-90 °C) and high pressure (500-700 MPa) for holding times up to 13 min inactivated these highly resistant spores much faster than a heat treatment alone. At Tmax = 85 °C and 600 MPa the spores of T. macrosporus and A. fischeri were inactivated by 5.0 and 5.5 log10 after 13 and 7 min, respectively. At Tmax = 85 °C the heat treatment alone did not reduce the viability of these spores up to 60 min of treatment. At Tmax = 90 °C the holding time of the combined pressure-heat treatment could be reduced to obtain the same degree of inactivation of the heat resistant fungi. In addition, treated and untreated ascospores in strawberry puree were stored for 14 days at room temperature to evaluate delayed outgrowth of spores. Untreated ascospores of A. fischeri were activated by storage in the puree. However, at conditions combining high pressure ≥ 600 MPa with Tmax ≥ 85 °C for 13 min, heat resistant fungi were successfully inactivated. This research showed that a combination of moderate heat and pressure can drastically improve the effectiveness to inactivate heat-resistant ascospores in a high-acid fruit product compared to a heat treatment, potentially resulting in a better product quality.

Induction of secondary metabolite production by fungal co-culture of Talaromyces pinophilus and Paraphaeosphaeria sp.

Fungal co-culture is a strategy to induce the production of secondary metabolites by activating cryptic genes. We discovered the production of a new compound, talarodone A (1), along with five known compounds 2-6 in co-culture of Talaromyces pinophilus and Paraphaeosphaeria sp. isolated from soil collected in Miyazaki Prefecture, Japan. Among them, the productions of penicidones C (2) and D (3) were enhanced 27- and sixfold, respectively, by the co-culture. The structure of 3 should be represented as a γ-pyridol form with the reported chemical shifts, but not as a γ-pyridone form, based on DFT calculation.

Laboratory Maintenance and Growth of Talaromyces marneffei.

Talaromyces marneffei is an important opportunistic human pathogen endemic to Southeast Asia. It is one of a number of pathogenic fungi that exhibits thermally controlled dimorphism. At 25°C, T. marneffei grows in a multicellular, filamentous hyphal form that can differentiate to produce dormant spores called conidia. These conidia are the likely infectious agent. At 37°C, T. marneffei grows as a uninucleate yeast that divides by fission. The yeast cells are the pathogenic form of this fungus. The protocols described here explain how to grow T. marneffei in the two vegetative growth forms in vitro, grow yeast cells inside mammalian macrophages, produce conidial stocks, and store strains both short and long term. © 2020 by John Wiley & Sons, Inc. Basic Protocol 1: Growth of the vegetative hyphal form on solid medium Alternate Protocol 1: Growth of the vegetative hyphal form in liquid suspension Basic Protocol 2: Growth of the vegetative yeast form on solid medium Alternate Protocol 2: Growth of the vegetative yeast form in liquid suspension Basic Protocol 3: Growth for production of dormant conidia Support Protocol: Preparation of Miracloth filter tubes Basic Protocol 4: Growth of Talaromyces marneffei in mammalian macrophages Basic Protocol 5: Storage of Talaromyces marneffei strains Alternate Protocol 3: Lyophilization of Talaromyces marneffei strains.

Natural trypanocidal product produced by endophytic fungi through co-culturing.

Endophytic fungi live inside vegetal tissues without causing damage to the host plant and may provide lead compounds for drug discovery. The co-culture of two or more endophytic fungi can trigger silent gene clusters, which could lead to the isolation of bioactive compounds. In this study, two endophytic strains isolated from Handroanthus impetiginosus leaves, identified as Talaromyces purpurogenus H4 and Phanerochaete sp. H2, were grown in mixed and axenic cultures. The meroterpenoid austin was detected only in the extracts from the mixed culture. Once isolated, austin displayed very interesting trypanocidal activity, with an IC50 value of 36.6 ± 1.2 µg/mL against Trypanosoma cruzi in the epimastigote form. The results obtained highlight the importance of the co-culturing of endophytic fungi to obtain natural bioactive products. The findings also enhance our understanding of the ecological relationships between endophytic fungi.

Methylcitrate cycle gene MCD is essential for the virulence of Talaromyces marneffei.

Talaromyces marneffei (T. marneffei), which used to be known as Penicillium marneffei, is the causative agent of the fatal systemic mycosis known as talaromycosis. For the purpose of understanding the role of methylcitrate cycle in the virulence of T. marneffei, we generated MCD deletion (ΔMCD) and complementation (ΔMCD+) mutants of T. marneffei. Growth in different carbon sources showed that ΔMCD cannot grow on propionate media and grew slowly on the valerate, valine, methionine, isoleucine, cholesterol, and YNB (carbon free) media. The macrophage killing assay showed that ΔMCD was attenuated in macrophages of mice in vitro, especially at the presence of propionate. Finally, virulence studies in a murine infection experiment revealed attenuated virulence of the ΔMCD, which indicates MCD is essential for T. marneffei virulence in the host. This experiment laid the foundation for the further study of the specific mechanisms underlying the methylcitrate cycle of T. marneffei and may provide suitable targets for new antifungals.

Unique processes yielding pure azaphilones in Talaromyces atroroseus.

Azaphilones are a class of fungal pigments, reported mostly in association with Monascus species. In Asian countries, they are used as food colourants under the name of "red yeast rice" and their production process is well described. One major limitation of current production techniques of azaphilones is that they always occur in a mixture of yellow, orange and red pigments. These mixtures are difficult to control and to quantify. This study has established a controlled and reproducible cultivation protocol to selectively tailor production of individual pigments during a submerged fermentation using another fungal species capable of producing azaphilone pigments, Talaromyces atroroseus, using single amino acids as the sole nitrogen source. The produced azaphilone pigments are called atrorosins and are amino acid derivatives of the known azaphilone pigment Penicillium purpurogenum-orange (PP-O), with the amino acid used as nitrogen source incorporated into the core skeleton of the azaphilone. This strategy was successfully demonstrated using 18 proteinogenic amino acids and the non-proteinogenic amino acid ornithine. Two cultivation methods for production of the pure serine derivative (atrorosin S) have been further developed, with yields of 0.9 g/L being obtained. Yielding pure atrorosins through switching from KNO3 to single amino acids as nitrogen source allows for considerably easier downstream processing and thus further enhances the commercial relevance of azaphilone producing fungal cell factories.

Atrorosins: a new subgroup of Monascus pigments from Talaromyces atroroseus.

A new series of azaphilone pigments named atrorosins have been isolated from the filamentous fungus Talaromyces atroroseus. Atrorosins have a similar azaphilone scaffold as the orange Monascus pigment PP-O, with a carboxylic acid group at C-1, but are unique by their incorporation of amino acids into the isochromene system. Despite that the atrorosin precursor PP-O, during fermentation, was initially produced as two isomers (3:2, cis:trans ratio), the atrorosins were surprisingly almost exclusively (99.5%) produced as the cis-form, possibly due to steric interactions with the incorporated amino acid. When grown on complex media, a whole range of atrorosins is produced, whereas individual atrorosins can be produced selectively during fermentation by supplementing with the desired primary amine-containing compound.

Effects of UV Irradiation on Penicillium Strains Isolated from a Bread Plant and the Application to Bakery Products.

We tested treatement with UV irradiation for controlling the growth of bread mold. First, we analyzed the sterilizing effect of a dose of approximately 25 mJ/cm2 radiation on nine Penicillium and two Talaromyces strains that were isolated from a bread-manufacturing plant. The P. chermesinum and P. paneum strains were sterilized completely at that dose, while it was only partially effective against P. corylophilum. P. chrysogenum and P. decumbens were sterilized at a dose of approximately 120 mJ/cm2, while T. amestolkiae was sterilized at approximately 150 mJ/cm2. Sterilization of T. cecidicola and P. hispanicum required more than 200 mJ/cm2 of radiation. These results suggest that UV resistance varies depending on the species and the strains. We also carried out UV irradiation of bread at 70 mJ/cm2: a dose at which the taste of bread is not affected; we observed that mold growth was delayed visibly compared to the non-irradiated bread. These results suggest that UV irradiation at 70 mJ/cm2 is effective at delaying mold growth, though it does not cause complete sterilization. This method should prove useful for extending the shelf-life of bread.

The Talaromyces pinophilus species complex.

A sample of isolates from Talaromyces pinophilus (55 isolates) and closely related species (76 isolates) was sequenced at four loci, the data were analyzed using maximum likelihood analysis and the GCPSR. The isolates were subjected to growth studies on the recommended media for description of Talaromyces species. On the basis of the combined data, five new species were segregated out of T. pinophilus and placed in newly described species. The T. pinophilus species complex contains ten species. The three other new species, Talaromyces argentinensis, T. californicus and T. louisianensis were not a part of the T. pinophilus species complex but occurred in Talaromyces sect. Talaromyces. T. argentinensis produces a teleomorphic state and is phylogenetically and morphologically distinct from other Talaromyces species.

A unique aspartyl protease gene expansion in Talaromyces marneffei plays a role in growth inside host phagocytes.

Aspartyl proteases are a widely represented class of proteolytic enzymes found in eukaryotes and retroviruses. They have been associated with pathogenicity in a range of disease-causing microorganisms. The dimorphic human-pathogenic fungus Talaromyces marneffei has a large expansion of these proteases identified through genomic analyses. Here we characterize the expansion of these genes (pop - paralogue of pep) and their role in T. marneffei using computational and molecular approaches. Many of the genes in this monophyletic family show copy number variation and positive selection despite the preservation of functional regions and possible redundancy. We show that the expression profile of these genes differs and some are expressed during intracellular growth in the host. Several of these proteins have distinctive localization as well as both additive and epistatic effects on the formation of yeast cells during macrophage infections. The data suggest that this is a recently evolved aspartyl protease gene family which affects intracellular growth and contributes to the pathogenicity of T. marneffei.

Feasibility studies with lignin blocking additives in enhancing saccharification and cellulase recovery: Mutant UV-8 of T. verruculosus IIPC 324 a case study.

The process economics of fermentable sugar production is dependent on the performance of cellulase cocktail on realistic lignocellulosic biomass and their capability to be recovered and recycled. Feasibility studies were conducted to enhance the digestibility of acid pretreated sugarcane bagasse using novel cellulase cocktail obtained from stable mutant UV-8 of Talaromyces verruculosus IIPC 324 in presence of lignin blocking additives. PEG 6000 was shortlisted as the best additive as it could simultaneously enhance saccharification and overall cellulase recoveries namely cellobiohydrolase, endoglucanase and cellobiase. Addition of 0.3 g PEG 6000/g acid-insoluble lignin content, resulted in 55% and 49.2% saccharification yields in terms of reducing sugars and glucose respectively using this cellulase cocktail (25 mg protein/g cellulose content) after 72 h from acid pretreated sugarcane bagasse loaded at 7.5%. The study also suggested that the endoglucanase of this mutant was unique with high desorption capability as 85% activity was observed in the saccharified broth devoid of any lignin blocking additive. At its optimum concentration, PEG 6000 was able to retain 94 ± 0.79% cellobiohydrolase I and 97.97 ± 1.16% cellobiase enzyme in the saccharified broth which were otherwise lost in residual biomass by ∼80%, in the absence of this polymeric additive. These results suggest that PEG 6000 was the most promising facilitator for recycling of cellulases obtained from mutant UV-8 of Talaromyces verruculosus IIPC 324 in particular. It paved a way towards the production of cheaper fermentable sugars which serve as a starting raw material for the production of green chemicals and fuels.

Inactivation of stress-resistant ascospores of Eurotiales by industrial sanitizers.

Different fungi, including the genera Aspergillus (Neosartorya), Paecilomyces (Byssochlamys) and Talaromyces, produce (asco)spores that survive pasteurization treatments and are regarded as the most stress-resistant eukaryotic cells. The sensitivity of the ascospores to treatments with industrial sanitizers containing chlorine dioxide and iodine (iodophors) has never been assessed before. Ascospores of 4 species of Eurotiales were tested and showed clear variations in sensitivity. The most resilient species, T. macrosporus and Pae. variotii (=B. spectabilis) survive 75, but not 200 ppm chlorine dioxide solution treatments. These species were able to survive 75 ppm iodine solution treatments, but relatively low amounts of ascospores (100-1000 spores) could be inactivated after 16 h of treatment. Inactivated spores did not show any sign of germination after 7 days following treatment on growth medium. As judged by microscopy, iodine inactivation resulted in visibly distorted ascospores. For the interpretation of results, the state of dormancy or activation of ascospores is highly important.

Role of autophagy in regulating the immune response of dendritic cells to Talaromyces marneffei infection.

Autophagy can regulate antimicrobial immunity. However, it is unknown whether autophagy mediates the immune response of dendritic cells (DCs) to Talaromyces marneffei (T. marneffei) infection. Therefore, to explore the relationship between autophagy and multiplication of T. marneffei and investigate whether ERK1/2 signaling pathway regulates activation of autophagy and TNF-α and IFN-γ secretion by intracellular signaling mechanisms during T. marneffei infection in human DCs. DCs were infected with T. marneffei for different times. First, we found that T. marneffei induced activation of autophagy and ERK1/2 in human DCs. Second, the inhibition of ERK1/2 suppressed activation of autophagy in T. marneffei-infected human DCs. Third, the suppression of ERK1/2 and autophagy decreased TNF-α and IFN-γ production and increased the proliferation of T. marneffei. These data suggest that ERK pathway plays vital regulatory roles in activation of autophagy and subsequent cytokine production during T. marneffei infection. Our data further indicate that autophagy is important in the regulation of the DC immune response to T. marneffei infection, thereby extending our understanding of host immune responses to the fungus.