PDB Medium Research Articles

Secretory Laccase from Pestalotiopsis Species CDBT-F-G1 Fungal Strain Isolated from High Altitude: Optimization of Its Production and Characterization

Microorganisms producing laccases may be used for the pretreatment of lignocellulosic biomass to recover fermentable sugar. Very few fungi and other microbes growing in high altitudes have been tested for this purpose. As part of this study, we have collected soil samples from different parts of the Kathmandu Valley and the Rautah at district of Nepal (1600 to 2303 m above sea level) and successfully cultured 53 different isolates of microorganisms. Among the 53 isolates obtained 30 were Actinomycetes, 20 were Streptomycetes, and three were fungi). These isolates were tested for laccase expression using guaiacol, tannic acid, and 1-naphthol as substrates. Twelve of the 53 isolates tested positive for the expression of laccase. Among the laccase- positive isolates, a fungal species designated as CDBT-F-G1was found to produce high levels of laccase. This isolate was identified as Pestalotiopsis species based on 18S rRNA sequencing. Pestalotiopsis spp. CDBT-F-G1 isolate grows efficiently in PDB media containing 1% Kraft lignin at pH 5 and 30 °C and secretes 20 ± 2 U/mL laccase in culture medium. Further optimization of growth conditions reveled that addition of (i) metal salts, e.g., 1 mM magnesium sulfate (51 ± 25 U/mL); (ii) agitation of cultures at 200 rpm (51 ± 9U/mL); (iii) surfactants, e.g., 0.75 mM Tween 80 (54 ± 14 U/mL); (iv) 40% dissolved O2 (57 ± 2 U/mL) and inducers, e.g., 1 mM gallic acid (69 ± 11 U/mL), further promote laccase production by Pestalotiopsis spp. CDBT-F-G1 isolate. On the other hand, 0.1 mM cysteine inhibited laccase production. The secretory laccase obtained from fermentation broth of CDBT-F-G1 was partially purified by ammonium sulfate (13-fold purification with specific activity 26,200 U/mg) and acetone (14-fold purification with specific activity 31,700 U/mg) precipitation methods. The enzyme has an approximate molecular mass of 43 kDa, pH and temperature optima werepH6 and 60 °C, respectively. Vmax and Km were 100 μmol/min and 0.10 mM, respectively, with ABTS as the substrate. Given the above characteristics, we believe Pestalotiopsis spp. CDBT-F-G1 strain native to high altitudes of Nepal could be used to pretreat lignocellulosic biomass to efficiently recover fermentable sugars.

Effects of Different Inducer on the Accumulation of Essential Oil from Endophytic Fungi of Cinnamomum longepaniculatum

The aim of this study was to investigate the effect of different inducer on the accumulation of essential oil in C. longepaniculatum endophytic fungus. The effect of the essential oil accumulation in the C. longepaniculatum fungus was studied by adding different inducer with an C. longepaniculatum endophytic fungus (serial number 2J1) with the ability of producing essential oil. The results show that the accumulation of essential oil can be greatly promoted by adding CaSO4 1.5 mg/L, H2O2 9 mmol/L, SA 15 mg/L and MnSO4 3 mmol/L in the PDB medium; compared with CK in the same condition increased 75.56%, 47.18%, 38.35%, 53.95% respectively; reached 0.0934 mg/L, 0.0783 mmol/L, 0.0736 mmol/L, 0.0819 mg/L; through the orthogonal experiment, the combinations 2.0 mg/L CaSO4, 12 mmol/L H2O2, 5 mg/L SA and 7 mmol/L MnSO4 were significantly different from other combinations; the yield reached 0.1847 mg/L and increased by 247.18%, indicating that adding inducer was an effective way to promote the accumulation of essential oil of endophytic fungi. This provides a reference for the artificial regulation of the secondary metabolites of C. longepaniculatum and the enhancement of subsequent microorganisms.

Cyclodepsipeptides and Sesquiterpenes from Marine-Derived Fungus Trichothecium roseum and Their Biological Functions.

On the basis of the ‘one strain, many compounds’ (OSMAC) strategy, chemical investigation of the marine-derived fungus Trichothecium roseum resulted in the isolation of trichomide cyclodepsipeptides (compounds 1–4) from PDB medium, and destruxin cyclodepsipeptides (compounds 5–7) and cyclonerodiol sesquiterpenes (compounds 8–10) from rice medium. The structures and absolute configurations of novel (compounds 1, 8, and 9) and known compounds were elucidated by extensive spectroscopic analyses, X-ray crystallographic analysis, and ECD calculations. All isolated compounds were evaluated for cytotoxic, nematicidal, and antifungal activities, as well as brine shrimp lethality. The novel compound 1 exhibited significant cytotoxic activities against the human cancer cell lines MCF-7, SW480, and HL-60, with IC50 values of 0.079, 0.107, and 0.149 μM, respectively. In addition, it also showed significant brine shrimp lethality, with an LD50 value of 0.48 μM, and moderate nematicidal activity against Heterodera avenae, with an LC50 value of 94.9 μg/mL. This study constitutes the first report on the cytotoxic and nematicidal potential of trichomide cyclodepsipeptides.

Enhancing the Production of D-Mannitol by an Artificial Mutant of Penicillium sp. T2-M10.

D-Mannitol belongs to a linear polyol with six-carbon and has indispensable usage in medicine and industry. In order to obtain more efficient D-mannitol producer, this study has screened out a stable mutant Penicillium sp. T2-M10 that was isolated from the initial D-mannitol-produced strain Penicillium sp.T2-8 via UV irradiation as well as nitrosoguanidine (NTG) induction. The mutant had a considerable enhancement in yield of D-mannitol based on optimizing fermentation. The production condition was optimized as the PDB medium with 24g/L glucose for 9days. The results showed that the production of D-mannitol from the mutant strain T2-M10 increased 125% in contrast with the parental strain. Meanwhile, the fact that D-mannitol is the main product in the mutant simplified the process of purification. Our finding revealed the potential value of the mutant strain Penicillium sp. T2-M10 to be a D-mannitol-producing strain.

Isolasi dan Uji Aktivitas Antimikroba Kapang Endofit dari Kayu Ulin (Eusideroxylon zwageri Teijsm & Binn.) (Isolation and Antimicrobial Activity of Endophytic Fungi from Ulin (Eusideroxylon zwageri Teijsm & Binn.))

Endophytic fungi are reported to be potential as antibacterial, antifungal, antioxidant, anticancer, and antivirus. One of the source of endophytic fungus is ulin (Eusideroxylon zwageri Teijsm & Binn.) which produced the same antimicrobial compound with ulin wood itself. The purpose of this research was to isolate endophytic caps from ulin wood branch, and tested the antimicrobial and antioxidant activity. The isolates were fermented on PDB media (Potato Dextrose Broth), and then tested the antimicrobial activity on Eschericiacoli, Pseudomonas aeruginosa, and Enterococcus aerogenes). In this research, there were two endofit isolates (white/PT and greenish black/HT). HT endophytic isolate exhibited strong antibacterial activity against the two pathogens (P. aeruginosa and E. coli) shown in 12 mm inhibition zone, while PT endophytic isolate exhibited the E. coli in 11.5 mm inhibition zone. The highest antioxidant activity found in HT endophytic shell was 47.47%. Keywords : antimicrobial, endophytic, Eusideroxylon zwageri, fungi, ulin

The Possible Mechanisms Involved in Citrinin Elimination by Cryptococcus podzolicus Y3 and the Effects of Extrinsic Factors on the Degradation of Citrinin.

Citrinin (CIT) is a toxic secondary metabolite produced by fungi belonging to the Penicillium, Aspergillus, and Monascus spp. This toxin has been detected in many agricultural products. In this study, a strain Y3 with the ability to eliminate CIT was screened and identified as Cryptococcus podzolicus, based on the sequence analysis of the internal transcribed spacer region. Neither uptake of CIT by cells nor adsorption by cell wall was involved in CIT elimination by Cryptococcus podzolicus Y3. The extracellular metabolites of Cryptococcus podzolicus Y3 stimulated by CIT or not showed no degradation for CIT. It indicated that CIT elimination was attributed to the degradation of intracellular enzyme(s). The degradation of CIT by C. podzolicus Y3 was dependent on the type of media, yeast concentration, temperature, pH, and initial concentration of CIT. Most of the CIT was degraded by C. podzolicus Y3 in NYDB medium at 42 h but not in PDB medium. The degradation rate of CIT was the highest (94%) when the concentration of C. podzolicus Y3 was 1 × 10⁸ cells/ml. The quantity of CIT degradation was highest at 28°C, and there was no degradation observed at 35°C. The study also showed that acidic condition (pH 4.0) was the most favorable for CIT degradation by C. podzolicus Y3. The degradation rate of CIT increased to 98% as the concentration of CIT was increased to 20 μg/ml. The toxicity of CIT degradation product(s) toward HEK293 was much lower than that of CIT.

Tenebrio molitor-mediated entomopathogenic fungal library construction for pest management

Entomopathogenic fungi are soil-dwelling microorganisms that infect host insects, and some have been used as biological control agents. Construction of an entomopathogenic fungal library provides a strong platform for the development of highly effective biopesticides, but the traditional method using antibiotic media has low isolation efficiency. Herein, to increase the efficiency of isolation, a Tenebrio molitor pathogenicity-based fungal collection method was used for the construction of a library. The isolation efficiency using the T. molitor larvae-based pathogenicity assay was 55.4%, which was significantly higher than the method of antibiotic medium with dodine (6.0%). We named it the Jeonbuk (Chonbuk) National University Entomopathogenic Fungi (JEF) library. It consisted of 279 isolates belonging to 12 genera and 29 species. Main features of the species-representative 15 isolates were characterized in terms of morphology, virulence, conidiogenesis, thermotolerance and production of biologically active materials such as virulence-related enzymes. Some of the species-representative isolates showed higher virulence against T. molitor, Riptortus pedestris and Plutella xylostella in laboratory conditions. The production of enzymes such as chitinase, Pr1 protease and lipase all related to pathogenesis in LB medium was higher than in the SDB and PDB media. In the thermotolerance assay, a Purpureocillium lilacinum isolate showed higher thermotolerance than other isolates. This work reports an efficient library method using T. molitor for building a library for development of fungal biopesticides for pest management.

Toxic metals accumulation in Trichoderma asperellum and T. harzianum

Heavy metal contamination represents an important environmental issue due to the toxic effects of metals on different organisms. Filamentous fungi play an important impact in the bioremediation of heavy metal-contaminated wastewater and soil. The purpose of this investigation was to observe fungal uptake behavior toward heavy metal. For this aim Trichoderma asperellum TS141 and T. harzianum TS103 at growth period were screened for their tolerance and uptake capability of cadmium (Cd), lead (Pb) and nickel (Ni) at different concentrations (0, 25, 50, 100, and 200 mg/L) in PDB media (potato dextrose broth as a complex medium). Results showed that both fungi were able to survive at the maximum concentration of 200 mg/L of the heavy metals, and remove them. T. asperellum had a better uptake capacity for Cd compared to Pb and Ni in the highest metal concentration in media. Maximum removal efficiency of Pb (68.4%) at 100 mg/L and Ni (78%) at 200 mg/L was performed by T. asperellum. For Cd, the highest removal efficiency (82.1%) was recorded by T. harzianum at 200 mg/L Cd in aqueous solution. The uptake of Cd was highly dependent on pH of solution than Pb and Ni so that the optimal pH of Cd uptake was 9 for T. asperellum and 4 for T. harzianum. Also, optimal temperature was 35°C for Cd and Pb uptake in both fungi, whereas for Ni uptake was 30 and 35°C in T. harzianum and T. asperellum, respectively. We propose that T. asperellum TS141 and T. harzianum TS103 can be used as a bioremediation agent for metal remediation from wastewater and heavy metal-contaminated soils.

Evaluation of bioactivity against some pathogenic bacteria and oxidation for fungal secondary metabolites of Fusarium solani isolated from soil

The study included isolate and diagnose fungus Fusarium solani of the local soil and purified and development in the PDB medium and the filtrate extracted using a solvent (Ethyl acetate) to obtain the fungal secondary metabolites extract. This extract has shown bioactivity against both reference isolates (E.coli (ATCC25922) and S.aureus(NCTC6571)) and pathogenic isolates S.pyogenes, K. pneumonia and S.typhimurium using agar disk diffusion technique , The diameters of the inhibition zones of fungal secondary metabolites24.0 mm against E.coli and 31.5 mm against S.aureus,and 34.0 mm against K.pneumoniae and 18.0 mm against S.pyogenes and 33.5mm against S.typhimurium. The test revealed the minimum inhibitory concentration (MIC) of the fungal extract less inhibitor concentration towards our isolation S.aureus was 6.25 µg/ ml also less deadly concentration (MBC).Also against isolate S.aureus was 12.5µg/ ml. also fungal extract showed no toxicity toward human red blood cells. Tested the antioxidant of the fungal extract by measuring its ability to be reduced iron duo ions (Fe ?) have shown high rates of inhibition. The metabolic product of soil fungi can be an alternative source of commercial anti-bacterial.

Enzyme activity and expression pattern of intra- and extracellular chitinase and β-1,3-glucanase of Wickerhamomyces anomalus EG2 using glycol chitin and glucan-containing high polymer complex

We investigated cell growth and activity of intra- and extracellular chitinase, β-1,3-glucanase, and chitin deacetylase with SDS‐PAGE by incubating W. anomalus EG2 in PDB and YPD media for 24h in presence of different concentrations (0%, 0.1%, 0.3%, and 0.5%) of colloidal chitin. Maximum cell growth was observed in both PDB and YPD media without colloidal chitin. In the absence of colloidal chitin, maximum extracellular β-1,3-glucanase activity of 32.96 and 47.28 units/mL was reported at 18h in PDB medium and 6h in YPD medium, respectively. In addition, extracellular chitinase was unaffected by various concentrations of carboxymethyl chitin in both PDB and YPD media. In the absence of colloidal chitin, maximum intracellular chitinase activity was indicated to be 9.82 and 9.86 units/mg protein in PDB and YPD media, respectively. Maximum intracellular β-1,3-glucanase activity reported was 17.34 units/mg protein in PDB medium containing 0.5% colloidal chitin and 15.0 units/mg protein in YPD medium containing 0.3% colloidal chitin. Five major isozymes, GN1, GN2, GN3, GN4, and GN5, of intracellular β-1,3-glucanase were detected with glucan-containing high polymer complex as a substrate with or without colloidal chitin.

Antifungal activity and expression patterns of extracellular chitinase and β-1,3-glucanase in Wickerhamomyces anomalus EG2 treated with chitin and glucan

In this study, the expression patterns of extracellular chitinase and β-1,3-glucanase from cultured Wickerhamomyces anomalus EG2 treated with chitin, glucan, and chemical chitinase inhibitors (kinetin, caffeine, and acetazolamide) were investigated using SDS-PAGE. Relationship between enzyme expression and antifungal activity from yeast plays a very important role for biocontrol of phytopathoges. To determine antifungal activity against phytopathogens, W. anomalus EG2 was shown to strongly inhibit hyphal growth of Fusarium oxysporum KACC 40032 and Rhizoctonia solani KACC 40111. Slight chitinase activity was observed 12 h after incubation in both PDB and YPD medium without colloidal chitin. The molecular weight of chitinase was approximately 124 kDa β-1,3-Glucanase isoenzyme (GN1 and GN2) was observed distinctly on SDS-PAGE gels when laminarin was used as a substrate. β-1,3-Glucanase isoenzyme was not observed when using glucan-containing high polymer complex (GHPC) as a substrate. Production of chitinase from W. anomalus EG2 was inhibited slightly by acetazolamide. Abnormal and cluster-shaped cells of W. anomalus EG2 were observed in both PDB and YPD medium treated with colloidal chitin. These results indicated that W. anomalus EG2 could be applied commercially as a biological control agent of phytopathogens and as a bioinhibitor of yeast cell growth.

An endophytic Basidiomycete, Grammothele lineata, isolated from Corchorus olitorius, produces paclitaxel that shows cytotoxicity.

Grammothele lineata, an endophyte isolated in our laboratory from jute (Corchorus olitorius acc. 2015) was found to be a substantial paclitaxel producer. Taxol and its related compounds, produced by this endophyte were extracted by growing the fungus in simple nutrient media (potato dextrose broth, PDB). Taxol was identified and characterized by different analytical techniques (TLC, HPLC, FTIR, LC-ESI-MS/MS) following its extraction by ethyl acetate. In PDB media, this fungus was found to produce 382.2 μgL-1 of taxol which is about 7.6 x103 fold higher than the first reported endophytic fungi, Taxomyces andreanae. The extracted taxol exhibited cytotoxic activity in an in vitro culture of HeLa cancer cell line. The fungal extract also exhibited antifungal and antibacterial activities against different pathogenic strains. This is the first report of a jute endophytic fungus harboring the capacity to produce taxol and also the first reported taxol producing species that belongs to the Basidiomycota phylum, so far unknown to be a taxol producer. These findings suggest that the fungal endophyte, Grammothele lineata can be an excellent source of taxol and can also serve as a potential species for chemical and genetic engineering to enhance further the production of taxol.

UJI EFEKTIVITAS EKSTRAK ASETON DAUN KAYU MANIS (Cinnamomum burmanni Blume) TERHADAP JAMUR FUSARIUM SOLANI PENYEBAB PENYAKIT BUSUK BATANG PADA BUAH NAGA (HYLOCEREUS SP.) SECARA IN VITRO

This research heads for examining the effectiveness of acetone extract of cinnamon leaf (Cinnamomum burmanni Blume) to the growth of Fusarium soloni fungus. Cinnamon leaf which was used in this research grows in Bedugul village Tabanan regency Bali province. The extract effectiveness experiment of cinnamon leaf to the fungus experiment was carried out by well difusion method in Biopesticide Laboratory of Agriculture Faculty Udayana University, by using the complete random plan consists of 6 treatments and 4 times refrains. The concentration treatment of acetone extract of cinnamon leaf obviously (P<0.05) can impede the growth of colony, biomass and establishment of experiment fungus spore i.e. Fusarium soloni in accordance with in-vitro by PDA and PDB media. Extract minimum blocked energy of cinnamon leaf in experiment fungus i.e. 0,5%. The extract of cinnamon leaf obviously can impedethe growth of colony, spore establishment and biomass establishment of experiment fungus. In extract concentration treatment 0.5% obviously can impede the colony growth of experiment fungus, spore establishment and fungus biomass establishment i.e. each of them in amount of 17,3%, 41,45% and 7,94% if they are compared by the control. The higher extract concentration then the blocked energy is the bigger.

Histone Methyltransferase aflrmtA gene is involved in the morphogenesis, mycotoxin biosynthesis, and pathogenicity of Aspergillus flavus

Arginine methyltransferases catalyze the posttranslational methylation of arginine, which is involved in a range of important biological processes. aflrmtA gene, an arginine methyltransferase was deleted from Aspergillus flavus in this study by homologous recombination. In morphogenesis assay, aflrmtA was found to down-regulate conidiation by regulating the activity of brlA and abaA genes. It was also found to increase sclerotia formation by up-regulating the expression of nsdC and nsdD genes. In mycotoxin biosynthesis, aflrmtA gene was found to significantly up-regulate the biosynthesis of AFB1 in PDA and PDB media by improving the expression of aflR, aflC and aflK, but it was of no effect in YES medium. aflrmtA was further found to be an important regulator of response to plasma membrane lesion, osmotic, and H2O2 - induced oxidative stresses. In pathogenicity analysis, aflrmtA was found to repress conidiation and up-regulate the AFB1 biosynthesis of A. flavus on peanut and corn seeds and also the activities of protease and lipase, but the activity of amylase was down-regulated. It was concluded that aflrmtA gene played important roles in the morphogenesis, mycotoxin biosynthesis and pathogenicity of A. flavus, and it could be a potential target in the prevention and control of crop contamination by A. flavus.

Evaluation of bioactivity against some pathogenic bacteria and oxidation for fungal secondary metabolites of Fusarium solani isolated from soil

The study included isolate and diagnose fungus Fusarium solani of the local soil and purified and development in the PDB medium and the filtrate extracted using a solvent (Ethyl acetate) to obtain the fungal secondary metabolites extract. This extract has shown bioactivity against both reference isolates (E.coli (ATCC25922) and S.aureus(NCTC6571)) and pathogenic isolates S.pyogenes, K. pneumonia and S.typhimurium using agar disk diffusion technique , The diameters of the inhibition zones of fungal secondary metabolites24.0 mm against E.coli and 31.5 mm against S.aureus,and 34.0 mm against K.pneumoniae and 18.0 mm against S.pyogenes and 33.5mm against S.typhimurium. The test revealed the minimum inhibitory concentration (MIC) of the fungal extract less inhibitor concentration towards our isolation S.aureus was 6.25 µg/ ml also less deadly concentration (MBC).Also against isolate S.aureus was 12.5µg/ ml. also fungal extract showed no toxicity toward human red blood cells. Tested the antioxidant of the fungal extract by measuring its ability to be reduced iron duo ions (Fe ?) have shown high rates of inhibition. The metabolic product of soil fungi can be an alternative source of commercial anti-bacterial.

Molecular Characterization and Antimicrobial Activity of an Endolichenic Fungus, Aspergillus sp. Isolated from Parmelia caperata of Similipal Biosphere Reserve, India.

Endolichenic fungi are microbes that inhabit healthy inner lichen tissues without any disease symptoms. They have been reported to produce new and interesting bioactive metabolites. In the present study, an endolichenic fungus frequently isolated from surface-sterilized lichen thallus of Parmelia caperata has been described. The fungus was identified as Aspergillus tubingensis based on morphological traits and ITS rDNA sequence. Crude metabolites extracted from the culture broth exhibited considerable antimicrobial activity against a panel of clinically significant human pathogens. The fungus showed optimum antimicrobial activity in PDB medium in day 7 of incubation period. PDB medium amended with 1% NaCl and at alkaline pH was found to be optimal for antimicrobial metabolites production. Enhanced activity was observed when the fungus was exposed briefly to a heat shock of 60°C during incubation. The metabolites showed optimum λ-max at 214nm with an absorbance value of 1.589. Molecular characterization of the isolate was carried out by ITS phylogeny and ITS2 secondary structure analyses. The phylogenetic trees based on both ITS rDNA and ITS2 sequences showed the isolate within the clade A. tubingensis. Considering the ubiquity and ambiguity in identifying Aspergillus species of different lifestyles, a method to differentiate pathogenic and endophytic Aspergillus at species level was developed using ITS2 secondary structure analysis. The results showed common folding pattern in the secondary structures with a helix and a 5' dangling end found to be highly conserved. Certain features in the secondary structure like multi-bulges and a symmetric interior loop were observed to be unique which distinguish our isolate from other A. tubingensis.

Secondary metabolites from fungus Quambalaria cyanescens

In this study, phytochemical and biological investigations for the fungus Quambalaria cyanescens were proceeded. This fungus was provided by Assiut University Mycological Center (Accession No. 4123). It was grown on PDB media and provided the isolation of one new compound; 3-butyl phenol (1), along with four known compounds: glycerol-monooleate (2), pyrrolopiperazine-2,5-dione (3), 3-isobutyl-pyrrolopiperazine-2,5-dione (4) and 2-(4'-hydroxyphenyl) ethanol (5). The structures of the isolated metabolites were elucidated based on spectroscopic and spectrometric techniques. All the isolated compounds were evaluated for their antimicrobial, antimalarial and antileishmanial activities.

Reduction of Cr(VI) using indigenous Aspergillus spp. isolated from heavy metal contaminated sites

Fifteen (15) fungal morphospecies isolated from heavy metal contaminated sites were assayed for Cr(VI)-tolerance in nutrient scarce PDB medium. Of the 15 isolates, Aspergillus sp. MSG7 and Aspergillus sp. BP3 tolerated 1440 mg/L Cr(VI) and 1800 mg/L for Aspergillus sp. BP2 and Aspergillus sp. BP5. Further, in vitro Cr(VI) reduction using mycelial balls in reactor tubes with 1/10 strength PDB were assayed at varying Cr(VI) concentrations (90, 180, 360, 720 and 1440 mg/L) at pH 2.0 and at varying pH (1.0 – 4.0) with 360 mg/L Cr(VI). Reactor tubes were incubated at room temperature with shaking at 150 rpm and residual Cr(VI) was quantified using UV-Vis spectrophotometer at OD540nm following 1,5-Diphenylcarbazide (DPCZ) method. Isolates showed Cr(VI) reduction of 30–65% with culture factors as not significant (p > 0.05). This signified that isolates were able to tolerate and reduce Cr(VI) within the utilized pH levels and Cr(VI) concentration effectively. The findings indicates the potential biotechnological application of the four fungal isolates in reducing Cr(VI) in broth medium

Postharvest application of a novel chitinase cloned from Metschnikowia fructicola and overexpressed in Pichia pastoris to control brown rot of peaches

Metschnikowia fructicola strain AP47 is a yeast antagonist against postharvest pathogens of fruits. The yeast was able to produce chitinase enzymes in the presence of pathogen cell wall. A novel chitinase gene MfChi (GenBank accession number HQ113461) was amplified from the genomic DNA of Metschnikowia fructicola AP47. Sequence analysis showed lack of introns, an open reading frame (ORF) of 1098bp encoding a 365 amino acid protein with a calculated molecular weight of 40.9kDa and a predicted pI of 5.27. MfChi was highly induced in Metschnikowia fructicola after interaction with Monilinia fructicola cell wall, suggesting a primary role of MfChi chitinase in the antagonistic activity of the yeast. The MfChi gene overexpressed in the heterologous expression system of Pichia pastoris KM71 and the recombinant chitinase showed high endochitinase activity towards 4-Nitrophenyl β-d-N,N′,N″-triacetylchitotriose substrate. The antifungal activity of the recombinant chitinase was investigated against Monilinia fructicola and Monilinia laxa in vitro and on peaches. The chitinase significantly controlled the spore germination and the germ tube length of the tested pathogens in PDB medium and the mycelium diameter in PDA. The enzyme, when applied on peaches cv. Redhaven, successfully reduced brown rot severity. This work shows that the chitinase MfChi could be developed as a postharvest treatment with antimicrobial activity for fruit undergoing a short shelf life, and confirms that P. pastoris KM71 is a suitable microorganism for cost-effective large-scale production of recombinant chitinases.

One strain-many compounds (OSMAC) method for production of polyketides, azaphilones, and an isochromanone using the endophytic fungus Dothideomycete sp.

Polyketides 1–6 were produced by a one strain-many compounds (OSMAC) approach using the endophytic fungus Dothideomycete sp. CRI7 as a producer. Metabolite production of the fungus Dothideomycete sp. CRI7 was sensitive to sources of potato and malt extract used for the preparation of PDB and Czapek malt media, respectively. Three hitherto unknown metabolites were obtained from the fungus CRI7 grown in PDB medium prepared from a commercial potato powder instead of fresh tubers of potato, while three others were obtained from the fungus cultivated in Czapek malt medium. Moreover, a source of malt extract used in the Czapek malt medium was found to influence metabolite production by the fungus CRI7. Structure elucidation of these compounds was achieved by analysis of spectroscopic data, as well as by single crystal X-ray analysis. Two of the compounds showed weak cytotoxic activity, while the remainders were inactive toward the cell lines tested. One compound exhibited radical scavenging activity with an IC50 value of 21.7μM, and inhibited aromatase with an IC50 value of 12.3μM.