Transcriptomics analysis and fed-batch regulation of high astaxanthin-producing Phaffia rhodozyma/Xanthophyllomyces dendrorhous obtained through adaptive laboratory evolution.

Astaxanthin has high utilization value in functional food because of its strong antioxidant capacity. However, the astaxanthin content of Phaffia rhodozyma is relatively low. Adaptive laboratory evolution is an excellent method to obtain high-yield strains. TiO2 is a good inducer of oxidative stress. In this study, different concentrations of TiO2 were used to domesticate P. rhodozyma, and at a concentration of 1000 mg/L of TiO2 for 105 days, the optimal strain JMU-ALE105 for astaxanthin production was obtained. After fermentation, the astaxanthin content reached 6.50 mg/g, which was 41.61% higher than that of the original strain. The ALE105 strain was fermented by batch and fed-batch, and the astaxanthin content reached 6.81 mg/g. Transcriptomics analysis showed that the astaxanthin synthesis pathway, and fatty acid, pyruvate, and nitrogen metabolism pathway of the ALE105 strain were significantly upregulated. Based on the nitrogen metabolism pathway, the nitrogen source was adjusted by ammonium sulphate fed-batch fermentation, which increased the astaxanthin content, reaching 8.36 mg/g. This study provides a technical basis and theoretical research for promoting industrialization of astaxanthin production of P. rhodozyma. ONE-SENTENCE SUMMARY: A high-yield astaxanthin strain (ALE105) was obtained through TiO2 domestication, and its metabolic mechanism was analysed by transcriptomics, which combined with nitrogen source regulation to further improve astaxanthin yield.

Characteristics of biological control and mechanisms of Pseudomonas chlororaphis zm-1 against peanut stem rot.

BACKGROUND: Peanut stem rot is a serious plant disease that causes great economic losses. At present, there are no effective measures to prevent or control the occurrence of this plant disease. Biological control is one of the most promising plant disease control measures. In this study, Pseudomonas chlororaphis subsp. aurantiaca strain zm-1, a bacterial strain with potential biocontrol properties isolated by our team from the rhizosphere soil of Anemarrhena asphodeloides, was studied to control this plant disease. METHODS: We prepared extracts of Pseudomonas chloroaphis zm-1 extracellular antibacterial compounds (PECEs), determined their antifungal activities by confrontation assay, and identified their components by UPLC-MS/MS. The gene knockout strains were constructed by homologous recombination, and the biocontrol efficacy of P. chlororaphis zm-1 and its mutant strains were evaluated by pot experiments under greenhouse conditions and plot experiments, respectively. RESULTS: P. chlororaphis zm-1 could produce extracellular antifungal substances and inhibit the growth of Sclerotium rolfsii, the main pathogenic fungus causing peanut stem rot. The components of PECEs identified by UPLC-MS/MS showed that three kinds of phenazine compounds, i.e., 1-hydroxyphenazine, phenazine-1-carboxylic acid (PCA), and the core phenazine, were the principal components. In particular, 1-hydroxyphenazine produced by P. chlororaphis zm-1 showed antifungal activities against S. rolfsii, but 2-hydroxyphenazine did not. This is quite different with the previously reported. The extracellular compounds of two mutant strains, ΔphzH and ΔphzE, was analysed and showed that ΔphzE did not produce any phenazine compounds, and ΔphzH no longer produced 1-hydroxyphenazine but could still produce PCA and phenazine. Furthermore, the antagonistic ability of ΔphzH declined, and that of ΔphzE was almost completely abolished. According to the results of pot experiments under greenhouse conditions, the biocontrol efficacy of ΔphzH dramatically declined to 47.21% compared with that of wild-type P. chlororaphis zm-1 (75.63%). Moreover, ΔphzE almost completely lost its ability to inhibit S. rolfsii (its biocontrol efficacy was reduced to 6.19%). The results of the larger plot experiments were also consistent with these results. CONCLUSIONS: P. chlororaphis zm-1 has the potential to prevent and control peanut stem rot disease. Phenazines produced and secreted by P. chlororaphis zm-1 play a key role in the control of peanut stem rot caused by S. rolfsii. These findings provide a new idea for the effective prevention and treatment of peanut stem rot.

The ectomycorrhizal fungus Pisolithus microcarpus encodes a microRNA involved in cross-kingdom gene silencing during symbiosis.

Small RNAs (sRNAs) are known to regulate pathogenic plant-microbe interactions. Emerging evidence from the study of these model systems suggests that microRNAs (miRNAs) can be translocated between microbes and plants to facilitate symbiosis. The roles of sRNAs in mutualistic mycorrhizal fungal interactions, however, are largely unknown. In this study, we characterized miRNAs encoded by the ectomycorrhizal fungus Pisolithus microcarpus and investigated their expression during mutualistic interaction with Eucalyptus grandis. Using sRNA sequencing data and in situ miRNA detection, a novel fungal miRNA, Pmic_miR-8, was found to be transported into E. grandis roots after interaction with P. microcarpus Further characterization experiments demonstrate that inhibition of Pmic_miR-8 negatively impacts the maintenance of mycorrhizal roots in E. grandis, while supplementation of Pmic_miR-8 led to deeper integration of the fungus into plant tissues. Target prediction and experimental testing suggest that Pmic_miR-8 may target the host NB-ARC domain containing transcripts, suggesting a potential role for this miRNA in subverting host signaling to stabilize the symbiotic interaction. Altogether, we provide evidence of previously undescribed cross-kingdom sRNA transfer from ectomycorrhizal fungi to plant roots, shedding light onto the involvement of miRNAs during the developmental process of mutualistic symbioses.

Application of CRISPR/Cas9 Tools for Genome Editing in the White-Rot Fungus Dichomitus squalens.

Dichomitus squalens is an emerging reference species that can be used to investigate white-rot fungal plant biomass degradation, as it has flexible physiology to utilize different types of biomass as sources of carbon and energy. Recent comparative (post-) genomic studies on D. squalens resulted in an increasingly detailed knowledge of the genes and enzymes involved in the lignocellulose breakdown in this fungus and showed a complex transcriptional response in the presence of lignocellulose-derived compounds. To fully utilize this increasing amount of data, efficient and reliable genetic manipulation tools are needed, e.g., to characterize the function of certain proteins in vivo and facilitate the construction of strains with enhanced lignocellulolytic capabilities. However, precise genome alterations are often very difficult in wild-type basidiomycetes partially due to extremely low frequencies of homology directed recombination (HDR) and limited availability of selectable markers. To overcome these obstacles, we assessed various Cas9-single guide RNA (sgRNA) ribonucleoprotein (RNP) -based strategies for selectable homology and non-homologous end joining (NHEJ) -based gene editing in D. squalens. We also showed an induction of HDR-based genetic modifications by using single-stranded oligodeoxynucleotides (ssODNs) in a basidiomycete fungus for the first time. This paper provides directions for the application of targeted CRISPR/Cas9-based genome editing in D. squalens and other wild-type (basidiomycete) fungi.

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

BACKGROUND: 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. METHODS AND RESULTS: 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. CONCLUSION: 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.

Crowdsourced analysis of fungal growth and branching on microfluidic platforms.

Fungal hyphal growth and branching are essential traits that allow fungi to spread and proliferate in many environments. This sustained growth is essential for a myriad of applications in health, agriculture, and industry. However, comparisons between different fungi are difficult in the absence of standardized metrics. Here, we used a microfluidic device featuring four different maze patterns to compare the growth velocity and branching frequency of fourteen filamentous fungi. These measurements result from the collective work of several labs in the form of a competition named the "Fungus Olympics." The competing fungi included five ascomycete species (ten strains total), two basidiomycete species, and two zygomycete species. We found that growth velocity within a straight channel varied from 1 to 4 µm/min. We also found that the time to complete mazes when fungal hyphae branched or turned at various angles did not correlate with linear growth velocity. We discovered that fungi in our study used one of two distinct strategies to traverse mazes: high-frequency branching in which all possible paths were explored, and low-frequency branching in which only one or two paths were explored. While the high-frequency branching helped fungi escape mazes with sharp turns faster, the low-frequency turning had a significant advantage in mazes with shallower turns. Future work will more systematically examine these trends.

A robust KASP marker for selection of four pairs of linked leaf rust and stripe rust resistance genes introgressed on chromosome arm 5DS from different wheat genomes.

Stripe rust and leaf rust are among the most devastating diseases of wheat, limiting its production globally. Wheat wild relatives harbour genetic diversity for new genes and alleles for all major wheat diseases. However, the use of this genetic variation from wild progenitor and non-progenitor species has been limited in the breeding programs. Reasons include limited recombination of donor and recipient genomes and the lack of tertiary gene pool markers. Here, we describe the development of a SNP based marker from the flow-sorted and sequenced Aegilops umbellulata chromosome 5U which can be used for marker assisted selection of four pair of alien leaf rust and stripe rust resistance genes. Lr57-Yr40_CAPS16 marker was reported earlier to be linked with alien leaf and stripe rust resistance genes introgressed on wheat chromosome 5DS. Due to its dominant nature and laborious to work with, a new SNP-based KASP marker, XTa5DS-2754099_kasp23, was developed from the same CAPS marker contig. XTa5DS-2754099_kasp23 was tested in Aegilops umbellulata, Ae. geniculata, Ae. peregrina and Ae. caudata derived alien introgression lines, which harbour four pairs of linked leaf and stripe rust genes; Lr76-Yr70, Lr57-Yr40, LrP- YrP, LrAc-YrAc, respectively. This KASP marker was found to be effective for the selection of the aforesaid four pairs of leaf rust and stripe rust resistance genes. Further, we tested and validated XTa5DS-2754099_kasp23 on commercial varieties and advanced breeding lines from four countries (India, Egypt, Australia and UK) including hexaploid and durum wheat. Our results provide evidence that KASP marker, XTa5DS-2754099_kasp23 can be used in marker-assisted selection of the four pairs of rust resistance alien genes in wheat breeding programmes.

The Endophytic Fungus Piriformospora indica Reprograms Banana to Cold Resistance.

Banana (Musa spp.), one of the most important fruits worldwide, is generally cold sensitive. In this study, by using the cold-sensitive banana variety Tianbaojiao (Musa acuminate) as the study material, we investigated the effects of Piriformospora indica on banana cold resistance. Seedlings with and without fungus colonization were subjected to 4 °C cold treatment. The changes in plant phenotypes, some physiological and biochemical parameters, chlorophyll fluorescence parameters, and the expression of eight cold-responsive genes in banana leaves before and after cold treatment were measured. Results demonstrated that P. indica colonization reduced the contents of malondialdehyde (MDA) and hydrogen peroxide (H2O2) but increased the activities of superoxide dismutase (SOD) and catalase (CAT) and the contents of soluble sugar (SS) and proline. Noteworthily, the CAT activity and SS content in the leaves of P. indica-colonized banana were significant (p < 0.05). After 24 h cold treatment, the decline in maximum photochemistry efficiency of photosystem II (Fv/Fm), photochemical quenching coefficient (qP), efficient quantum yield [Y(II)], and photosynthetic electron transport rate (ETR) in the leaves of P. indica-colonized banana was found to be lower than in the non-inoculated controls (p < 0.05). Moreover, although the difference was not significant, P. indica colonization increased the photochemical conversion efficiency and electron transport rate and alleviated the damage to the photosynthetic reaction center of banana leaves under cold treatment to some extent. Additionally, the expression of the most cold-responsive genes in banana leaves was significantly induced by P. indica during cold stress (p < 0.05). It was concluded that P. indica confers banana with enhanced cold resistance by stimulating antioxidant capacity, SS accumulation, and the expression of cold-responsive genes in leaves. The results obtained from this study are helpful for understanding the P. indica-induced cold resistance in banana.

Characterization of chitin-glucan complex from Tremella fuciformis fermentation residue and evaluation of its antibacterial performance.

Submerged fermentation of fungi is an efficient way to obtain extracellular polysaccharides, however, in this process, excess discarded biomass is produced. In this study, Tremella fuciformis mycelia were reused as the raw material to isolate a novel fungal chitin-glucan complex (CGC-TFM) using alkaline extraction. Characteristic analysis revealed that the CGC-TFM consisted of glucosamine/acetylglucosamine and glucose (GlcN:Glc = 26:74 mol%), indicating a reference to the ß polymorphism of chitin-glucan complex, with the molecular weight and crystallinity index of 256 ± 3.0 kDa and 54.25 ± 1.04%, respectively. Fourier transform infrared spectroscopy, X-ray diffraction, nuclear magnetic resonance, and scanning electron microscopy analyses confirmed that the chitin portion of the CGC-TFM exhibited a typical ß configuration and N-acetylation degree of 70.52 ± 2.09%. Furthermore, the CGC-TFM exhibited good thermal stability and effective Escherichia coli inhibition ability, indicating that it could be applied as a potential food packaging material.

A cell surface-exposed protein complex with an essential virulence function in Ustilago maydis.

Plant pathogenic fungi colonizing living plant tissue secrete a cocktail of effector proteins to suppress plant immunity and reprogramme host cells. Although many of these effectors function inside host cells, delivery systems used by pathogenic bacteria to translocate effectors into host cells have not been detected in fungi. Here, we show that five unrelated effectors and two membrane proteins from Ustilago maydis, a biotrophic fungus causing smut disease in corn, form a stable protein complex. All seven genes appear co-regulated and are only expressed during colonization. Single mutants arrest in the epidermal layer, fail to suppress host defence responses and fail to induce non-host resistance, two reactions that likely depend on translocated effectors. The complex is anchored in the fungal membrane, protrudes into host cells and likely contacts channel-forming plant plasma membrane proteins. Constitutive expression of all seven complex members resulted in a surface-exposed form in cultured U. maydis cells. As orthologues of the complex-forming proteins are conserved in smut fungi, the complex may become an interesting fungicide target.

Modelling the motion of organelles in an elongated cell via the coordination of heterogeneous drift-diffusion and long-range transport.

 Cellular distribution of organelles in living cells is achieved via a variety of transport mechanisms, including directed motion, mediated by molecular motors along microtubules (MTs), and diffusion which is predominantly heterogeneous in space. In this paper, we introduce a model for particle transport in elongated cells that couples poleward drift, long-range bidirectional transport and diffusion with spatial heterogeneity in a three-dimensional space. Using stochastic simulations and analysis of a related population model, we find parameter regions where the three-dimensional model can be reduced to a coupled one-dimensional model or even a one-dimensional scalar model. We explore the efficiency with which individual model components can overcome drift towards one of the cell poles to reach an approximately even distribution. In particular, we find that if lateral movement is well mixed, then increasing the binding ability of particles to MTs is an efficient way to overcome a poleward drift, whereas if lateral motion is not well mixed, then increasing the axial diffusivity away from MTs becomes an efficient way to overcome the poleward drift. Our three-dimensional model provides a new tool that will help to understand the mechanisms by which eukaryotic cells organize their organelles in an elongated cell, and in particular when the one-dimensional models are applicable.

Cost-effective optimized scleroglucan production by Sclerotium rolfsii ATCC 201126 at bioreactor scale. A quantity-quality assessment.

Exopolysaccharide (EPS) secretion by Sclerotium rolfsii ATCC 201126 in submerged cultures, already identified as high-osmolarity responsive, was assessed by reducing C-source without compromising EPS yields. A designed medium with 80 g sucrose L-1 (MOPT80) was tested at 3 L-bioreactor scale at different temperature, agitation, aeration and pH (uncontrolled vs. controlled) values. Optimal operative conditions (200 rpm, 28 °C, 0.5 vvm and initial pH -pHi- 4.5) were validated, as well as the possibility to work at pHi 5.5 to reduce biomass production. Purified EPSs produced in MOPT80 at optimal and other valid operative conditions exhibited refined grade (<1 % proteins and ash, 3-4 % reducing sugars, 87-99 % total sugars). EPS purity, MW and rheological parameters led to discourage pH controlled at 4.5. Relatively constant MW (6-8 × 106 Da) and outstanding viscosifying ability were found. Polyphasic EPS analysis (titre, purity, macromolecular features and rheological fitness) would support to properly select production conditions.

Differences in aluminum tolerance and immobilization between two indigenous ectomycorrhizal fungi Lactarius deliciosus and Pisolithus tinctorius from Southwest China's forest stands.

Aluminum (Al) toxicity severely decreases plant growth and productivity in acidic soil globally. Ectomycorrhizal (ECM) fungi can promote host plant's Al-tolerance by acting as a physical barrier or bio-filter. However, little information is available on the role of ECM fungus on Al immobilization with respect to Al-tolerance. This present study aimed to screen a promising indigenous ECM fungus with high Al-tolerance and to understand its role in Al immobilization related to Al-tolerance. Two ECM fungal strains (Lactarius deliciosus 2 and Pisolithus tinctorius 715) isolated from forest stands in Southwest China were cultured in vitro with 0.0, 1.0 or 2.0 mM Al addition for 21 days to compare their Al accumulation and Al-tolerance. Meanwhile, fungal mycelia were incubated in 0.037 mM Al3+ solutions, and then Al3+ concentrations in the solution were determined at time 2, 5, 10, 20, 40, 60, 120, 180, and 240 min, and the Al3+ immobilization characteristics were evaluated using the pseudo-first order, pseudo-second order and intraparticle diffusion models. Results showed that 1.0 or 2.0 mM Al3+ addition significantly increased fungal biomass production by 23% or 41% in L. deliciosus 2, not in P. tinctorius 715. Fungal Al3+ concentrations in L. deliciosus 2 and P. tinctorius 715 were significantly increased by 293% and 103% under 2.0 mM than under 1.0 mM Al3+ addition. The pH values in the culture solution were significantly decreased by 0.43 after 21 d fungus growth but no changes between these two fungi under the same Al3+ addition. Fungal Al3+ immobilization showed a three-stage trend with initially a rapid rate followed a relatively slower rate until reaching equilibrium. The pseudo-second order model was the best (R2 = 0.98 and 0.99 for L. deliciosus 2 and P. tinctorius 715) to fit the experimentally observed data among the three models. Compared to P. tinctorius 715, L. deliciosus 2 also had greater intercept value, cation exchange capacity (CEC), and extracellular Al3+ proportion in fungal mycelia. Additionally, bio-concentration on Al3+, active site numbers for Al3+, boundary layer thickness, CEC, and immobilization on the cell wall in fungal mycelia were involved in ECM fungal Al-tolerance. These results show that both ECM fungi are Al-tolerant while L. deliciosus 2 is a promising indigenous ECM isolate with higher Al-tolerance in Southwest China, and they can be hence applied to the afforestation and ecological restoration in acidic soil.

Synthesis of silver nanoparticles using white-rot fungus Anamorphous Bjerkandera sp. R1: influence of silver nitrate concentration and fungus growth time.

Currently, silver nanoparticles (AgNPs) constitute an interesting field of study in medicine, catalysis, optics, among others. For this reason, it has been necessary to develop new methodologies that allow a more efficient production of AgNPs with better antimicrobial and biological properties. In this research growth time effects Anamorphous Bjerkandera sp. R1 and the silver nitrate (AgNO3) concentration over AgNPs synthesis were studied. Through the protocol used in this work, it was found that the action of the capping proteins on the surface of the mycelium played a determining role in the reduction of the Ag+ ion to Ag0 nanoparticles producing a particle size that oscillated between 10 and 100 nm. The progress of the reaction was monitored using visible UV-Vis spectroscopy and the synthesized AgNPs were characterized by scanning electron microscopy (SEM), transmission electron microscopy (TEM) and Fourier transform infrared radiation (FTIR) spectroscopy. The best synthetic properties were found at 1 mM of AgNO3 concentration, growth time of 8 days, and reaction time of 144 h. Nanometals obtention from microorganisms could be considered as a new method of synthesis, due to reducing abilities of metal ions through its enzymatic system and represents low-cost synthesis that reduces the generation of harmful toxic wastes.

Optimization of culture medium for Sanghuangporus vaninii and a study on its therapeutic effects on gout.

The increasing incidence of gout poses a very challenging management problem. However, the currently available drugs often have various toxic side effects. As a traditional edible and medicinal macrofungus, Sanghuangporus vaninii presents high medical research value. Therefore, to improve fermentation efficiency and identify novel anti-gout drugs, we optimized the culture medium of S. vaninii with lignin and further investigated its anti-gout effects. The results indicated that 0.06 g/L of lignin was most favorable for S. vaninii growth. In the hyperuricemia cell model, we found that S. vaninii could significantly induce the downregulation of xanthine oxidoreductase and the upregulation of hypoxanthine-guanine phosphoribosyltransferase. Furthermore, following oral administration of the extracts, the serum uric acid levels of mice with hyperuricemia were effectively reduced. In a gouty arthritis rat model, S. vaninii also achieved strong suppression of synovial swelling, indicating its anti-inflammatory activity. In addition, the antioxidant assays suggested that S. vaninii shows a strong free radical scavenging capacity and can effectively alleviate cellular oxidative stress. This activity further enhances its anti-inflammatory activity and reduces the incidence of comorbidities. In summary, our results provide the basis for the utilization of S. vaninii to develop anti-gout drugs.

Microwave-Assisted One-Pot Lipid Extraction and Glycolipid Production from Oleaginous Yeast Saitozyma podzolica in Sugar Alcohol-Based Media.

Glycolipids are non-ionic surfactants occurring in numerous products of daily life. Due to their surface-activity, emulsifying properties, and foaming abilities, they can be applied in food, cosmetics, and pharmaceuticals. Enzymatic synthesis of glycolipids based on carbohydrates and free fatty acids or esters is often catalyzed using certain acyltransferases in reaction media of low water activity, e.g., organic solvents or notably Deep Eutectic Systems (DESs). Existing reports describing integrated processes for glycolipid production from renewables use many reaction steps, therefore this study aims at simplifying the procedure. By using microwave dielectric heating, DESs preparation was first accelerated considerably. A comparative study revealed a preparation time on average 16-fold faster than the conventional heating method in an incubator. Furthermore, lipids from robust oleaginous yeast biomass were successfully extracted up to 70% without using the pre-treatment method for cell disruption, limiting logically the energy input necessary for such process. Acidified DESs consisting of either xylitol or sorbitol and choline chloride mediated the one-pot process, allowing subsequent conversion of the lipids into mono-acylated palmitate, oleate, linoleate, and stearate sugar alcohol esters. Thus, we show strong evidence that addition of immobilized Candida antarctica Lipase B (Novozym 435®), in acidified DES mixture, enables a simplified and fast glycolipid synthesis using directly oleaginous yeast biomass.

The damage and tolerance mechanisms of Phaffia rhodozyma mutant strain MK19 grown at 28 °C.

BACKGROUND: Phaffia rhodozyma has many desirable properties for astaxanthin production, including rapid heterotrophic metabolism and high cell densities in fermenter culture. The low optimal temperature range (17-21 °C) for cell growth and astaxanthin synthesis in this species presents an obstacle to efficient industrial-scale astaxanthin production. The inhibition mechanism of cell growth at > 21 °C in P. rhodozyma have not been investigated. RESULTS: MK19, a mutant P. rhodozyma strain grows well at moderate temperatures, its cell growth was also inhibited at 28 °C, but such inhibition was mitigated, and low biomass 6 g/L was obtained after 100 h culture. Transcriptome analysis indicated that low biomass at 28 °C resulted from strong suppression of DNA and RNA synthesis in MK19. Growth inhibition at 28 °C was due to cell membrane damage with a characteristic of low mRNA content of fatty acid (f.a.) pathway transcripts (acc, fas1, fas2), and consequent low f.a. CONTENT: Thinning of cell wall and low mannose content (leading to loss of cell wall integrity) also contributed to reduced cell growth at 28 °C in MK19. Levels of astaxanthin and ergosterol, two end-products of isoprenoid biosynthesis (a shunt pathway of f.a. biosynthesis), reached 2000 µg/g and 7500 µg/g respectively; ~2-fold higher than levels at 21 or 25 °C. Abundance of ergosterol, an important cell membrane component, compensated for lack of f.a., making possible the biomass production of 6 g/L for MK19 at 28 °C. CONCLUSIONS: Inhibition of growth of P. rhodozyma at 28 °C results from blocking of DNA, RNA, f.a., and cell wall biosynthesis. In MK19, abundant ergosterol made possible biomass production 6 g/L at 28 °C. Significant accumulation of astaxanthin and ergosterol indicated an active MVA pathway in MK19 at 28 °C. Strengthening of the MVA pathway can be a feasible metabolic engineering approach for enhancement of astaxanthin synthesis in P. rhodozyma. The present findings provide useful mechanistic insights regarding adaptation of P. rhodozyma to 28 °C, and improved understanding of feasible metabolic engineering techniques for industrial scale astaxanthin production by this economically important yeast species.

Investigation on mycelial growth requirements of Cantharellus cibarius under laboratory conditions.

The golden chanterelle represents one of the commonly found, edible mushrooms that is highly valued in various cuisines. The present study focused on assessing the requirements of Cantharellus cibarius such as pH, temperature, as well as the carbon and nitrogen sources for mycelial growth. Optimization of the growth parameters was carried out by one-factor-at-a-time method. The optimal pH and temperature were determined to be 6.0 and 22.5 °C, respectively. Among the various carbon sources studied, sucrose at a concentration of 2% gave maximum mycelial growth and proved to be the most suitable one. Amongst the nitrogen sources studied, peptone, ammonium sulphate, and sodium nitrate, gave the maximum mycelial growth at an optimized concentration of 0.5%. In the presence of beef extract and yeast extract, a change in colony pigmentation from yellow to dark grey was observed. Finally, the carbon to nitrogen ratio of 2:0.5 proved to be optimal for mycelial growth. This study is the first report on the optimisation of in vitro growth requirements of C. cibarius.

Selection of suitable reference genes for quantitative real time PCR in different Tulasnella isolates and orchid-fungus symbiotic germination system.

Under natural conditions, mycorrhizal symbiosis accompanies nearly the entire life cycle of orchids from seed germination through to flowering and fruiting. Tulasnella-like orchid mycorrhizal fungi are the most common mycorrhizal fungi found in association with orchid species. Presently suitable reference genes have not been systematically selected for the quantification of gene expression via Real-Time Quantitative Reverse Transcription PCR (RT-qPCR). We evaluated 12 candidate Tulasnella genes in nine different Tulasnella isolates and in the Dendrobium-fungal symbiotic germination associations followed by statistical analysis using the programs Bestkeeper, geNorm, and Normfinder to analyze the expression stability of the individual genes. The results showed that the EF2, UBC, and PP2A genes had the highest rankings with relatively stable expression levels across the different genotypes and during the symbiotic seed germination process by the three programs, and may be suitable for RT-qPCR normalization. Furthermore, the gene encoding C-5 Sterol desaturase (C5SD) was selected to verify the reliability of EF2, UBC, and PP2A expression during the Tulasnella-Dendrobium symbiotic seed germination process. This study is the first systematic exploration of optimal reference genes for gene expression studies during the colonization of orchid seeds by the mycorrhizal fungus Tulasnella.

Phosphoinositide signaling plays a key role in the regulation of cell wall reconstruction during the postharvest morphological development of Dictyophora indusiata.

The potential signaling mechanism of Dictyophora indusiata during postharvest morphological development was investigated through quantitative phosphoproteomic analyses. A total of 1566 phosphorylation sites changed significantly (872 upregulated and 694 downregulated) in the mature stage compared with those in the peach-shaped stage of D. indusiata. Bioinformatics analysis showed that the upregulated differentially phosphorylated proteins were mainly involved in the "phosphatidylinositol signaling system" and "mitogen-activated protein kinase signaling pathway-yeast", while the downregulated differentially phosphorylated proteins were related mainly to "starch and sucrose metabolism". Further mining of the phosphoproteome data revealed that upregulated phosphoinositide signaling activated the cell wall integrity pathway and then regulated the synthesis of the main components of the cell wall. The results suggested that phosphoinositide signaling could be a potential target pathway for the regulation of the postharvest morphological development of D. indusiata.