Perfiles de utilización de sustratos carbonados de Cladorrhinum (Ascomycota) / Carbon-substrate utilization profiles by Cladorrhinum (Ascomycota)

Abstract Fungi from the genus Cladorrhinum (Ascomycota) are promising agents in the biocontrol of phytopathogens, in the promotion of plant growth, and in the production of enzymes with technological application. We analyzed comparatively the ability of 5 native strains of Cladorrhinum samala and Cladorrhinum bulbillosum with reference strains belonging to the same genus. We used 95 individual carbon sources available in microplates from the Biolog® FF system. Although most of the strains mainly used soluble carbohydrates, the metabolic profile was highly dependent upon each isolate and it revealed intraspecific physiological variability in Cladorrhinum species.

Resumen Los hongos del género Cladorrhinum (Ascomycota) son agentes prometedores en el biocontrol de fitopatógenos, la promoción del crecimiento de las plantas y la producción de enzimas con aplicación tecnológica. En este trabajo se analizaron comparativamente las habilidades de 5 cepas nativas pertenecientes a las especies Cladorrhinum samala y Cladorrhinum bulbillosum con cepas de referencia del mismo género. Se usaron 95 fuentes individuales de carbono, disponibles en microplacas de Biolog® FF system. Aunque la mayoría de las cepas utilizaron principalmente carbohidratos solubles, el perfil metabólico fue altamente dependiente de cada aislamiento y reveló variabilidad fisiológica intraespecífica en las especies de Cladorrhinum.

Expression and purification of the transcription factor StMsn2 from Setosphaeria turcica in Escherichia coli

Background: In Saccharomyces cerevisiae, Msn2, which acts as a key transcription factor downstream the MAPKHOG cascade pathway, also regulates the expression of genes related to stress responses. However, little is known about the regulation mechanisms of the transcription factor in Setosphaeria turcica. Results: In this study, a zinc finger DNA-binding protein, designated as StMSN2, was cloned from S. turcica. Sequencing results showed that StMSN2 had a 1752 bp open reading frame (ORF), which was interrupted by an intron (135 bp) and encoded a putative 538-amino acid protein. Phylogenetic analysis further revealed that StMsn2 was more closely related to Msn2 of Aspergillus parasiticus. StMSN2 was cloned into the pET-28a vector with His (Histidine) tags and induced with 1 mM IPTG (isopropyl-ß-D-thiogalactoside) at 37°C. The recombinant His-tagged StMsn2 was purified, and a band of size approximately 58.8 kDa was obtained. The high specificity of the polyclonal antibody Msn2-2 was detected with the StMsn2 protein from S. turcica and prokaryotic expression system, respectively. Conclusions: A new gene, named StMSN2, with 1617 bp ORF was cloned from S. turcica and characterized using bioinformatics methods. StMsn2 was expressed and purified in a prokaryotic system. A polyclonal antibody, named Msn2-2, against StMsn2 with high specificity was identified.

The improvement of bioactive secondary metabolites accumulation in Rumex gmelini Turcz through co-culture with endophytic fungi

Abstract Aspergillus sp., Fusarium sp., and Ramularia sp. were endophytic fungi isolated from Rumex gmelini Turcz (RGT), all of these three strains could produce some similar bioactive secondary metabolites of their host. However the ability to produce active components degraded significantly after cultured these fungi alone for a long time, and were difficult to recover. In order to obtain more bioactive secondary metabolites, the co-culture of tissue culture seedlings of RGT and its endophytic fungi were established respectively, and RGT seedling was selected as producer. Among these fungi, Aspergillus sp. showed the most significant enhancement on bioactive components accumulation in RGT seedlings. When inoculated Aspergillus sp. spores into media of RGT seedlings that had taken root for 20 d, and made spore concentration in co-culture medium was 1 × 104 mL-1, after co-cultured for 12 d, the yield of chrysophaein, resveratrol, chrysophanol, emodin and physcion were 3.52-, 3.70-, 3.60-, 4.25-, 3.85-fold of the control group. The extreme value of musizin yield was 0.289 mg, which was not detected in the control groups. The results indicated that co-culture with endophytic fungi could significantly enhance bioactive secondary metabolites production of RGT seedlings.

Comparative proteomic analyses of Hyphozyma roseonigra ATCC 20624 in response to sclareol

Abstract Sclareol is an important intermediate for ambroxide synthesis industries. Hyphozyma roseonigra ATCC 20624 was the only reported strain capable of degrading sclareol to the main product of sclareol glycol, which is the precursor of ambroxide. To date, knowledge is lacking about the effects of sclareol on cells and the proteins involved in sclareol metabolism. Comparative proteomic analyses were conducted on the strain H. roseonigra ATCC 20624 by using sclareol or glucose as the sole carbon source. A total of 79 up-regulated protein spots with a >2.0-fold difference in abundance on 2-D gels under sclareol stress conditions were collected for further identification. Seventy spots were successfully identified and finally integrated into 30 proteins. The up-regulated proteins under sclareol stress are involved in carbon metabolism; and nitrogen metabolism; and replication, transcription, and translation processes. Eighteen up-regulated spots were identified as aldehyde dehydrogenases, which indicating that aldehyde dehydrogenases might play an important role in sclareol metabolism. Overall, this study may lay the fundamentals for further cell engineering to improve sclareol glycol production.

Efficient production of pullulan by Aureobasidium pullulans grown on mixtures of potato starch hydrolysate and sucrose

Abstract Pullulan is a natural exopolysaccharide with many useful characteristics. However, pullulan is more costly than other exopolysaccharides, which limits its effective application. The purpose of this study was to adopt a novel mixed-sugar strategy for maximizing pullulan production, mainly using potato starch hydrolysate as a low-cost substrate for liquid-state fermentation by Aureobasidium pullulans. Based on fermentation kinetics evaluation of pullulan production by A. pullulans 201253, the pullulan production rate of A. pullulans with mixtures of potato starch hydrolysate and sucrose (potato starch hydrolysate:sucrose = 80:20) was 0.212 h−1, which was significantly higher than those of potato starch hydrolysate alone (0.146 h−1) and mixtures of potato starch hydrolysate, glucose, and fructose (potato starch hydrolysate:glucose:fructose = 80:10:10, 0.166 h−1) with 100 g L−1 total carbon source. The results suggest that mixtures of potato starch hydrolysate and sucrose could promote pullulan synthesis and possibly that a small amount of sucrose stimulated the enzyme responsible for pullulan synthesis and promoted effective potato starch hydrolysate conversion effectively. Thus, mixed sugars in potato starch hydrolysate and sucrose fermentation might be a promising alternative for the economical production of pullulan.

Enhancement of botrallin and TMC-264 production in liquid culture of endophytic fungus Hyalodendriella sp. Ponipodef12 after treatments with metal ions

Background: Hyalodendriella sp. Ponipodef12, an endophytic fungus from a poplar hybrid, was a high producer of botrallin and TMC-264 with various bioactivities. In this study, the influences of eight metal ions (i.e.,Mn2+,Na+, Mg2+,Zn2+,Cu2+,Fe2+,Fe3+ and Al3+) on botrallin and TMC-264 production in liquid culture of the endophytic fungus Hyalodendriella sp. Ponipodef12 were investigated. Results: Three most effective metal ions (Zn2+,Cu2+ and Mg2+) along with their optimum concentrations were screened. The optimum addition time and concentrations of Zn2+,Cu2+ and Mg2+ were further obtained respectively for improving botrallin and TMC-264 production. The combination effects of Zn2+,Cu2+ and Mg2+ on the production of botrallin and TMC-264 by employing statistical method based on the central composite design (CCD) and response surface methodology (RSM) were evaluated, and two quadratic predictive models were developed for botrallin and TMC-264 production. The yields of botrallin and TMC-264, which were predicted as 144.12 mg/L and 36.04 mg/L respectively, were validated to be 146.51 mg/L and 36.63 mg/L accordingly with the optimum concentrations of Zn2+ at 0.81 mmol/L, Cu2+ at 0.20 mmol/L, and Mg2+ at 0.13 mmol/L in medium. Conclusion: The results indicated that the enhancement of botrallin and TMC-264 accumulation in liquid culture of the endophytic fungus Hyalodendriella sp. Ponipodef12 by the metal ions and their combination should be an effective strategy.

Medium optimization for palmarumycin C13 production in liquid culture of endophytic fungus Berkleasmium sp. Dzf12 using response surface methodology

Background: Berkleasmium sp. Dzf12, an endophytic fungus from Dioscorea zingiberensis, was a high producer of palmarumycin C13 with various bioactivities. In the present study, the experimental designs based on statistics were employed to evaluate and optimize the medium for palmarumycin C13 production in mycelia liquid culture of Berkleasmium sp. Dzf12. Results: Among various carbon and nitrogen sources, glucose, peptone and yeast extract were found to be the most favourable for palmarumycin C13 production based on the one-factor-at-a-time experiments. After Plackett-Burman test on the medium, glucose, peptone and yeast extract were further verified to be the most significant factors to stimulate palmarumycin C13 accumulation. These three factors (i.e., glucose, peptone and yeast extract) were then optimized through the experiments of central composite design (CCD) and analysis of response surface methodology (RSM). The optimized medium compositions for palmarumycin C13 production were determined as 42.5 g/l of glucose, 6.5 g/l of peptone, 11.0 g/l of yeast extract, 1.0 g/l of KH2PO4, 0.5 g/l of MgSO4 x 7H2O, 0.05 g/l of FeSO4 x 7H2O, and pH 6.5. Under the optimal culture conditions, the maximum palmarumycin C13 yield of Berkleasmium sp. Dzf12 was increased to 318.63 mg/l, which was about 2.5-fold in comparison with that (130.44 mg/l) in the basal medium. Conclusions: The results indicate that the optimum production of palmarumycin C13 in Berkleasmium sp. Dzf12 liquid culture can be achieved by addition of glucose, peptone and yeast extract with their appropriate concentrations in the modified Sabouraud medium.

Epicoccum nigrum and Cladosporium sp. for the treatment of oily effluent in an air-lift reactor

The metalworking industry is responsible for one of the most complex and difficult to handle oily effluents. These effluents consist of cutting fluids, which provide refrigeration and purification of metallic pieces in the machining system. When these effluents are biologically treated, is important to do this with autochthonous microorganisms; the use of these microorganisms (bioaugmentation) tends to be more efficient because they are already adapted to the existing pollutants. For this purpose, this study aimed to use two indigenous microorganisms, Epicoccum nigrum and Cladosporium sp. for metalworking effluent treatment using an air-lift reactor; the fungus Aspergillus niger (laboratory strain) was used as a reference microorganism. The original effluent characterization presented considerable pollutant potential. The color of the effluent was 1495 mg Pt/L, and it contained 59 mg/L H2O2, 53 mg/L total phenols, 2.5 mgO2/L dissolved oxygen (DO), and 887 mg/L oil and grease. The COD was 9147 mgO2/L and the chronic toxicity factor was 1667. Following biotreatment, the fungus Epicoccum nigrum was found to be the most efficient in reducing (effective reduction) the majority of the parameters (26% COD, 12% H2O2, 59% total phenols, and 40% oil and grease), while Cladosporium sp. was more efficient in color reduction (77%).

Two Cases of Mushroom Poisoning by Podostroma Cornu-Damae

Podostroma cornu-damae is a rare fungus that houses a fatal toxin in its fruit body. In this case report, two patients collected and boiled the wild fungus in water, which they drank for one month. One patient died, presenting with desquamation of the palms and soles, pancytopenia, severe sepsis and multiple organ failure. The other patient recovered after one month of conservative care after admission. We found a piece of Podostroma cornu-damae in the remaining clusters of mushrooms. Mushroom poisoning by Podostroma cornu-damae has never been previously reported in Korea.

Polygalacturonase inhibitor protein from fruits of anthracnose resistant and susceptible varieties of Chilli (Capsicum annuum L).

Chilli fruit is highly susceptible to anthracnose infection at the stage of harvest maturity, due to which the fruit yield in the leading commercial variety Byadgi is severely affected. Field studies on screening of several varieties for resistance to anthracnose have shown that a variety of chilli AR-4/99K is resistant to anthracnose infection. In many crops, resistance to fungal attack has been correlated with PGIP activity in developing fruits based on which transgenic varieties have been developed with resistance to fungi. The present study was carried out to determine whether anthracnose resistance in AR-4/99K was due to the increased levels of PGIP alone and/ or due to differences, if any, in the properties of PGIP. Hence, a comparative study of the properties of polygalacturonase inhibitor protein (PGIP) isolated from fruits of anthracnose resistant chilli var AR-4/99K and a susceptible variety Byadgi was conducted with the objective of utilizing the information in genetic transformation studies. Both the PGIPs from anthracnose resistant and susceptible varieties of chilli exhibited similarities in the elution pattern on Sephadex gel, DEAE cellulose, PAGE and SDS-PAGE. The two PGIPs were active over a wide range of pH and temperature. Both PGIPs showed differential inhibitory activity against polygalacturonase (PG) secreted by Colletotrichum gleosporoides, C. capsici, C. lindemuthianum, Fusarium moniliforme and Sclerotium rolfsii. The inhibitory activity of PGIP from both resistant and susceptible varieties was the highest (82% and 76%, respectively) against the PG from Colletotrichum capsici, a pathogen causing anthracnose rot of chilli, while the activity was lower (1.27 to 12.3%) on the other fungal PGs. Although PGIP activity decreased with fruit maturation in both the varieties, the resistant variety maintained a higher activity at 45 days after flowering (DAF) as compared to the susceptible variety which helped it to overcome the infection by anthracnose as against the susceptible variety (Byadgi) in which PGIP activity was drastically reduced at maturity. The molecular mass of PGIP as determined by SDS-PAGE was found to be 37 kDa. N-terminal sequence analysis of the PGIP showed the first six amino acid residues from N-terminal end were Asp-Thr-His-Lys-Ser-Glu (DTHKSE), respectively. The similarities in properties of the two PGIPs support the earlier findings that resistance of AR-4/99K to anthracnose fungus is a result of its higher PGIP activity at maturity.

Nitrogen fixation in lichens is important for improved rock weathering.

It is known that cyanobacteria in cyanolichens fix nitrogen for their nutrition.However, specific uses of the fixed nitrogen have not been examined. The present study shows experimentally that a mutualistic interaction between a heterotrophic N2 fixer and lichen fungi in the presence of a carbon source can contribute to enhanced release of organic acids, leading to improved solubilization of the mineral substrate. Three lichen fungi were isolated from Xanthoparmelia mexicana, a foliose lichen, and they were cultured separately or with a heterotrophic N2 fixer in nutrient broth media in the presence of a mineral substrate. Cells of the N2-fixing bacteria attached to the mycelial mats of all fungi, forming biofilms. All biofilms showed higher solubilizations of the substrate than cultures of their fungi alone. This finding has bearing on the significance of the origin and existence of N2-fixing activity in the evolution of lichen symbiosis. Further, our results may explain why there are N2-fixing photobionts even in the presence of non- fixing photobionts (green algae) in some remarkable lichens such as Placopsis gelida. Our study sheds doubt on the idea that the establishment of terrestrial eukaryotes was possible only through the association between a fungus and a phototroph.

Extracellular amylase(s) production by fungi Botryodiplodia theobromae and Rhizopus oryzae grown on cassava starch residue.

The fungi Botryodiplodia theobromae and Rhizopus oryzae produce extracellular amylase when grown on a liquid medium containing 2% (WN) soluble starch or cassava starch residue(CSR) (as starch equivalent), a waste generated after extraction of starch from cassava, as the sole carbon source. Using CSR as the sole carbon source, the highest amylase activity of 3.25 and 3.8 units (mg, glucose released x ml(-1) x h(-1)) were obtained in shake flask cultures during the late stationary phase of growth of B. theobromae and R. oryzae, respectively. These values were slightly lower than the values obtained using soluble starch as the carbon source. Maximum enzyme synthesis in CSR incorporated medium occurred at the growth temperature of 30 degrees C and pH 6.0. Presence of inorganic NH4+ salts like ammonium acetate and ammonium nitrate in culture medium yielded more amylase than the other nitrogen sources. Amylase(s) production in the controlled environment of a Table-Top glass Jar Fermenter (2-L capacity) was 4.8 and 5.1 units for B. theobromae and R. oryzae, respectively using CSR as the carbon substrate. It is concluded that CSR, a cheap agricultural waste obtained after starch extraction from cassava could replace soluble starch as carbon substrate for commercial production of fungal amylase(s).

Culture filtrate of Lasiodiplodia theobromae restricts the development of natural resistance in Brassica nigra plants.

Culture filtrate of Lasiodiplodia theobromae increased respiration rate, phenylalanine ammonia lyase activity, and levels of hydrogen peroxide, lipid peroxides and salicylic acid in B. nigra plants. Salicylic acid (SA) level increased for 1 hr of interaction and reduced later. Development of systemic acquired resistance (SAR) was found restricted in plants infected with L. theobromae due to deficiency of SA, which is a major signal for development of SAR. Exogenously supplied SA did develop resistance and plant death was delayed. It was hypothesized that deficiency of SA could be due to jasmonic acid produced by fungus that inhibits SA biosynthesis.