Production of pectolytic and cellulolytic enzymes byfusarium oxysporum andF. moniliforme under different cultivation conditions

Six-day incubation was most suitable for production of pectolytic and cellulolytic enzymes byFusarium on different culture media. Czapek’s medium favoured maximum production of polygalacturonase (PG) and cellulase (C_x), peptone dextrose gave highest yields of pectin methyl galacturonase (PMG) withF. oxysporum. Cole’s medium was found to be poor for the enzyme production by both organisms. A positive correlation was observed between the growth rate of the pathogenic forms and their enzyme production. InF. oxysporum the PG secretion was maximum at pH 4.5 and inF. moniliforme at pH 5.0. PMG production optimum was at pH 5.5. No PG and PMG were produced above pH 7. InF. oxysporum the C_x activity was highest at pH 5.5 and inF. moniliforme at pH 4.5. Maximum PG and PMG activities were recorded at 35 °C in both pathogens. The C_x activity of both organisms was maximum at 45 °C but some carboxymethyl cellulose hydrolysis was found even at 60 °C.     

Effects of concentrated carbon dioxide on the fruiting of several cultivated basidiomycetes (II)

Edible basidiomycetesFlammulia velutipes andPleurotus ostreatus were cultivated in the usual manner on media based on sawdust and rice bran, and the cultures were exposed to slowly flowing CO₂-enriched air (550 (control), 3,000, 6,000, and 9,000µl/l) for seven days at different stages of cultivation. When the cultures were exposed at the primordium stage (less than 10 mm in length), length and yield of fruit-bodies increased and pileus expansion was slightly inhibited inF. velutipes, while inP. ostreatus length increased, yield decreased, and pileus expansion was greatly inhibited. When the cultures with fruit-bodies larger than 10 mm were exposed, length and yield were insensitive and pileus expansion was greatly inhibited inF. velutipes, while inP. ostreatus length was insensitive, but pileus expansion was heavily damaged by trumpet-like deformation and yield decreased. The different action of CO₂ on the two species appeared to be due to the different anatomical structures of their fruit-bodies.     

Effect of the fungicide chlorothalonil (Bravo) on some metabolic activities of aquatic fungi

Physiological responses ofAchlya proliferoides, Saprolegnia ferax andDictyuchus sterilis as affected by the fungicide chlorothalonil (Bravo) were determined. Glucose consumption differed in dependence on the organisms used. Ammonia and peptide nitrogen secretion were stimulated inS. ferax but inhibited in the other two organisms. All doses of the fungicide used decreased phosphorus absorption and increased acid phosphatase activity. The lowest concentrations (30 ppm) of the fungicide increased DNA, RNA and protein synthesis while inhibition was observed at moderate or high concentrations. Aspartate aminotransferase and alanine aminotransferase activities were inhibited inA. proliferoides, stimulated inS. ferax but remained similar to that of the control inD. sterilis.     

Effect of L-cystine on macromolecular changes during spore and parasporal crystal formation inBacillus thuringiensis var.thuringiensis

High concentration of L-cystine (0.25%) when present in a glucose-mineral salt medium inhibited sporulation-specific events like protease production, calcium uptake and dipicolinic acid synthesis inBacillus thuringiensis var.thuringiensis. In addition, the enzymes of the Krebs cycle from aconitase onwards were completely inhibited by a high concentration of cystine. At a low concentration of cystine (0.05%), none of the above mentioned macromolecular changes were affected. Lipid synthesis monitored by [1,2¹⁴ C]-acetate incorporation into lipid as well as into whole cells was completely inhibited.     

Purification and characterization of an extracellular keratinase from a hornmeal-degrading <Emphasis Type="Italic">Bacillus subtilis</Emphasis> MTCC (9102)

Native proteolytic microorganisms were isolated from the hornmeal, which is a product obtained by treatment of horns and hoofs with steam under high pressure. Keratinolytic activities of these organisms were screened in mineral salt medium with 1% hornmeal. Bacillus subtilis MTCC (9102), a keratinase-producing organism causing extensive degradation of hornmeal has been identified. Keratinase was purified (45-fold) by ion exchange, and gel filtration chromatography. Among the keratinases produced by the various organisms, keratinase from the Bacillus subtilis strain reported by us was found to have a molecular weight range between 64 and 69 kDa and high activity in the pH range between 5 and 7, with maximum activity at pH 6.0 and at an optimum temperature of 40°C. It remained stable up to 70°C. The keratinase activity was completely inhibited by ethylenediamine tetraacetic acid (EDTA), and 1 10-phenanthroline, and remained unaffected by phenylmethanesulfonyl fluoride (PMSF, relative activity: 93%), whereas iodoacetamide inhibited considerably. Zinc, magnesium, calcium, manganese, and nickel were found to enhance the enzyme activity, whereas mercury and copper inhibited its activity completely. The keratinolytic metalloprotease from native Bacillus subtilis differed from the other serine proteases. It may have potential applications in the bioconversion of keratinous wastes and eco-friendly dehairing in the leather industry.     

Abscisic acid controls embryo growth potential and endosperm cap weakening during coffee (<Emphasis Type="Italic">Coffea arabica</Emphasis> cv. Rubi) seed germination

The mechanism and regulation of coffee seed germination were studied in Coffea arabica L. cv. Rubi. The coffee embryo grew inside the endosperm prior to radicle protrusion and abscisic acid (ABA) inhibited the increase in its pressure potential. There were two steps of endosperm cap weakening. An increase in cellulase activity coincided with the first step and an increase in endo--mannanase (EBM) activity with the second step. ABA inhibited the second step of endosperm cap weakening, presumably by inhibiting the activities of at least two EBM isoforms and/or, indirectly, by inhibiting the pressure force of the radicle. The increase in the activities of EBM and cellulase coincided with the decrease in the force required to puncture the endosperm and with the appearance of porosity in the cell walls as observed by low-temperature scanning electronic microscopy. Tissue printing showed that EBM activity was spatially regulated in the endosperm. Activity was initiated in the endosperm cap whereas later during germination it could also be detected in the remainder of the endosperm. Tissue printing revealed that ABA inhibited most of the EBM activity in the endosperm cap, but not in the remainder of the endosperm. ABA did not inhibit cellulase activity. There was a transient rise in ABA content in the embryo during imbibition, which was likely to be responsible for slow germination, suggesting that endogenous ABA also may control embryo growth potential and the second step of endosperm cap weakening during coffee seed germination.     

Characterization of a novel cellulose synthesis inhibitor

The physiological effects of an experimental herbicide and cellulose synthesis inhibitor, N2-(1-ethyl-3-phenylpropyl)-6-(1-fluoro-1-methylethyl)-1,3,5-triazine-2,4-diamine, called AE F150944, are described. In the aminotriazine molecular class, AE F150944 is structurally distinct from other known cellulose synthesis inhibitors. It specifically inhibits crystalline cellulose synthesis in plants without affecting other processes that were tested. The effects of AE F150944 on dicotyledonous plants were tested on cultured mesophyll cells of Zinnia elegans L. cv. Envy, which can be selectively induced to expand via primary wall synthesis or to differentiate into tracheary elements via secondary wall synthesis. The IC₅₀ values during primary and secondary wall synthesis in Z. elegans were 3.91×10^–8 M and 3.67×10^–9 M, respectively. The IC₅₀ in suspension cultures of the monocot Sorghum halapense (L.) Pers., which were dividing and synthesizing primary walls, was 1.67×10^–10 M. At maximally inhibitory concentrations, 18–33% residual crystalline cellulose synthesis activity remained, with the most residual activity observed during primary wall synthesis in Z. elegans. Addition to Z. elegans cells of two other cellulose synthesis inhibitors, 1 M 2,6-dichlorobenzonitrile and isoxaben, along with AE F150944 did not eliminate the residual cellulose synthesis, indicating little synergy between the three inhibitors. In differentiating tracheary elements, AE F150944 inhibited the deposition of detectable cellulose into patterned secondary wall thickenings, which was correlated with delocalization of lignin as described previously for 2, 6-dichlorobenzonitrile. Freeze-fracture electron microscopy showed that the plasma membrane below the patterned thickenings of AE F150944-treated tracheary elements was depleted of cellulose-synthase-containing rosettes, which appeared to be inserted intact into the plasma membrane followed by their rapid disaggregation. AE F150944 also inhibited cellulose-dependent growth in the rosette-containing alga, Spirogyra pratensis, but it did not inhibit cellulose synthesis in Acetobacter xylinum or Dictyostelium discoideum, both of which synthesize cellulose via linear terminal complexes. Therefore, AE F150944 may inhibit crystalline cellulose synthesis by destabilizing plasma membrane rosettes.Abbreviations AE F150944 N2-(1-ethyl-3-phenylpropyl)-6-(1-fluoro-1-methylethyl)-1,3,5-triazine-2,4-diamine - CBI cellulose biosynthesis inhibiting - CGA CGA 325615, 1-cyclohexyl-5-(2,3,4,5,6-pentafluorophenoxy)-14,2,4,6-thiatriazin-3-amine - DCB 2,6-dichlorobenzonitrile - TE tracheary element     

The flocculation of wine yeasts: biochemical and morphological characteristics inZygosaccharomyces — flocculation inZygosaccharomyces

The floc-forming ability of flocculent strains ofZygosaccharomyces bailii andZ fermentati, isolated from musts, was tested for susceptibility to proteinase and sugar treatments.Z. fermentati was found highly resistant to the proteolytic enzymes tested, whereasZ. baili was only trypsin-resistant.The inhibition of flocculation by sugars distinguished two types: inZ. fermentati flocculation was completely inhibited by mannose, inZ. bailli by various sugars.By SEM observation, the cell surface ofZygosaccharomyces revealed the presence of a column structure, resulting from fusion of vesicles present on the cell surface.     

Characterization of urease from the phototrophic bacteriumRhodobacter capsulatus E1F1

Rhodobacter capsulatus E1F1 showed high cytosolic urease activity when growing on urea, purines, and purine metabolites as nitrogen source. Molecular mass ofR. capsulatus enzyme is similar to that of other bacteria and greatly differs from that of jack bean. Kinetic parameters of partially purifiedR. capsulatus enzyme resemble those described in other bacterial ureases. The activity was inhibited by metal-chelating agents and by mercurials. Urease fromR. capsulatus E1F1 was negligible in nitrogen-starved cells or in cells cultured with nitrate, ammonium, or amino acids. Moreover, ammonium inhibited both the urea uptake and the urease activity expression inR. capsulatus cells.     

一株链霉菌21-1的分离鉴定及其对禾谷镰刀菌的抑菌活性

【背景】由禾谷镰刀菌(Fusarium graminearum)引起的小麦赤霉病严重威胁我国的小麦生产。【目的】筛选对禾谷镰刀菌具有拮抗能力的链霉菌菌株,为生防菌剂开发提供理论基础。【方法】利用平板对峙法筛选对禾谷镰刀菌具有拮抗能力的链霉菌;通过形态特征、生理生化特征和16S rRNA基因序列分析对其进行鉴定;通过病原菌菌丝生长、孢子产生及萌发抑制试验分析其发酵液的抑菌活性;利用人工接种试验测定该菌株发酵液的防病效果。【结果】筛选到一株对禾谷镰刀菌具有较强拮抗活性的链霉菌21-1,抑菌率为59.5%。依据形态特征、生理生化特性和16S rRNA基因序列分析,将该菌株鉴定为黄三素链霉菌(Streptomycesflavotricini)。菌株21-1发酵液能够抑制禾谷镰刀菌的菌丝生长、孢子产生及萌发过程,而且可以降低禾谷镰刀菌菌丝中可溶性蛋白质的含量,并增加丙二醛的含量。菌株21-1可以产生蛋白酶及纤维素酶。菌株21-1菌液10倍稀释液对小麦赤霉病的防效最佳,为70.1%。此外,菌株21-1发酵液对其他8种植物病原菌均有较好的抑制作用。【结论】菌株21-1对禾谷镰刀菌有较好的抑菌活性,具...     

Chlorate and nitrate reduction in the phototrophic bacteriaRhodobacter capsulatus andRhodobacter sphaeroides

Chlorate or trimethylamine-N-oxide (TMAO) added to phototrophic cultures ofRhodobacter sphaeroides DSM 158 increased both the growth rate and the growth yield although this stimulation was not observed in the presence of tungstate. This strain, exhibited basal activities of nitrate, chlorate, and TMAO reductases independently of the presence of these substrates in the culture medium, and nitrate reductase (NR) activity was competitively inhibited by chlorate. Phototrophic growth ofRhodobacter capsulatus B10, a strain devoid of NR activity, was inhibited only by 100 mM chlorate. However, growth of the nitrate-assimilatingR. capsulatus strains E1F1 and AD2 was sensitive to 10mm chlorate, and their NR activities were not inhibited by chlorate. Both NR and chlorate reductase (CR) activities of strain E1F1 were induced in the presence of nitrate or chlorate respectively, whereas strain AD2 showed basal levels of these activities in the absence of the substrates. A basal TMAO reductase (TR) activity was also observed when these strains ofR. capsulatus were cultured in the absence of this electron acceptor. These results suggest that chlorate and TMAO can be used as ancillary oxidants byRhodobacter strains and that a single enzyme could be responsible for nitrate and chlorate reduction inR. sphaeroides DSM 158, whereas these reactions are catalyzed by two different enzymes inR. capsulatus E1F1 and AD2.     

Regulation of the cellulase biosynthesis inAspergillus terreus

Synthesis of cellulase inAspergillus terreus GTC826 was induced by glucose, xylose and cellobiose at up to 5.0 mg/ml but beyond this concentration they repressed enzyme synthesis. Cycloheximide at 0.2 mM prevented this induction.     

Purification and characterization of a novel endo-β-1,4-glucanase from the thermoacidophilic <Emphasis Type="Italic">Aspergillus terreus</Emphasis>

An endo-β-1,4-glucanase from a thermoacidophilic fungus, Aspergillus terreus M11, was purified 18-fold with 14% yield and a specific activity of 67 U mg⁻¹ protein. The optimal pH was 2 and the cellulase was stable from pH 2 to 5. The cellulase had a temperature optimum of 60°C measured over 30 min and retained more than 60% of its activity after heating at 70°C for 1 h. The molecular mass of the cellulase was about 25 kDa. Its activity was inhibited by 77% by Hg²⁺ (2 mM) and by 59% by Cu²⁺ (2 mM).     

Fungus fruit body lytic enzyme from a myxomycete,Badhamia utricularis

Chitinase,β-1,3-glucanase, cellulase, xylanase and protease activity were detected in a crude enzyme preparation obtained from a slime mold (Badhamia utricularis) which was grown on autoclaved mycelia ofPholiota nameko in a petri dish. The optimal pH of the enzyme preparation for lytic activity against fruit bodies ofLentinus edodes was 4.0, and those ofβ-1,3-glucanase and cellulase were the same. On the other hand, chitinase and protease showed optimal activity at pH 5.0 and 8.0, respectively. The lytic activity was stable below 40°C but completely inactivated at 70°C, and was most stable at pH 5.0. The studies of the optimal pH, thermal stability, and pH stability, and isoelectric focusing analysis of the enzyme preparation suggest that chitinase,β-1,3-glucanase and cellulase activities may be responsible for lysis of fruit bodies of some mushrooms. The crude enzyme preparation from the slime mold lysed fruit bodies of several mushrooms more efficiently than did commercial lytic enzymes preparations (Driselase and Usukizyme).     

Biocontrol activity of salt tolerant Streptomyces isolates against phytopathogens causing root rot of sugar beet

Biological control of fungi causing root rot on sugar beet by native Streptomyces isolates (C and S2) was evaluated in this study. The dry weight and colony forming unit (CFU) of S2 and C increased when 300 mM NaCl was added to medium. The in vitro antagonism assays showed that both isolates had inhibitory effect against Rhizoctonia solani AG-2, Fusarium solani and Phytophthora drechsleri. In dual culture, Streptomyces isolate C inhibited mycelial growth of R. solani, F. solani and P. drechsleri 45%, 53% and 26%, respectively. NaCl treatment of medium increased biocontrol activity of soluble and volatile compounds of isolate C and S2. After salt treatment, growth inhibition of R. solani, F. solani and P. drechsleri by isolate C increased up to 59%, 70% and 79%, respectively. To elucidate the mode of antagonism, protease, chitinase, beta glucanase, cellulase, lipase and α-amylase activity and siderophore and salicylic acid (SA) production were evaluated. Both isolates showed protease, chitinase and α-amylase activity. Also, biosynthesis of siderophore was detectable for both isolates. Production of siderophore and activity of protease and α-amylase increased after adding salt for both isolates. In contrast, chitinase activity decreased significantly. Production of SA, beta glucanase and lipase by isolate S2 and biosynthesis of cellulase by isolate C were observed in presence and absence of NaCl. Soil treatment with Streptomyces isolate C inhibited root rot of sugar beet caused by P. drechsleri, R. solani and F. solani. Results of this study showed that these two Streptomyces isolates had potential to be utilized as biocontrol agent against fungal diseases especially in saline soils.     

Purification and characterization of extracellular lipases from <Emphasis Type="Italic">Pseudomonas monteilii</Emphasis> TKU009 by the use of soybeans as the substrate

A lipase-producing bacterium was isolated and identified as Pseudomonas monteilii TKU009. A lipase (F2) and lipase-like materials (F1) were purified from the culture supernatant of P. monteilii TKU009 with soybean powder as the sole carbon/nitrogen source. The molecular mass of F1 and F2 was estimated to be 44 kDa by SDS-PAGE and gel filtration. The optimum pH, optimum temperature, and pH and thermal stabilities of F2 were 7, 40°C, 8–11, and 50°C; and of F1 were 6, 40°C, 6–7, and 50°C, respectively. F2 was completely inhibited by EDTA and slightly by Mg²⁺, Fe²⁺, Mn²⁺, and SDS. F1 was completely inhibited by EDTA and Fe²⁺ and strongly by Zn²⁺, Mn²⁺, Ca²⁺, Mg²⁺, and SDS. The activities of both the enzymes were enhanced by the addition of non-ionic surfactants Triton X–100 and Tween 40, especially for F1. F2 preferably acted on substrates with a long chain (C10–C18) of fatty acids, while F1 showed a broad spectrum on those with chain length of C4–C18. The marked activity of F2 in organic solvents makes it an ideal choice for application in a water-restricted medium including organic synthesis. Li-June Ming is a visiting Professor at the National Cheng Kung University.     

Differential expression of sialylglycoconjugates and sialidase activity in distinct morphological stages of<Emphasis Type="Italic"> Fonsecaea pedrosoi</Emphasis>

The expression of sialoglycoconjugates in Fonsecaea pedrosoi conidia, mycelia, and sclerotic cells was analyzed using influenza A and C virus strains, sialidase treatment, and lectin binding. Conidium and mycelium whole cells were recognized by Limax flavus (LFA), Maackia amurensis (MAA), and Sambucus nigra (SNA) lectins, denoting the presence of surface sialoglycoconjugates containing 2,3- and 2,6-sialylgalactosyl sequences. Sialidase-treated conidia reacted more intensively with peanut agglutinin (PNA), confirming the occurrence of sialyl-galactosyl linkages. Conidial cells agglutinated in the presence of influenza A and C virus strains, which confirmed the results obtained from lectin-binding experiments and revealed the presence of sialoglycoconjugates bearing 9-O-acetyl-N-acetylneuraminic acid (Neu5,9Ac₂) surface structures. Western blotting analysis with peroxidase-labeled LFA demonstrated the occurrence of sialylglycoproteins in protein extracts from conidia and mycelia, with molecular masses corresponding to 56 and 40 kDa. An additional band of 77 kDa was detected in conidial extracts, suggesting an association between sialic acid expression and morphogenesis. Synthesis of sialic acids was correlated with sialidase expression, since both conidial and mycelial morphological stages presented secreted and cell-associated enzyme activity. Sialoglycoconjugates were not detected in F. pedrosoi sclerotic cells from in vitro and in vivo sources, which also do not express sialidase activity. The surface sialyl residues in F. pedrosoi are apparently involved in the fungal interaction with immune effector cells, since sialidase-treated conidia were less resistant to phagocytosis by human neutrophils from healthy individuals. These findings suggest that sialic acid expression in F. pedrosoi varies according to the morphological transition and may protect infecting propagules against immune destruction by host cells.     

Cellulolytic activity of four Fusarium spp.

Fusarium lini, F. lycopersici, F. pallidoroseum and F. semitectum grown in shake flasks produced, respectively, 0.19, 0.33, 0.13 and 0.09 units filter-paper cellulase/ml. Trichoderma reesei, in comparison, produced 0.8 U/ml.The authors are with Defence Food Research Laboratory, Mysore-570 011, India.     

Formation of sperm cells inGalium mollugo (Rubiaceae),Trichodiadema setuliferum (Aizoaceae), andAvena sativa (Poaceae)

The formation of sperm cells has been examined ultrastructurally in the tricellular pollen grains ofGalium mollugo L. (Rubiaceae).Trichodiadema setuliferum Schwantes (Aizoaceae), andAvena sativa L. (Poaceae). After detachement from the intine the generative cell of all three species lies free within the vegetative cytoplasm. The two sperm cells are built inTrichodiadema andAvena by a single separating wall, while inGalium mollugo two independent walls are formed. However, both mechanisms separate the two male gametes completely.     

拟松材线虫纤维素酶活性与其致病性关系

【目的】研究拟松材线虫是否分泌纤维素酶以及纤维素酶与其致病力的关系。【方法】对拟松材线虫的不同致病性种群以及松材线虫的虫体蛋白提取液、分泌液的纤维素酶活性进行定量测定,比较群体间纤维素同工酶谱型差异。【结果】拟松材线虫也含有纤维素酶,并向体外分泌;且不同致病性种群的纤维素酶活性与其致病性有一定相关性,致病性越强纤维素酶活性越强。【结论】纤维素酶活性大小是导致拟松材线虫不同种群间致病性差异的重要原因,这对于全面认识松树线虫萎蔫病的致病机理有重要意义。