Twenty-eight isolates of Trichoderma belonging to four different species were screened in vitro for their antagonistic ability against Fusarium oxysporum f.sp. dianthi causing carnation wilt. Three different levels of antagonism observed in dual plate assay were further confirmed by cell-free culture filtrate experiments. Isolates showing class I level of antagonism produced maximum lytic enzymes, chitinases and beta-1,3-glucanases. Genetic variability of 25 selected isolates was assessed by random amplified polymorphic DNA technique and the amplified products were correlated for their level of antagonism. Unweighed pair-group method with arithmetical averages cluster analysis revealed prominent inter-and intraspecific genetic variation among the isolates. Based on their genetic relationship, the isolates were mainly distributed into 3 major groups representing T. atroviride, T. pseudokoningii and T. harzianum, with 20-35% interspecific dissimilarity. However, the polymorphism shown by the isolates did not correlate to their level of antagonism.

• MeSH
• antibióza MeSH
• chitinasy genetika   metabolismus   MeSH
• fungální proteiny genetika   metabolismus   MeSH
• Fusarium fyziologie   MeSH
• fylogeneze MeSH
• genetická variace MeSH
• nemoci rostlin mikrobiologie   MeSH
• polymorfismus délky restrikčních fragmentů MeSH
• půdní mikrobiologie MeSH
• Trichoderma fyziologie   genetika   izolace a purifikace   klasifikace   MeSH

• MeSH
• 1-methyl-3-isobutylxanthin farmakologie   MeSH
• celulasa biosyntéza   MeSH
• dibutyryl cyklický AMP farmakologie   MeSH
• glukosa farmakologie   MeSH
• Trichoderma enzymologie   MeSH

A mannanase-coding gene was cloned from Sphingobacterium sp. GN25 isolated from the feces of Grus nigricollis. The gene encodes a 371-residue polypeptide (ManAGN25) showing less than 74 % identity with a number of hypothetical proteins and putative glucanases and mannanases. Before experiment's performance, ManAGN25 was predicted to be a low-temperature active mannanase based on the molecular characterization, including (1) ManAGN25 shared the highest identity of 41.1 % with the experimentally verified low-temperature active mannanase (ManAJB13) from Sphingomonas sp. JB13; (2) compared with their mesophilic and thermophilic counterparts, ManAGN25 and ManAJB13 had increased number of amino acid residues around their catalytic sites; (3) these increased number of amino acid residues built longer loops, more α-helices, and larger total accessible surface area and packing volume. Then the experiments of biochemical characterization verified that the purified recombinant ManAGN25 is a low-temperature active mannanase: the enzyme showed apparently optimal activity at 35-40 °C and retained 78.2, 44.8, and 15.0 % of its maximum activity when assayed at 30, 20, and 10 °C, respectively; the half-life of the enzyme was approximately 60 min at 37 °C; the enzyme presented a K m of 4.2 mg/ml and a k cat of 0.4/s in McIlvaine buffer (pH 7.0) at 35 °C using locust bean gum as the substrate; and the activation energy for hydrolysis of locust bean gum by the enzyme was 36.0 kJ/mol. This study is the first to report the molecular and biochemical characterizations of a mannanase from a strain.

• MeSH
• bakteriální proteiny chemie   genetika   izolace a purifikace   metabolismus   MeSH
• beta-mannosidasa chemie   genetika   izolace a purifikace   metabolismus   MeSH
• kinetika MeSH
• konformace proteinů MeSH
• molekulární sekvence - údaje MeSH
• nízká teplota MeSH
• sekvence aminokyselin MeSH
• sekvenční seřazení MeSH
• Sphingobacterium chemie   enzymologie   genetika   MeSH
• stabilita enzymů MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH

The production of phytase and associated feed enzymes (phosphatase, xylanase, CMCase, alpha-amylase and beta-glucosidase) was determined in a thermotolerant fungus Mucor indicus MTCC 6333, isolated from composting soil. Solid-substrate culturing on wheat bran and optimizing other culture conditions (C and N sources, level of N, temperature, pH, culture age, inoculum level), increased the yield of phytase from 266 +/- 0.2 to 513 +/- 0.4 nkat/g substrate dry mass. The culture extract also contained 112, 194, 171, 396, and 333 nkat/g substrate of phosphatase, xylanase, CMCase, beta-glucosidase and alpha-amylase activities, respectively. Simple 2-step purification employing anion exchange and gel filtration chromatography resulted in 21.9-fold purified phytase. The optimum pH and temperature were pH 6.0 and 70 degrees C, respectively. The phytase was thermostable under acidic conditions, showing 82% residual activity after exposure to 60 degrees C at pH 3.0 and 5.0 for 2 h, and displayed broad substrate specificity. The Km was 200 nmol/L and v(lim) of 113 nmol/s per mg protein with dodecasodium phytate as substrate. In vitro feed trial with feed enzyme resulted in the release of 1.68 g inorganic P/kg of feed after 6 h of incubation at 37 degrees C.

• MeSH
• 6-fytasa chemie   izolace a purifikace   metabolismus   MeSH
• alfa-amylasy metabolismus   MeSH
• buněčná stěna metabolismus   MeSH
• celulasa metabolismus   MeSH
• endo-1,4-beta-xylanasy metabolismus   MeSH
• fermentace MeSH
• gelová chromatografie MeSH
• hydrolasy metabolismus   MeSH
• koncentrace vodíkových iontů MeSH
• krmivo pro zvířata MeSH
• kultivační média chemie   MeSH
• Mucor enzymologie   izolace a purifikace   růst a vývoj   MeSH
• potravní vláknina mikrobiologie   MeSH
• průmyslová mikrobiologie MeSH
• stabilita enzymů MeSH
• substrátová specifita MeSH
• teplota MeSH

Radial growth, asexual sporulation, and cleistothecia formation as well as extracellular chitinase and proteinase formation of Aspergillus nidulans were monitored in surface cultures in order to study the physiological role of extracellular hydrolase production in carbon-stressed cultures. We set up carbon-stressed and carbon-overfed experimental conditions by varying the starting glucose concentration within the range of 2.5 and 40 g/L. Glucose starvation induced radial growth and hydrolase production and enhanced the maturation of cleistothecia; meanwhile, glucose-rich conditions enhanced mycelial biomass, conidia, and cleistothecia production. Double deletion of chiB and engA (encoding an extracellular endochitinase and a β-1,3-endoglucanase, respectively) decreased conidia production under carbon-stressed conditions, suggesting that these autolytic hydrolases can support conidia formation by releasing nutrients from the cell wall polysaccharides of dead hyphae. Double deletion of prtA and pepJ (both genes encode extracellular proteases) reduced the number of cleistothecia even under carbon-rich conditions except in the presence of casamino acids, which supports the view that sexual development and amino acid metabolism are tightly connected to each other in this fungus.

• MeSH
• Aspergillus nidulans enzymologie   růst a vývoj   MeSH
• celulasa genetika   MeSH
• chitinasy genetika   MeSH
• fungální proteiny genetika   MeSH
• glukosa chemie   MeSH
• hydrolasy genetika   MeSH
• hyfy růst a vývoj   MeSH
• kultivační média MeSH
• mutace MeSH
• proteasy genetika   MeSH
• regulace genové exprese enzymů MeSH
• regulace genové exprese u hub * MeSH
• spory hub růst a vývoj   MeSH
• vývojová regulace genové exprese MeSH
• Publikační typ
• časopisecké články MeSH

This work aimed to evaluate in vitro antagonistic activities and mechanisms of endophytic yeasts against phytopathogenic fungi. A total of 407 yeast strains isolated from tissue of rice, corn, and sugarcane leaves were evaluated for their antagonistic activities against ten phytopathogenic fungi. Only strains of Wickerhamomyces anomalus and Kodamaea ohmeri were found to inhibit the growth of phytopathogenic fungi. Wickerhamomyces anomalus (seven strains) showed antagonistic activity against Curvularia lunata (cause of dirty panicle disease of rice), three Fusarium moniliforme strains (cause of bakanae disease of rice, stalk rot disease of corn, and red rot disease of sugarcane), and Rhizoctonia solani (cause of sheath blight disease of rice). Whereas four Kodamae ohmeri strains inhibited growth of F. moniliforme (cause of bakanae disease of rice). Antagonistic mechanisms of W. anomalus were based on the production of volatile organic compounds (VOCs) (mainly 3-methyl-1-butyl acetate and 3-methyl-1-butanol), fungal cell wall-degrading enzymes (β-1,3-glucanase and chitinase), and siderophores as well as phosphate and zinc oxide solubilization. As for K. ohmeri, the production of VOCs (mainly 3-methyl-1-butanol), β-1,3-glucanase and chitinase were hypothesized as possible mechanisms. The antagonistic activity of W. anomalus against these phytopathogenic fungi and of K. ohmeri against F. moniliforme, and the analysis of the VOCs produced by K. ohmeri are reported for the first time. Two W. anomalus strains, DMKU-RE13 and DMKU-CE52, were evaluated for controlling rice sheath blight disease caused by R. solani in rice plants in the greenhouse and found to suppress the disease 55.2-65.1%, whereas 3% validamycin suppressed this disease by 88.5%.

• MeSH
• antibióza * MeSH
• biologická ochrana MeSH
• endofyty izolace a purifikace   fyziologie   MeSH
• houby růst a vývoj   patogenita   MeSH
• kvasinky izolace a purifikace   fyziologie   MeSH
• listy rostlin mikrobiologie   MeSH
• nemoci rostlin ekonomika   mikrobiologie   prevence a kontrola   MeSH
• zemědělské plodiny ekonomika   mikrobiologie   MeSH
• Publikační typ
• časopisecké články MeSH
• Geografické názvy
• Thajsko MeSH

Preparations with elicitation activity were obtained from the mycelium of Leptosphaeria maculans , a fungal pathogen of oilseed rape (Brassica napus). Crude delipidated and deproteinized extract from fungal cell walls induced expression of pathogenesis related gene 1 (PR1), hydrogen peroxide accumulation, and enhanced resistance of B. napus plants toward infection by L. maculans. Elicitation activity significantly decreased after treatment of a crude extract with α- or β-glucanase. Monosaccharide composition analysis of a crude extract purified by ion-exchange chromatography revealed glucose (∼58 mol %), mannose (∼22 mol %), and galactose (∼18 mol %) as the major sugars. FT-IR and NMR spectra confirmed the presence of both carbohydrate and polypeptide components in the purified product. Correlation NMR experiments defined trisaccharide bound to O-3 of serine residue α-D-Glcp-(1→2)-β-D-Galf-(1→6)-α-D-Manp-(1→3)-L-Ser. Terminal α-D-Glcp and (1→6)-β-D-glucan were also detected. The obtained results strongly support the conclusion that these carbohydrates induce defense response in B. napus plants.

• MeSH
• Ascomycota chemie   růst a vývoj   imunologie   MeSH
• Aspergillus niger enzymologie   MeSH
• Brassica napus účinky léků   imunologie   metabolismus   mikrobiologie   MeSH
• buněčná stěna chemie   MeSH
• buněčné extrakty chemie   izolace a purifikace   farmakologie   MeSH
• chemie zemědělská metody   MeSH
• down regulace MeSH
• fungální proteiny analýza   chemie   metabolismus   MeSH
• fungicidy průmyslové chemie   izolace a purifikace   farmakologie   MeSH
• glykosidhydrolasy biosyntéza   genetika   metabolismus   MeSH
• glykosidy analýza   chemie   metabolismus   MeSH
• hydrolýza MeSH
• mycelium chemie   růst a vývoj   imunologie   MeSH
• odolnost vůči nemocem účinky léků   MeSH
• oligosacharidy analýza   chemie   metabolismus   MeSH
• peptidové fragmenty analýza   chemie   metabolismus   MeSH
• peroxid vodíku metabolismus   MeSH
• rostlinné proteiny biosyntéza   genetika   metabolismus   MeSH
• semenáček účinky léků   imunologie   metabolismus   mikrobiologie   MeSH
• upregulace účinky léků   MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH

Biocontrol fungi are widely used to promote plant growth and pest control. Four fungi were isolated from Cremastra appendiculata tubers and screened for plant growth-promoting and antagonistic effects. Based on the morphological characterization and ITS, 18S rRNA and 28S rRNA gene sequencing analysis, the fungi were identified to be related to Colletotrichum gloeosporioides (DJL-6), Trichoderma tomentosum (DJL-9), Colletotrichum godetiae (DJL-10) and Talaromyces amestolkiae (DJL-15). The growth inhibition tests showed that the four isolates had different inhibitory effects on Colletotrichum fructicola, Alternaria alternata and Alternaria longipes, among which DJL-9 showed the highest inhibitory activity. Their culture filtrates (especially that of DJL-15) can also inhibit pathogens. Four isolates were positive for the production of indole-3-acid (IAA) and β-1,3-glucanase and possessed proteolytic activity but were negative for the production of iron siderophore complexes. The four fungi showed strong nitrogen fixation and potassium dissolution abilities. In addition to DJL-9 being able to solubilize phosphate, DJL-10 was able to produce chitinase and cellulase. Pot experiments indicated that the four fungi increased the germination rate of C. appendiculata and soybean seeds and increased soybean radicle growth and plant biomass. Among them, DJL-6 had a better growth-promoting effect. Therefore, we successfully screened the biocontrol potential of endophytes from C. appendiculata, with a focus on preventing fungal diseases and promoting plant growth, and selected strains that could provide nutrients and hormones for plant growth.

• MeSH
• endofyty MeSH
• fosfáty MeSH
• houby * MeSH
• nemoci rostlin mikrobiologie   MeSH
• semena rostlinná MeSH
• vývoj rostlin * MeSH
• Publikační typ
• časopisecké články MeSH

Rutinosidases (α-l-rhamnosyl-β-d-glucosidases) catalyze the cleavage of the glycosidic bond between the aglycone and the disaccharide rutinose (α-l-rhamnopyranosyl-(1→6)-β-d-glucopyranose) of specific flavonoid glycosides such as rutin (quercetin 3-O-rutinoside). Microbial rutinosidases are part of the rutin catabolic pathway, enabling the microorganism to utilize rutin and related plant phenolic glycosides. Here, we report the first three-dimensional structure of a rutinosidase determined at 1.27-Å resolution. The rutinosidase from Aspergillus niger K2 (AnRut), a member of glycoside hydrolase family GH-5, subfamily 23, was heterologously produced in Pichia pastoris. The X-ray structure of AnRut is represented by a distorted (β/α)8 barrel fold with its closest structural homologue being an exo-β-(1,3)-glucanase from Candida albicans (CaExg). The catalytic site is located in a deep pocket with a striking structural similarity to CaExg. However, the entrance to the active site of AnRut has been found to be different from that of CaExg - a mostly unstructured section of ~ 40 residues present in CaExg is missing in AnRut, whereas an additional loop of 13 amino acids partially covers the active site of AnRut. NMR analysis of reaction products provided clear evidence for a retaining reaction mechanism of AnRut. Unexpectedly, quercetin 3-O-glucoside was found to be a better substrate than rutin, and thus, AnRut cannot be considered a typical diglycosidase. Mutational analysis of conserved active site residues in combination with in silico modeling allowed identification of essential interactions for enzyme activity and helped to reveal further details of substrate binding. The protein sequence of AnRut has been revised. DATABASES: The nucleotide sequence of the rutinosidase-encoding gene is available in the GenBank database under the accession number MN393234. Structural data are available in the PDB database under the accession number 6I1A. ENZYME: α-l-Rhamnosyl-β-d-glucosidase (EC 3.2.1.168).

• MeSH
• Aspergillus niger enzymologie   MeSH
• fungální proteiny chemie   genetika   metabolismus   MeSH
• glykosidhydrolasy chemie   genetika   metabolismus   MeSH
• katalytická doména MeSH
• konformace proteinů MeSH
• krystalografie rentgenová MeSH
• molekulární modely MeSH
• mutace MeSH
• oxidace-redukce MeSH
• rutin chemie   metabolismus   MeSH
• sekvence aminokyselin MeSH
• sekvenční homologie MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH