Age Of Mycelium Research Articles

Multidimensional discriminant analysis of species, strains and culture age of closely related entomopathogenic fungi using reflectance spectroscopy

The diversity of fungal strains is influenced by genetic and environmental factors, growth conditions and mycelium age, and the spectral features of fungal mycelia are associated with their biochemical, physiological, and structural traits. This study investigates whether intraspecific differences can be detected in two closely related entomopathogenic species, namely Cordyceps farinosa and Cordyceps fumosorosea, using ultraviolet A to shortwave infrared (UVA-SWIR) reflectance spectra. Phylogenetic analysis of all strains revealed a high degree of uniformity among the populations of both species. The characteristics resulting from variation in the species, as well as those resulting from the age of the cultures were determined. We cultured fungi on PDA medium and measured the reflectance of mycelia in the 350–2500 nm range after 10 and 17 days. We subjected the measurements to quadratic discriminant analysis (QDA) to identify the minimum number of bands containing meaningful information. We found that when the age of the fungal culture was known, species represented by a group of different strains could be distinguished with no more than 3–4 wavelengths, compared to 7–8 wavelengths when the age of the culture was unknown. At least 6–8 bands were required to distinguish cultures of a known species among different age groups. Distinguishing all strains within a species was more demanding: at least 10 bands were required for C. fumosorosea and 21 bands for C. farinosa. In conclusion, fungal differentiation using point reflectance spectroscopy gives reliable results when intraspecific and age variations are taken into account.

Biosynthesis of silver nanoparticles using nitrate reductase from Aspergillus terreus N4 and their potential use as a non-alcoholic disinfectant.

Green technology has been developed for the quick production of stabilized silver nanoparticles (AgNPs), with the assistance of nitrate reductase from an isolated culture of Aspergillus terreus N4. The organism's intracellular and periplasmic fractions contained nitrate reductase, with the former demonstrating the highest activity of 0.20 IU/g of mycelium. When the fungus was cultivated in a medium comprising 1.056% glucose, 1.836% peptone, 0.3386% yeast extract, and 0.025% KNO3, the greatest nitrate reductase productivity of 0.3268 IU/g was achieved. Statistical modeling via response surface methodology was used to optimize the enzyme production. The periplasmic and intracellular enzyme fractions were found to convert Ag+ to Ag0, initiating synthesis within 20min, with predominant nanoparticle sizes between 25 and 30nm. By normalizing the effects of temperature, pH, AgNO3 concentration, and mycelium age with a variable shaking period for enzyme release, the production of AgNPs with the periplasmic fraction was optimized. The synthesis of nanoparticles occurred at temperatures of 30, 40, and 50°C, with the highest yield observed at 40 and 50°C during shorter incubation periods. Similarly, the nanoparticles were synthesized at pH levels of 7.0, 8.0, and 9.0, with the greatest production observed at pH 8.0 and 9.0 at lower incubation periods. The antimicrobial activity of AgNPs was demonstrated against common foodborne pathogens, including Staphylococcus aureus and Salmonella typhimurium, indicating their potential as non-alcoholic disinfectants.

Effects of growth Medium, temperature and mycelium age on CHV-1 accumulation and transmission

Some mycoviruses such as Cryphonectria hypovirus 1 (CHV-1) have a potential application for biocontrol of Cryphonectria parasitica, the causal agent of chestnut blight, but little is known about the factors that regulate the fungus-virus interaction, especially when the concentration of the virus is low as it is usual in the wider environment in England. In this study, four CHV-1 infected isolate types of C. parasitica were tested for parameters relating to hypovirulence by inoculating onto potato dextrose agar (PDA), apples, and sweet chestnut branch fragments. Isolate L-6 (CHV-1 subtype I, haplotype E-5), together with the positive control isolate M784 (same haplotype), presented the highest hypovirulence values. The lowest hypovirulence was observed with isolate M784 previously kept in the cold at 8 °C, followed by isolate Db-1 (same haplotype but naturally mutated). The differences in hypovirulence were much more pronounced when inoculations took place using mycelial plugs taken from the edge of plate colonies as opposed to the centre of a colony. The same strains were used to further evaluate the effects of the growth medium (PDA or plant material), optimum and cold temperatures, and mycelium age (centre or edge plugs) on the respective virus concentration. Virus RNA was extracted and amplified by RT-PCR and the concentration of the CHV-1 DNA band (394 bp) measured. Virus concentration decreased when its host was growing on PDA when compared to its host growing on plant material. Virus concentration was reduced further if held in the cold temperature for 7 days, and the virus concentration decreased even further after an additional incubation period at 25 °C. Virus isolate L-6, sub-cultured from the colony centre, was the only one that recovered to its original concentration following incubation for 7 days on sweet chestnut wood fragments. The concentration of the virus was more stable in the centre of the plates than that found at the edge of a plate colony. Together with the expected 394 bp CHV-1 amplicon, another 90 bp band was occasionally simultaneously amplified using the same RT-PCR procedure. This band was quantified, and sequenced, finding no homologs. It is first time this satellite amplicon has been described and its concentration related with hypovirulence attributes of the virus infected C. parasitica, such as reduced fungal growth, and positive correlation with donor and recipient CHV-1 band load in donor-recipient transmission experiments. We discuss our findings in terms of previous parameters related to hypovirulence.

Changes of Physiological Characteristic Indexes in the Aging Process of the Liquid Spawn of Auricularia auricula

In order to explore the change rule of physiological indexes during the aging process of the liquid spawn of Auricularia auricula, this test chooses three species of Auricularia auricula(Hei29, Heiweibanjin and Heiweidanpian), which treasured the activities of antioxidant enzymes, antioxidant substances, membrane peroxidation degree and reactive oxygen concentration during different mycelia age. The conclusion showed that: at the same mycelial age, the vitality of Heiweidanpian was the strongest, Hei29 followed, and Heiweibanjin was the weakest; with the increase of mycelia age, Catalase (CAT) activity, Ascorbic Acid(ASA) content, Malondialdehyde (MDA) content and O2- production rate of the three kinds of Auricularia auricula increased continuously. Among them, ASA content, MDA content and O2- production rate increased significantly, while Superoxide Dismutase (SOD) activity decreased significantly; although the Proline (Pro) content and electrical conductivity of the three kinds of Auricularia auricula showed different trends, which decreased significantly at 14 to 16d and increased significantly at 8 to 10d, respectively, and H2O2 content increased significantly at 12 to 16d. It can be seen that SOD activity, CAT activity, ASA content, MDA content and O2- production rate have significant regular changes during the aging process of the liquid spawn of Auricularia auricula. The quantitative analysis found that when the MDA content is 1.42~1.72 μmol·g-1, the H2O2 content is 3.4~4.3 μmol·g-1 or the O2- production rate is 9.5~12.1 μmol·g-1·min-1, it can be considered Auricularia auricula just started to age or the aging degree is relatively low.

Rapid characterization of aquatic hyphomycetes by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry

ABSTRACTProtein fingerprinting using matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-­TOF MS) is a rapid, reliable, and economical method to characterize isolates of terrestrial fungi and other microorganisms. The objective of our study was to evaluate the suitability of MALDI-TOF MS for the identification of aquatic hyphomycetes, a polyphyletic group of fungi that play crucial roles in stream ecosystems. To this end, we used 34 isolates of 21 aquatic hyphomycete species whose identity was confirmed by spore morphology and internal transcribed spacer (ITS1-5.8S-ITS2 = ITS) nuc rDNA sequencing. We tested the efficiency of three protein extraction methods, including chemical and mechanical treatments using 13 different protocols, with the objective of producing high-quality MALDI-TOF mass spectra. In addition to extraction protocols, mycelium age was identified as a key parameter affecting protein extraction efficiency. The dendrogram based on mass-spectrum similarity indicated good and relevant taxonomic discrimination; the tree structure was comparable to that of the phylogram based on ITS sequences. Consequently, MALDI-TOF MS could reliably identify the isolates studied and provided greater taxonomic accuracy than classical morphological methods. MALDI-TOF MS seems suited for rapid characterization and identification of aquatic hyphomycete species.

Simple and efficient genomic DNA extraction protocol for molecular characterisation of Phytophthora colocasiae causing taro leaf blight

Genomic DNA extraction protocol with relatively high quantity and purity is prerequisite for the successful molecular identification and characterisation of plant pathogens. Conventional DNA extraction methods are often time-consuming and yield only very poor quantity of genomic DNA for samples with higher mycelial age. In our laboratory, we have aimed at establishing an efficient DNA isolation procedure, exclusively for the oomycete pathogen Phytophthora colocasiae causing serious leaf blight disease in taro. For this a phenol free protocol was adopted, which involves SDS/Proteinase K-based inactivation of protein contaminants, extraction of nucleic acids using chloroform: isoamyl alcohol and later precipitation of genomic DNA using isopropanol and sodium acetate. The purity of the isolated DNA was analysed by A260/280 and A260/230 spectrophotometric readings and confirmed by restriction digestion with restriction enzyme Eco RI. In this study, a comparative assessment was done with CTAB method and the commercial genomic DNA purification kit (Thermo Fisher Scientific, Fermentas, EU). The extracted DNA was found to be suitable for further downstream applications like ITS amplification of the rDNA ITS region and PCR amplification with species-specific primers.

Assessment of in vitro growth and predation of Arthrobotrys oligospora to Haemonchus contortus under the influence of media type and age of mycelium

Corn meal agar followed by potato dextrose agar was found the best medium for supporting in vitro growth of Chhattisgarh isolate of Arthrobotrys oligospora. Media type clearly affected the ring structure and trophic hyphal development, though attachment of Haemonchus contortus larvae to adhesive hyphae of A. oligospora was not affected. Initially a part of active thirds stage larvae of H. contortus was attached to hyphae within a period of 4 days in all media types except in nutrient agar at 26°C. However, the capture and immobility of the infective larvae were very sluggish. Growth, capture and development of trophic hyphae inside the H. contortus larval body in water agar indicated that development of trapping devices increased when few energy sources are available. Age of mycelium of A. oligospora isolate had significant influence on deployment of all the trapping structures, where maximum predatory effect was found in one month-old fungal mycelium. In colonies in which nematodes mainly encountered aged hyphae, significant reduction in attachment, capture and development of trophic hyphae was found.

Mycoremediation of Potentially Toxic Trace Elements—a Biological Tool for Soil Cleanup: A Review

Anthropogenic and geogenic activities release potentially toxic trace elements (PTEs) that impact human health and the environment. Increasing environmental pollution stresses the need for environmentally friendly remediation technologies. Physico-chemical treatments are effective, but are costly and generate secondary pollution on- or off-site. Phytoremediation is a biological treatment that provides positive results for PTE eradication with few limitations. Mycoremediation, a type of bioremediation to use macrofungi (mushrooms) for PTE extraction from polluted sites, is the best option for soil cleanup. This review highlights the scope, mechanisms, and potentials of mycoremediation. Mushrooms produce a variety of extracellular enzymes that degrade polycyclic aromatic hydrocarbons (PAHs), polychlorinated biphenyls (PCBs), pesticides, dyes, and petroleum hydrocarbons into simpler compounds. Cadmium (Cd), lead (Pb), mercury (Hg), chromium (Cr), copper (Cu), zinc (Zn), and iron (Fe) have been effectively extracted by Phellinus badius, Amanita spissa, Lactarius piperatus, Suillus grevillei, Agaricus bisporous, Tricholoma terreum, and Fomes fomentarius, respectively. Mycoremediation is affected by environmental and genetic factors, such as pH, substrate, mycelium age, enzyme type, and ecology. The bioaccumulation factor (BAF) can make clear the effectiveness of a mushroom for the extraction of PTEs from the substrate. Higher BAF values of Cd (4.34), Pb (2.75), Cu (9), and Hg (95) have been reported for Amanita muscaria, Hypholoma fasciculare, Russula foetens, and Boletus pinophilus, respectively, demonstrating their effectiveness and suitability for mycoremediation of PTEs.

DEVELOPMENT OF ECOLOGICALLY SAFE TECHNOLOGY OF WASTE MYCELIA RECYCLING

Chitosan is a cationic polymer derived by deacetylation of chitin obtained from crustaceans. Biodegradable and mucoadhesion properties of chitosan have recently led to increasing the interest. Chitosan can be used as raw material for the manufacture of films, membranes and fibers, in such branches as medicine, agriculture, veterinary medicine, biotechnology, cosmetics and pulp, and paper industry. Previously it was investigated that in Aspergillus Niger cell wall constituents, chitin comprises of 42 % and also researchers confirmed that the chitosan content of fungi depends on fungal strains, mycelial age, cultivation medium and conditions [1]. In the paper the results of the study of physical and chemical properties of obtained samples of chitosan are shown. The influence of initial parameters of the process on the quality of chitosan is investigated. Use of the biomass to produce chitosan on the basis of the developed methodology is shown. It is found that the resulting chitosan is characterized by low values of ash content, moisture content and the value is within 75–82 %. A further development of the scientific basis for the creation of an efficient, competitive and environmentally safe technologies for utilization mycelial biomass of the fungus Aspergillus Niger with the production of a valuable product is chitosan, which is in contrast to the known allows to reduce production costs by 10–80 % (by using not concentrated solutions of chemicals and low temperature process. This leads to reducing the cost of reagents and electricity. The calculation was performed on indicators such as net present value, internal rate of return and payback period.) The use of the developed technological schemes in practice allows utilization of mycelial waste with the aim of obtaining from them valuable product of chitosan, and to ensure the improvement of ecological situation in the region.

Improvement of hypocrellin production by a new fungal source and optimization of cultivation conditions

ABSTRACTHypocrellins is regarded as the new generation of potential photosensitizer in the photodynamic therapy. However, the current production of HA in large quantity is limited, whether extracted from the fruit bodies of Hypcrella bambusae and Shiraia bambusicola, or its fermented mycelia. To break through the bottleneck, we screened over 350 fungal strains from the uncommon moso bamboo (Phyllostachys edulis) seeds, and acquired 14 isolates of Shiraia sp. Hypocrellins were generally extracted from the stroma of S. bambusicola, and however the hypocrellin-producing strains in our study were identified as endophytic fungi, where several of them exhibited significantly high content of hypocrellins. One of them, a newly isolated Shiraia sp. strain ZZZ816 was selected for further research. The hypocrellin production conditions of the strain ZZZ816 under submerged fermentation were optimized by one-factor-at-a-time and orthogonal experiment design. The optimum parameters for the quid fermentation were determined as follows: incubation time (144 h), initial pH (6.0), volume of medium (100/250 mL), rotary speed (130 rpm), mycelial age (60 h) and inoculation level (10%). The optimum culture medium constituents were determined as follows: yeast extract 20g/L, malt sugar 40g/L, FeSO4·H2O 0.5g/L, urea 4.0g/L, MgS04·7H20 0.5g/L and initial pH 5.0. Using submerged fermentation of the optimum production conditions, the maximum content of hypocrellin A from the strain ZZZ816 was 921.6 mg/L, more than the other industrial strains published in the previous reports. The results demonstrated that Shiraia sp. strain ZZZ816 could enhance the hypocrellin production tremendously with optimizing the fermentation progress.

Sclerotinia sclerotiorum virulence is affected by mycelial age via reduction in oxalate biosynthesis

Sclerotinia sclerotiorum is one of the most devastating necrotrophic phytopathogens. Virulence of the hyphae of this fungus at different ages varies significantly. Molecular mechanisms underlying this functional distinction are largely unknown. In this study, we confirmed the effect of mycelial culture time/age on virulence in two host plants and elucidated its molecular and morphological basis. The virulence of the S. sclerotiorum mycelia in plants dramatically decreases along with the increase of the mycelial age. Three-day-old mycelia lost the virulence in plants. Comparative proteomics analyses revealed that metabolism pathways were comprehensively reprogrammed to suppress the oxalic acid (OA) accumulation in old mycelia. The oxaloacetate acetylhydrolase (OAH), which catalyzes OA biosynthesis, was identified in the S. sclerotiorum genome. Both gene expression and protein accumulation of OAH in old mycelia were strongly repressed. Moreover, in planta OA accumulation was strikingly reduced in old mycelia-inoculated plants compared with young vegetative mycelia-inoculated plants. Furthermore, supply with 10 mmol L−1 OA enabled the old mycelia infect the host plants, demonstrating that loss of virulence of old mycelia is mainly caused by being unable to accumulate OA. Additionally, aerial mycelia started to develop from 0.5-day-old vegetative mycelia and dominated over 1-day-old mycelia grown on potato dextrose agar plates. They were much smaller in hypha diameter and grew significantly slower than young vegetative mycelia when subcultured, which did not maintain to progenies. Collectively, our results reveal that S. sclerotiorum aerial hyphae-dominant old mycelia fail to accumulate OA and thereby lose the virulence in host plants.

Basidiospore and Protoplast Regeneration from Raised Fruiting Bodies of Pathogenic Ganoderma boninense.

Ganoderma boninense, a phytopathogenic white rot fungus had sought minimal genetic characterizations despite huge biotechnological potentials. Thus, efficient collection of fruiting body, basidiospore and protoplast of G. boninense is described. Matured basidiocarp raised under the glasshouse conditions yielded a total of 8.3 × 104 basidiospores/ml using the low speed centrifugation technique. Mycelium aged 3-day-old treated under an incubation period of 3 h in lysing enzyme from Trichoderma harzianum (10 mg/ml) suspended in osmotic stabilizer (0.6 M potassium chloride and 20 mM dipotassium phosphate buffer) yielded the highest number of viable protoplasts (8.9 × 106 single colonies) among all possible combinations tested (regeneration media, age of mycelium, osmotic stabilizer, digestive enzyme and incubation period).

Effect of LED Blue Light on Penicillium digitatum and Penicillium italicum Strains.

Studies on the antimicrobial properties of light have considerably increased due in part to the development of resistance to actual control methods. This study investigates the potential of light-emitting diodes (LED) blue light for controlling Penicillium digitatum and Penicillium italicum. These fungi are the most devastating postharvest pathogens of citrus fruit and cause important losses due to contaminations and the development of resistant strains against fungicides. The effect of different periods and quantum fluxes, delaying light application on the growth and morphology of P. digitatum strains resistant and sensitive to fungicides, and P. italicum cultured at 20°C was examined. Results showed that blue light controls the growth of all strains and that its efficacy increases with the quantum flux. Spore germination was always avoided by exposing the cultures to high quantum flux (700 μmol m(-2) s(-1) ) for 18 h. Continuous light had an important impact on the fungus morphology and a fungicidal effect when applied at a lower quantum flux (120 μmol m(-2) s(-1) ) to a growing fungus. Sensitivity to light increased with mycelium age. Results show that blue light may be a tool for P. digitatum and P. italicum infection prevention during handling of citrus fruits.

Studies on chityn level in vegetative mycelium of some mycorrhizal fungi

Chitin content was investigated in vegetative mycelium of the <i>Basidiomycetes</i> and <i>Ascomycetes</i> mycorrbizal fungi grown in vitro. It was demonstrated that the content varies between species and even between strains of a given species, and it changes with the age of mycelium. The chitin content increased with age in the mycelium of fungi of <i>Basidiomycetes</i> while it decreased in fungi of <i>Ascomycetes</i>. Essential effect was found of nitrogen source on chitin content in the mycelium. Less chitin was determined in the mycelium grown on nitrate than in that grown on urea or ammonia salts.

Arachidonic acid synthesis from biodiesel‐derived waste by Mortierella alpina

The synthesis of arachidonic acid (15–20% of lipid) from biodiesel‐derived waste by Mortierella alpina strains was demonstrated. An inverse correlation between lipid and arachidonic acid production was revealed in media with different concentrations of carbon substrate. A method of increasing the arachidonic acid content of M. alpina lipid based on the additional incubation of harvested mycelium was developed. The effect of mycelium age, time of incubation, and temperature on the fatty acid composition of lipid was studied. Under optimal conditions (incubation of a harvested 7‐day mycelium at RT for 4–7 days), the arachidonic acid content of lipid in M. alpina strains NRRL‐A‐10995 and LPM‐301 grown on biodiesel‐derived waste and in the pure‐glycerol grown strain LPM‐301 increased by 162–199% of initial level (from 14.6 to 29.0%, from 19.8 to 36.5%, and from 35.0 to 56.8%, respectively).Practical applications: Arachidonic acid (AA) has found wide applications in medicine, pharmacology, diet, and infant nutrition as a precursor of several key eicosanoid hormones and pharmacologically active metabolites, as well as in agriculture as an elicitor of plant resistance to phytopathogens. Microbiological processes for AA production usually use carbohydrate substrates. Results of this study indicate that AA can be produced by Mortierella alpina strains from biodiesel‐derived waste, inexpensive and renewable carbon substrate.

Factors affecting the production and regeneration of protoplasts: the case of the mycorrhizal fungus Tulasnella calospora from roots of orchids

Protoplast isolation is relevant for many different applications and has been principally used in procedures involving genetic manipulation. In this study, the age of mycelium, osmotic stabilizers, enzyme, incubation temperature and incubation time were evaluated in terms of their effects on protoplast yield. The young mycelia (3 d) of Tulasnella calospora were digested for 6 h at 30°C in a mixture of 1.2 mol·L−1 MgSO4 + 10 mmol·L−1 K2HPO4 as the osmotic stabilizer, with a 1.0% lysing enzyme and 1.5% driselase: more than 106 protoplasts mL−1 were obtained. When collected by density gradient centrifugation, the concentration of protoplasts can reach 107–108 protoplasts mL−1, an amount suitable enough for experiments of transformation in fungi. For every 105 protoplasts, about 15–25 protoplasts can regenerate after 24–36 h cultivation in a liquid medium and after 8–10 d in an agar medium. This study produced an efficient method for protoplast production, reverting them into a typical mycelia morphology using a Tulasnella calospora isolate.

Phenotypic characterization of crypticDiplodiaspecies by MALDI-TOF MS and the bias of mycelium age

Summary Detection and identification of fungal cryptic species has been facilitated by DNA sequencing. However, the examination of some phenotypic traits is fundamental for the confirmation of genetic results. MALDI-TOF mass spectrometry has shown remarkable reliability in the recognition of species-specific phenotypic markers and has already been tested in several species of fungi. However, even though there is no direct evidence, the age of the mycelium appears to influence the composition of the metabolites detected. In this study, we demonstrate that MALDI-TOF is a reliable technique to identify suitable metabolites to distinguish three monophyletic species of Diplodia (D. pinea, D. seriata and D. scrobiculata), thus supporting recent DNA results. Nevertheless, different collections of samples over a period of 3 months after inoculation also revealed that the MALDI-TOF spectra are highly dependent on mycelial age. More importantly, the species-specific markers emerge only after 1 month of mycelial growth. The methodological and biological implications of these findings are discussed.

Comparative Studies on the Induction of Trichoderma harzianum Mutanase by α-(1→3)-Glucan-Rich Fruiting Bodies and Mycelia of Laetiporus sulphureus

Mutanase (α-(1→3)-glucanase) is a little-known inductive enzyme that is potentially useful in dentistry. Here, it was shown that the cell wall preparation (CWP) obtained from the fruiting body or vegetative mycelium of polypore fungus Laetiporus sulphureus is rich in α-(1→3)-glucan and can be successfully used for mutanase induction in Trichoderma harzianum. The content of this biopolymer in the CWP depended on the age of fruiting bodies and increased along with their maturation. In the case of CWP prepared from vegetative mycelia, the amount of α-(1→3)-glucan depended on the mycelium age and also on the kind of medium used for its cultivation. All CWPs prepared from the individually harvested fruiting body specimens induced high mutanase activity (0.53–0.82 U/mL) in T. harzianum after 3 days of cultivation. As for the CWPs obtained from the hyphal mycelia of L. sulpureus, the maximal enzyme productivity (0.34 U/mL after 3 days of incubation) was recorded for CWP prepared from the 3 week-old mycelium cultivated in Sabouraud medium. Statistically, a high positive correlation was found between the total percentage content of α-(1→3)-glucan in the CWP and the mutanase activity.

Effects of growth medium, incubation temperature, and mycelium age on production of five major fatty acids by six species of Phytophthora

The effects of growth medium, incubation temperature, and mycelium age on the production of five major fatty acids by five isolates each of P. cactorum, P. citricola, P. citrophthora, P. cinnamomi, P. cryptogea, and P. nicotianae were investigated. Fatty acids were extracted from mycelia, and fatty acid methyl ester (FAME) profiles were analyzed by gas chromatography. Fifteen fatty acids were common in all species, but five of them comprised more than 75% of FAME profiles. These five fatty acids – myristic (14:0), palmitic (16:0), linoleic (18:2ω6c), oleic (18:1 ω9c), and eicosapentaenoic (20:5ω3c) acids – were used to investigate the effects of five liquid growth media (20% carrot broth, CB; 10% lima bean broth, LBB; 5% clarified V8 juice broth, V8B; ¼-strength potato dextrose broth, PDB; and ¼-strength Sabourand dextrose broth; SDB), six incubation temperatures (10, 15, 20, 25, 30, and 35°C), and four mycelium ages (4, 8, 12, and 16 days) on fatty acid production. The effect of medium on the relative percentages of five major fatty acids produced by the six species of Phytophthora was highly significant (P < 0.001). The effect of temperature on production of myristic acid was inconsistent and species-dependent. The production of palmitic and linoleic acids were promoted by higher temperature ( ≧ 25°C), whereas the production of oleic acid and eicosapentaenoic acid was promoted by lower temperatures (≦ 20°C). Palmitic acid production was greater in younger mycelia (four and eight days old), and oleic acid was greater in older mycelia (12 and 16 days). Effects of age on the production of linoleic and eicosapentaenoic acids were inconsistent among species.

English

EFY-21 (Ozonium&nbsp;sp.) is a newly isolated taxol-producing endophytic fungus fromTaxus chinensis&nbsp;var.&nbsp;mairei. In this study, an efficient PEG-mediated transformation of EFY-21 was established and conditions for transformation were evaluated.&nbsp;By the optimized enzyme system, mycelium age, digesting temperature and time,&nbsp;over 7 &times; 107&nbsp;ml protoplasts were obtained and protoplast regeneration frequency was more than 6%. Plasmid pV2 containing the hygromycin-B phosphotransferase gene driven by a fungal promoter (trpC) was used to transform EFY-21 and 50% PEG with 20 mM Ca2+&nbsp;was found to be suitable for the transformation. Southern blot analysis revealed that the transforming DNA was successfully integrated into the EFY-21 genome. By the optimized procedure, over two transformants per &mu;g DNA could be obtained. The establishment of efficient transformation system of taxol-producing endophytic fungus enables us to improve taxol production of the fungus by engineering the taxol biosynthetic pathway genes in the future. &nbsp; Key words:&nbsp;Endophytic fungus, taxol, PEG-mediated transformation, protoplast, regeneration,&nbsp;Ozonium&nbsp;sp.