Cutinase can degrade aliphatic and aromatic polyesters, as well as polyethylene terephthalate. Lack of commercially available cutinase calls for development of cost-effective production of efficient cutinase. In this study, eight cutinase genes were cloned from Sclerotinia sclerotiorum. The most active gene SsCut-52 was obtained by PCR combined with RT-PCR, expressed in Escherichia coli BL21 and purified by Ni-NTA affinity chromatography to study its characteristics and pathogenicity. Sscut-52 had a total length of 768 bp and 17 signal peptides at the N terminals. Phylogenetic analysis showed that its amino acid sequence had the highest homology with Botrytis keratinase cutinase and was closely related to Rutstroemia cutinase. Sscut-52 was highly expressed during the process of infecting plants by Sclerotinia sclerotiorum. Moreover, the expression level of Sscut-52 was higher than those of other cutinase genes in the process of sclerotia formation from mycelium. The heterologously expressed cutinase existed in the form of inclusion body. The renatured SsCut-52 was active at pH 4.0-10.0, and mostly active at pH 6.0, with a specific activity of 3.45 U/mg achieved. The optimum temperature of SsCut-52 was 20-30 ℃, and less than 60% of the activity could be retained at temperatures higher than 50 ℃. Plant leaf infection showed that SsCut-52 may promote the infection of Banlangen leaves by Sclerotinia sclerotiorum.
Ascomycota/genetics* ; Carboxylic Ester Hydrolases ; Cloning, Molecular ; Phylogeny

Gummy stem blight, a plant disease caused by Didymella bryoniae, is one of the major diseases in melon. The disease can seriously reduce melon yield and quality. However, little information is available on the genetics and functional genomics of the fungal pathogen. In this study, we developed an Agrobacterium-mediated transformation system for D. bryoniae by using a universal pathogenic isolate DB11 and the Agrobacterium tumefaciens strain C58C1 carrying plasmid pBIG2RHPH2 harboring the hygromycin B phosphotransferase gene (hph). Total 45 transformants could be obtained per 1 x 10(5) spores when 1 x 10(6) spores per milliliter of D. bryoniae spore suspension were cocultivated with Agrobacterium cells at OD600 = 0.15 for 48 h in the presence of induction medium (pH 5.2) containing acetosyringone at 200 microg/mL and selection medium contained 100 microg/mL of hygromycin B and 200 microg/mL of cefotaxime sodium, ampicillin and tetracycline, respectively. The transformants were stable when grown on PDA medium without hygromycin B for five times and were verified by PCR amplification with the hph primers and by Southern blot analysis with the hph probe. The transformation system will be useful for further studies of functional genes in D. bryoniae.
Agrobacterium tumefaciens ; genetics ; Ascomycota ; genetics ; Cucumis melo ; microbiology ; Plant Diseases ; microbiology ; Plants, Genetically Modified ; Transformation, Genetic

To develop a genetic transformation method of Aureobasidium pullulans and T-DNA insertion for high-efficient screening of polymalic acid (PMA) producing strain. Agrobacterium tumefaciens-AGL1, containing the selection genes encoding hygromycin B phosphotase or phosphinothricin acetyltranferase, was used to transform Aureobasidium pullulans CCTCC M2012223 and transformants were confirmed by colony PCR method. Transferred DNA (T-DNA) insertional mutants were cultured in microwell plate, and screened for high-titer PMA producing strain according to the pH response model. DNA walking was used to detect the insertion sites in the mutant. Results show that the selection markers could stably generated in the transformants, and 80 to 120 transformants could be found per 10(7) single cells. A high-titer PMA mutant H27 was obtained, giving a good PMA production caused by the disruption of phosphoglycerate mutase, that increased by 24.5% compared with the control. Agrobacterium tumefaciens-mediated transformation and high-efficient screening method were successfully developed, which will be helpful for genetic transformation of Aureobasidium pullulans and its functional genes discovery.
Agrobacterium tumefaciens ; Ascomycota ; genetics ; metabolism ; DNA, Bacterial ; Malates ; metabolism ; Polymerase Chain Reaction ; Polymers ; metabolism ; Transformation, Genetic

In this study, the authors cloned a glycosyltransferase gene PpUGT2 from Paris polyphylla var. yunnanensis with the ORF length of 1 773 bp and encoding 590 amino acids. The phylogenetic tree revealed that PpUGT2 belonged to the UGT80A subfamily and was named as UGT80A49 by the UDP-glycosyltransferase(UGT) Nomenclature Committee. The expression vector pET28a-PpUGT2 was constructed, and enzyme catalytic reaction in vitro was conducted via inducing protein expression and extraction. With UDP-glucose as sugar donor and diosgenin and pennogenin as substrates, the protein was found with the ability to catalyze the C-3 hydroxyl β-glycosylation of diosgenin and pennogenin. To further explore its catalytic characteristic, 15 substrates including steroids and triterpenes were selected and PpUGT2 showed its activity towards the C-17 position of sterol testosterone with UDP-glucose as sugar donor. Homology modelling and molecule docking of PpUGT2 with substrates predicted the key residues interacting with ligands. The re-levant residues of PpUGT2-ligand binding model were scanned to calculate the corresponding mutants, and the optimized mutants were obtained according to the changes in binding affinity of the ligand with protein and the surrounding residues within 5.0 Å of ligands, which had reference value for design of the mutants. This study laid a foundation for further exploring the biosynthetic pathway of polyphyllin as well as the structure of sterol glycosyltransferases.
Ligands ; Glycosyltransferases/genetics* ; Sterols ; Phylogeny ; Ascomycota ; Liliaceae/chemistry* ; Melanthiaceae ; Diosgenin ; Sugars ; Glucose ; Uridine Diphosphate

In order to obtain resistant mutants to nystatin, ultraviolet radiation and LiCl were used to mutagenize the protoplasts of taxol-producing fungi NCEU-1, and four positive mutants with high yield of taxol were screened out on nystatin flat. After further screening experiments on fermentation, a mutant strain--UL04-5 which was able to produce taxol with high yield and could be stably passed on in genetics was eventually found, it's ability to produce taxol was improved from 314.07 microg/L (strain NCEU-1) to 418.24 microg/L (strain U04-5).
Ascomycota ; drug effects ; genetics ; growth & development ; metabolism ; Fermentation ; Genetic Variation ; Mutagenesis ; Nystatin ; pharmacology ; Paclitaxel ; biosynthesis ; Protoplasts ; metabolism

Northern corn leaf blight, caused by the fungus Exserohirum turcicum Pass. (Leonard and Suggs), is one of the major diseases in most corn-growing areas of the world. Research on gene tagging of E. turcicum has been limited due to the lack of an efficient transformation system. Since E. turcicum produces and accumulates melamin in cell walls during vegetative growth, it is difficult to efficiently isolate its protoplast. To isolate the protoplast of this pathogen with a high frequency, the effects of cell wall degradation enzymes, including beta-1,3-glucanase (Fungase, Funcelase, Novozyme and Glucanex) and beta-glucuronidase (Driselase, Uskizyme and Kitalase), enzyme concentrations, combinations, strains and medium on the isolation frequency were tested. The isolation frequencies were high enough for transformation when the combinations of (Kitalase + Glucanex + Driselase), (Kitalase + Glucanex) or (Kitalase + Uskizyme) were used. Moreover, the isolation frequencies of protoplast were significantly affected by the cultural morphologies of strain and the growth stage of mycelia. Among the plasmids tested, only plasmid pAN71 is efficient for transformation of E. turcicum. This result will provide some useful information for gene tagging of E. turcicum and other species in Exserohirum.
Ascomycota ; cytology ; metabolism ; Cell Wall ; metabolism ; Fungal Proteins ; metabolism ; Glycoside Hydrolases ; metabolism ; Protoplasts ; cytology ; metabolism ; Transformation, Genetic ; genetics

OBJECTIVETo isolate and identify the anamorph of Shiraia bambusicola.

METHODFungus strains were isolated from mature ascospores and stroma. They were identified by means of morphological identification including colony and microscope characteristic, the molecular identification was done by 5.8S-ITS rDNA.

RESULTThe strains GZDXIFR-171 and GZDXIFR-181 were isolated and obtained with the separation of different methods, which had the same colony morphology. With the universal primers of the ITS1-5.8S rDNA-ITS2, the 5.8S-ITS rDNA sequence of GZDXIFR-171 and GZDXIFR-181 were obtained by the PCR amplification and sequencing. Compared with the published nucleotide sequence of 5.8S-ITS rDNA in NCBI (National Center for Biotechnology Information), GZDXIFR-171 and GZDXIFR-181 were highly identical with S. bambusicola.

CONCLUSIONThe isolated strains GZDXIFR-171 and GZDXIFR-181 were confirmed to be the true anamorph of S. bambusicola.

Ascomycota ; classification ; genetics ; isolation & purification ; DNA, Fungal ; genetics ; DNA, Ribosomal Spacer ; genetics ; Molecular Sequence Data ; Mycological Typing Techniques ; Phylogeny ; RNA, Ribosomal, 5.8S ; genetics

A new stem rot disease is found to occur naturally on Arabidopsis plants in greenhouses of Fuzhou, China. In order to identify its pathogen, we conducted a series of fungal isolation and purification, plant reinoculation, and ascus and ascospore induction from the sclerotia. The isolate caused typical water-soaked lesions after reinoculation and produced sclerotia both on Arabidopsis plants and culture medium plates, and the sclerotia could be induced to produce discal apothecia and 8 binucleate ascospores per ascus. These disease symptom and fungal morphology data revealed that the fungus Sclerotinia sclerotiorum (Lib.) de Bary was the pathogen for Arabidopsis stem rot. To confirm this, we further amplified its large subunit ribosomal DNA (LSU rDNA) by polymerase chain reaction (PCR), and compared the sequence with the known LSU rDNA sequences in GenBank. The results show that the sequence shares the highest identities with the LSU rDNAs of different S. sclerotiorum strains. Taking all these data together, we concluded that the fungus that caused the Arabidopsis stem rot is S. sclerotiorum (Lib.) de Bary. This is the first report that Arabidopsis is naturally infected by S. sclerotiorum.
Arabidopsis ; microbiology ; Ascomycota ; classification ; genetics ; isolation & purification ; pathogenicity ; Base Sequence ; China ; DNA, Fungal ; genetics ; DNA, Ribosomal ; genetics ; Phylogeny ; Plant Diseases ; microbiology ; Plant Stems ; microbiology

The antifungal, anti-bacterical, anti-brine shrimp activities of SD22 isolated from Paenibacillus daejeonensis Bacteria SS02 were studied. The separation steps included ultracentrifugation, ultrafiltration and (NH4)2SO4 fractional precipitation, further purification was performed by SephadexG-75 and DEAE-32 chromatography. Its molecular weight determined by SDS-PAGE was 56.0 kD and its isoelectfic point was 6.4. SD22 was thermostable to some extent and stable to ultraviolet, but sensitive to some of the enzyme. SD22 could kill most pathogens from propagation, such as Rhizoctonia cerealis, Sclerotinia sclerotiorum Physalospora piricala, Trichodema viride, Gliocladium viride, Curvularia leaf spot, Fusarium sp, Fusarium head blight, Beauveria Bassiana, Escherichia coli, Staphylococcus aureus, Bacillus subtilis , Candidal vaginitis, Fusarium oxysporum Schl. emend. Sayder & Hansem et al. The results will be helpful to find out a novel antifungal protein.
Antifungal Agents ; isolation & purification ; pharmacology ; Ascomycota ; drug effects ; Bacillus ; chemistry ; genetics ; Bacterial Proteins ; isolation & purification ; pharmacology ; Gliocladium ; drug effects ; Plants ; microbiology ; Rhizoctonia ; drug effects

To investigate the secondary metabolites of endophytic fungi Pericinia sp. F-31. Column chromatography on silica gel, Sephadex LH-20 and semi-preparative HPLC were used to separate and purify the compounds. Two compounds were isolated from the fermentation broth of Periconia sp. Their structures were identified as 5-(1-hydroxyhexyl) -6-methyl-2H-pyran-2-one (1) and 2-(3-hydroxy-4-methylphenyl) -propanoic acid (2). Compound 1 was a new lactone compound, compound 2 was new natural product, and the NMR data of compound 2 was reported for the first time.
Annona ; microbiology ; Ascomycota ; chemistry ; genetics ; isolation & purification ; metabolism ; Drugs, Chinese Herbal ; chemistry ; isolation & purification ; metabolism ; Endophytes ; chemistry ; genetics ; isolation & purification ; metabolism ; Lactones ; chemistry ; isolation & purification ; metabolism ; Mass Spectrometry ; Molecular Structure