Intracerebroventricular administration of the (1→6)-ß-d-glucan (lasiodiplodan) in male rats prevents d-penicillamine-induced behavioral alterations and lipoperoxidation in the cortex.

CONTEXT: Lasiodiplodan, an exocellular (1→6)-ß-d-glucan of molecular weight >1.4 × 106 Da produced by MMPI strain of Lasiodiplodia theobromae (Pat.) Griffon & Maubl. (Brotyosphaeriaceae) is known to exhibit anti-proliferative activity on breast cancer cells (MCF-7), anticoagulant activity when sulfonylated, and reduction in transaminase activity when administered in rats. OBJECTIVE: The effect of intracerebroventricular (I.C.V) injection of lasiodiplodan on neurotoxicity and behavioural changes induced by d-penicillamine was investigated. MATERIALS AND METHODS: Twenty-four male Wistar rats were initially separated in groups of six and treated with 0.15 µmol/µL of NaCl (Groups Ct and d-Pen) and 0.01 µg/µL of lasiodiplodan (Groups Las and Las + d-Pen). After 15 min, they received 6 µmol/µL of NaCl (Groups Ct and Las) and 2 µmol/µL of d-penicillamine (Groups d-Pen and Las + d-Pen). The animal behavior was observed in an open-field test for 60 min. Twenty-four h later, the animals were sacrificed and histopathological analysis and Thiobarbituric acid reactive substances (TBARS) production measurements were performed. RESULTS: Lasiodiplodan prevented neurotoxicity induced by d-penicillamine significantly reducing the production of TBARS (308%; p < 0.05), and behavioural signs; convulsive and pre-convulsive. No histopathological alterations in the cerebral cortex were observed. DISCUSSION AND CONCLUSION: The reduction of TBARS production and convulsive episodes suggests that the protector effect provided by lasiodiplodan passes thought an antioxidant path, possibly interfering in a cascade of neurochemical events, triggering cell death and convulsive episodes. These results demonstrated that lasiodiplodan can be effective in treating neurotoxicity, and reducing damage triggered by convulsions in neuropathies related to GABAergic system.

Carboxymethylation of (1 → 6)-ß-glucan (lasiodiplodan): Preparation, characterization and antioxidant evaluation.

D-Glucans possess immunomodulatory activities and potential for the development of new therapeutic agents. Biological activities can be enhanced in these biopolymers through chemical derivatization, e.g., carboxymethylation. This work presents the carboxymethylation, characterization and the evaluation of antioxidant activities of the exocellular (1 → 6)-ß-D-glucan produced by Lasiodiplodia theobromae MMPI. Thermal analysis indicated that the native and carboxymethylated polysaccharides presented four stages of mass-loss. The first stage occurred at 125°C (loss of water) with two consecutive events of mass loss (200-400°C) attributed to polymer degradation and the fourth stage between 425 and 620°C (final decomposition). Scanning electron microscopy analysis indicated that the gross morphological features of lasiodiplodan were ruptured following carboxymethylation. X-ray diffractometry analysis demonstrated that the native and carboxymethylated polysaccharides presented a non-crystalline structure. Carboxymethylation contributed to improving the polysaccharide's water solubility and antioxidant capacity.

Modulation of gene expression and cell cycle by botryosphaeran, a (1→3)(1→6)-ß-d-glucan in human lymphocytes.

There is growing interest in the anticancer and immunomodulatory potential of fungal ß-d-glucans. In the present study, the modulation of gene expression via RT-qPCR and cell cycle kinetics via flow cytometry were assessed in human normal and tumor (Jurkat) lymphocytes after treatment with botryosphaeran (a fungal (1→3)(1→6)-ß-d-glucan) from Botryosphaeria rhodina MAMB-05. Cell cultures were treated with botryosphaeran either alone, or in combination with doxorubicin (DXR), in a post-treatment protocol. The expression of genes involved in immunomodulatory processes, apoptosis and cell cycle control, as well as ß-d-glucans cell receptors were assessed. Flow cytometry analysis identified tetraploid Jurkat cells in G1 phase when treated with botryosphaeran combined with DXR. This antiproliferative effect in G1 may be associated with down-regulation of the expression of genes involved in the G1 checkpoint. The repression of the CCR5 gene following botryosphaeran treatment, either alone or in combination with DXR, in tumor lymphocytes indicates a possible affinity of this particular (1→3)(1→6)-ß-d-glucan for the receptor CCR5. Therefore, botryosphaeran action appears to be involved in the repression of genes related to the G1 phase of the cell cycle and possibly in the interaction of the botryosphaeran, either alone, or in combination with DXR, with the CCR5 receptor.

Biological activities of derivatized D-glucans: a review.

D-Glucans have triggered increasing interest in commercial applications in the chemical and pharmaceutical sectors because of their technological properties and biological activities. The glucans are foremost among the polysaccharide groups produced by microorganisms with demonstrated activity in stimulating the immune system, and have potential in treating human disease conditions. Chemical alterations in the structure of D-glucans through derivatization (sulfonylation, carboxymethylation, phosphorylation, acetylation) contributes to their increased solubility that, in turn, can alter their biological activities such as antioxidation and anticoagulation. This review surveys and cites the latest advances on the biological and technological potential of D-glucans following chemical modifications through sulfonylation, carboxymethylation, phosphorylation or acetylation, and discusses the findings of their activities. Several studies suggest that chemically modified d-glucans have potentiated biological activity as anticoagulants, antitumors, antioxidants, and antivirals. This review shows that in-depth future studies on chemically modified glucans with amplified biological effects will be relevant in the biotechnological field because of their potential to prevent and treat numerous human disease conditions and their clinical complications.

Metformin induces apoptosis and cell cycle arrest mediated by oxidative stress, AMPK and FOXO3a in MCF-7 breast cancer cells.

Recent studies have demonstrated that the anti-diabetic drug, metformin, can exhibit direct antitumoral effects, or can indirectly decrease tumor proliferation by improving insulin sensitivity. Despite these recent advances, the underlying molecular mechanisms involved in decreasing tumor formation are not well understood. In this study, we examined the antiproliferative role and mechanism of action of metformin in MCF-7 cancer cells treated with 10 mM of metformin for 24, 48, and 72 hours. Using BrdU and the MTT assay, it was found that metformin demonstrated an antiproliferative effect in MCF-7 cells that occurred in a time- and concentration-dependent manner. Flow cytometry was used to analyze markers of cell cycle, apoptosis, necrosis and oxidative stress. Exposure to metformin induced cell cycle arrest in G0-G1 phase and increased cell apoptosis and necrosis, which were associated with increased oxidative stress. Gene and protein expression were determined in MCF-7 cells by real time RT-PCR and western blotting, respectively. In MCF-7 cells metformin decreased the activation of IRß, Akt and ERK1/2, increased p-AMPK, FOXO3a, p27, Bax and cleaved caspase-3, and decreased phosphorylation of p70S6K and Bcl-2 protein expression. Co-treatment with metformin and H2O2 increased oxidative stress which was associated with reduced cell number. In the presence of metformin, treating with SOD and catalase improved cell viability. Treatment with metformin resulted in an increase in p-p38 MAPK, catalase, MnSOD and Cu/Zn SOD protein expression. These results show that metformin has an antiproliferative effect associated with cell cycle arrest and apoptosis, which is mediated by oxidative stress, as well as AMPK and FOXO3a activation. Our study further reinforces the potential benefit of metformin in cancer treatment and provides novel mechanistic insight into its antiproliferative role.

Lipase production by Botryosphaeria ribis EC-01 on soybean and castorbean meals: optimization, immobilization, and application for biodiesel production.

The effects of soybean and castorbean meals were evaluated separately, and in combinations at different ratios, as substrates for lipase production by Botryosphaeria ribis EC-01 in submerged fermentation using only distilled water. The addition of glycerol analytical grade (AG) and glycerol crude (CG) to soybean and castorbean meals separately and in combination, were also examined for lipase production. Glycerol-AG increased enzyme production, whereas glycerol-CG decreased it. A 2(4) factorial design was developed to determine the best concentrations of soybean meal, castorbean meal, glycerol-AG, and KH2PO4 to optimize lipase production by B. ribis EC-01. Soybean meal and glycerol-AG had a significant effect on lipase production, whereas castorbean meal did not. A second treatment (2(2) factorial design central composite) was developed, and optimal lipase production (4,820 U/g of dry solids content (ds)) was obtained when B. ribis EC-01 was grown on 0.5 % (w/v) soybean meal and 5.2 % (v/v) glycerol in distilled water, which was in agreement with the predicted value (4,892 U/g ds) calculated by the model. The unitary cost of lipase production determined under the optimized conditions developed ranged from US$0.42 to 0.44 based on nutrient costs. The fungal lipase was immobilized onto Celite and showed high thermal stability and was used for transesterification of soybean oil in methanol (1:3) resulting in 36 % of fatty acyl alkyl ester content. The apparent K m and V max were determined and were 1.86 mM and 14.29 µmol min(-1) mg(-1), respectively.

Sulfonation and anticoagulant activity of fungal exocellular ß-(1→6)-D-glucan (lasiodiplodan).

An exocellular ß-(1→6)-D-glucan (lasiodiplodan) produced by a strain of Lasiodiplodia theobromae (MMLR) grown on sucrose was derivatized by sulfonation to promote anticoagulant activity. The structural features of the sulfonated ß-(1→6)-D-glucan were investigated by UV-vis, FT-IR and (13)C NMR spectroscopy, and the anticoagulant activity was investigated by the classical coagulation assays APTT, PT and TT using heparin as standard. The content of sulfur and degree of substitution of the sulfonated glucan was 11.73% and 0.95, respectively. UV spectroscopy showed a band at 261 nm due to the unsaturated bond formed in the sulfonation reaction. Results of FT-IR and (13)C NMR indicated that sulfonyl groups were inserted on the polysaccharide. The sulfonated ß-(1→6)-D-glucan presented anticoagulant activity as demonstrated by the increase in dose dependence of APTT and TT, and these actions most likely occurred because of the inserted sulfonate groups on the polysaccharide. The lasiodiplodan did not inhibit the coagulation tests.

Toxicological assessment of ß-(1-->6)-glucan (lasiodiplodan) in mice during a 28-day feeding study by gavage.

Studies evaluating the toxicity caused by fungal exopolysaccharides of the ß-(1-->6)-D-glucan type are rare. In this study, the toxicological effects of sub-chronic treatments with lasiodiplodan (ß-(1-->6)-D-glucan from Lasiodiplodia theobromae MMPI) were evaluated in mice through the assessment of biochemical, hematological, and histopathological alterations. Thirty-two mice (16 male, 16 female) were used in this study divided in two groups; one group received lasiodiplodan (50 mg/kg body weight) daily for 28 days via gavage, and another (control group) received saline during the same period. Blood samples were collected via cardiac puncture for hematological and biochemical analyses. Liver, heart, kidney, and spleen were collected for histopathological analysis. Statistical analysis was performed through one-way analysis of variance and only p < 0.05 F-values were presented. Significant reduction in blood glucose in the male group (35%; p < 0.01), transaminases activity in both sexes (AST and ALT; ~35%; p < 0.05), and urea (20%; p < 0.01) in the female group was observed with the lasiodiplodan treatment. The results showed that sub-chronic treatments with lasiodiplodan did not generate hematological and histopathological alterations leading to signs of toxicity in healthy mice, independent of gender.

Lasiodiplodan, an exocellular (1→6)-ß-D: -glucan from Lasiodiplodia theobromae MMPI: production on glucose, fermentation kinetics, rheology and anti-proliferative activity.

Lasiodiplodan, an exopolysaccharide of the (1→6)-ß-D: -glucan type, is produced by Lasiodiplodia theobromae MMPI when grown under submerged culture on glucose. The objective of this study was to evaluate lasiodiplodan production by examining the effects of carbon (glucose, fructose, maltose, sucrose) and nitrogen sources (KNO(3), (NH(4))(2)SO(4), urea, yeast extract, peptone), its production in shake flasks compared to a stirred-tank bioreactor, and to study the rheology of lasiodiplodan, and lasiodiplodan's anti-proliferative effect on breast cancer MCF-7 cells. Although glucose (2.05 ± 0.05 g L(-1)), maltose (2.08 ± 0.04 g L(-1)) and yeast extract (2.46 ± 0.06 g L(-1)) produced the highest amounts of lasiodiplodan, urea as N source resulted in more lasiodiplodan per unit biomass than yeast extract (0.74 ± 0.006 vs. 0.22 ± 0.008 g g(-1)). A comparison of the fermentative parameters of L. theobromae MMPI in shake flasks and a stirred-tank bioreactor at 120 h on glucose as carbon source showed maximum lasiodiplodan production in agitated flasks (7.01 ± 0.07 g L(-1)) with a specific yield of 0.25 ± 0.57 g g(-1) and a volumetric productivity of 0.06 ± 0.001 g L(-1) h(-1). A factorial 2(2) statistical design developed to evaluate the effect of glucose concentration (20-60 g L(-1)) and impeller speed (100-200 rpm) on lasiodiplodan production in the bioreactor showed the highest production (6.32 g L(-1)) at 72 h. Lasiodiplodan presented pseudoplastic behaviour, and the apparent viscosity increased at 60°C in the presence of CaCl(2). Anti-proliferative activity of lasiodiplodan was demonstrated in MCF-7 cells, which was time- and dose-dependent with an IC(50) of 100 µg lasiodiplodan mL(-1).

Chemical modification of botryosphaeran: structural characterization and anticoagulant activity of a water-soluble sulfonated (1-->3)(1-->6)-ß-D-glucan.

The exopolysaccharide botryosphaeran (EPS(GLC); a (1--> 3)(1-->6)-ß-D-glucan from Botryosphaeria rhodina MAMB- 05) was sulfonated to produce a water-soluble fraction (EPS(GLC)-S) using pyridine and chlorosulfonic acid in formamid. This procedure was then repeated twice to produce another fraction (EPSGLC-RS) with a higher degree of substitution (DS, 1.64). The purity of each botryosphaeran sample (unsulfonated and sulfonated) was assessed by gel filtration chromatography (Sepharose CL-4B), where each polysaccharide was eluted as a single symmetrical peak. The structures of the sulfonated and re-sulfonated botryosphaerans were investigated using ultraviolet-visible (UV-Vis), Fourier-transform infrared (FT-IR), and (13)C nuclear magnetic resonance ((13)C NMR) spectroscopies. EPS(GLC) and EPS(GLC)-RS were also assayed for anticoagulation activity, and EPS(GLC)-RS was identified as an anticoagulant.

Thermal and rheological properties of a family of botryosphaerans produced by Botryosphaeria rhodina MAMB-05.

Differential scanning calorimetry (DSC), thermogravimetry (TG) and Fourier-transform infra-red spectroscopy (FT-IR) analyses were performed to investigate changes in the physico-chemical properties of botryosphaerans, a family of exopolysaccharides (EPS) produced by the fungus Botryosphaeria rhodina MAMB-05 grown on glucose (EPS(GLC)), sucrose (EPS(SUC)) and fructose (EPS(FRU)). A slight endothermic transition and small mass loss attributable to the removal of water of hydration were observed in the DSC and TG analyses, respectively, for the three EPS samples. The FT-IR spectra confirmed no structural changes occurred during thermal treatment. Viscometry was utilized to obtain information on the rheological behaviour of the EPS in aqueous solutions. The Power Law and Cross Equations determined the natural pseudoplastic characteristics of the EPS. Comparatively, results obtained for EPS produced when B. rhodina MAMB-05 was grown on each of the three carbohydrate sources demonstrated similar apparent viscosity values for EPS(GLC) and EPS(SUC), while EPS(FRU) displayed the lowest apparent viscosity of the three botryosphaerans, suggesting a higher degree of ramification and lower Mw. EPS(GLC) and EPS(SUC) possessed similar degrees of ramification. The slight differences found in their viscosities can be explained by the differences in the type of branching among the three botryosphaerans, thus varying the strength of intermolecular interactions and consequently, consistency and viscosity. The physico-chemical studies of botryosphaerans represent the originality of this work, and the knowledge of these properties is an important criterion for potential applications.

Hypoglycemic and Hypocholesterolemic Effects of Botryosphaeran from Botryosphaeria rhodina MAMB-05 in Diabetes-Induced and Hyperlipidemia Conditions in Rats.

Botryosphaeran, a water-soluble exopolysaccharide of the ß-(1 → 3;1 → 6)-D-glucan type that has been isolated from the culture medium of Botryosphaeria rhodina MAMB-05 grown in submerged fermentation using glucose as the sole carbon source, was previously demonstrated to be non-genotoxic in peripheral blood and bone marrow, and exhibited strong anticlastogenic activity. In the present study, the effects of botryosphaeran were investigated in streptozotocin-induced diabetic rats as well as in high-fat diet-fed hyperlipidemic Wistar rats. The plasma glucose level was reduced by 52% in the diabetic group of rats after administration of 12 mg botryosphaeran/kg body weight of the rats (b.w.)/day by gavage over 15 days. A reduction in the median ration intake was accompanied by an increase in the median body weight gain, as well as the efficiency of food conversion. These results demonstrate that botryosphaeran has protective effects by reducing the symptoms of cachexia in Diabetes mellitus. Botryosphaeran administered by gavage at a concentration of 12 mg botryosphaeran/kg b.w./day over 15 days also reduced the plasma levels of total cholesterol and low density lipoprotein-cholesterol by 18% and 27%, respectively, in hyperlipidemic rats. Based on these findings, we conclude that botryosphaeran possesses hypoglycemic and hypocholesterolemic properties in conditions of diabetes mellitus and hyperlipidemia, respectively, and may be used as an oral anti-diabetic agent.

Extracellular beta-glucosidase production by the yeast Debaryomyces pseudopolymorphus UCLM-NS7A: optimization using response surface methodology.

beta-Glucosidase production by Debaryomyces pseudopolymorphus UCLM-NS7A using a simple nutrient medium containing cellobiose was evaluated under several biochemical and physiological parameters in submerged fermentation. Enzyme induction was also examined using different carbon and nitrogen sources. Cellobiose and ammonium nitrate were the best C and N sources to enhance beta-glucosidase production. The addition of NaCl, MgSO(4), yeast extract, ethanol and Tween 80 to the nutrient medium before inoculation was also compared. A factorial design to optimize enzyme production was developed using four variables that most influenced beta-glucosidase production and data analyzed by the response surface method. Optimal conditions to produce beta-glucosidase in shake-flasks were 1.25% cellobiose, 0.05% Tween 80, 0.4% NH(4)NO(3) over 72 hours. In another factorial design to further increase enzyme production, a lab fermenter using prior-determined shake-flask optimized conditions resulted in higher beta-glucosidase titres at 72 hours, pH controlled at 6.25 and agitation of 200 rpm.

Sulfonation and anticoagulant activity of botryosphaeran from Botryosphaeria rhodina MAMB-05 grown on fructose.

Botryosphaeran (EPS(FRU)), an exopolysaccharide of the beta-(1-->3,1-->6)-d-glucan type with 31% branching at C-6, is produced by the fungus Botryosphaeria rhodina MAMB-05 when grown on fructose as carbon source. Botryosphaeran was derivatized by sulfonation to induce anticoagulant activity. The effectiveness of the sulfonation reaction by chlorosulfonic acid in pyridine was monitored by the degree of substitution and FT-IR analysis of the sulfonated EPS(FRU) (once sulfonated, EPS(FRUSULF); and re-sulfonated, EPS(FRURESULF)). Activated partial thromboplastin time (APTT) and thrombin time (TT) tests of EPS(FRURESULF) indicated significant in vitro anticoagulant activity that was dose-dependent. EPS(FRU) did not inhibit any of the coagulation tests.

On the diversity of the laccase gene: a phylogenetic perspective from Botryosphaeria rhodina (Ascomycota: Fungi) and other related taxa.

The present study is the first describing the sequencing of a fragment of the copper-oxidase domain of a laccase gene in the family Botryosphaeriaceae. The aim of this work was to assess the degree of genetic and evolutionary relationships of a laccase gene from Botryosphaeria rhodina MAMB-05 with other ascomycete and basidiomycete laccase genes. The 193-amino acid sequences of the copper-oxidase domain from several different fungi, insects, a plant, and a bacterial species were retrieved from GenBank and aligned. Phylogenetic analyses were performed using neighbor-joining, maximum parsimony, and Bayesian inference methods. The organisms studied clustered into five gene clades: fungi (ascomycetes and basidiomycetes), insects, plants, and bacteria. Also, the topologies showed that fungal laccases of the ascomycetes and basidiomycetes are clearly separated into two distinct clusters. This evidence indicated that B. rhodina MAMB-05 and other closely related ascomycetes are a new biological resource given the biotechnological potential of their laccase genes.

Three exopolysaccharides of the beta-(1-->6)-D-glucan type and a beta-(1-->3;1-->6)-D-glucan produced by strains of Botryosphaeria rhodina isolated from rotting tropical fruit.

Four exopolysaccharides (EPS) obtained from Botryosphaeria rhodina strains isolated from rotting tropical fruit (graviola, mango, pinha, and orange) grown on sucrose were purified on Sepharose CL-4B. Total acid hydrolysis of each EPS yielded only glucose. Data from methylation analysis and (13)C NMR spectroscopy indicated that the EPS from the graviola isolate consisted of a main chain of glucopyranosyl (1-->3) linkages substituted at O-6 as shown in the putative structure below: [carbohydrate structure: see text]. The EPS of the other fungal isolates consisted of a linear chain of (1-->6)-linked glucopyranosyl residues of the following structure: [carbohydrate structure: see text]. FTIR spectra showed one band at 891 cm(-1), and (13)C NMR spectroscopy showed that all glucosidic linkages were of the beta-configuration. Dye-inclusion studies with Congo Red indicated that each EPS existed in a triple-helix conformational state. beta-(1-->6)-d-Glucans produced as exocellular polysaccharides by fungi are uncommon.

Structural characterization of the cell wall D-glucans isolated from the mycelium of Botryosphaeria rhodina MAMB-05.

Three D-glucans were isolated from the mycelium of the fungus Botryosphaeria rhodina MAMB-05 by sequential extraction with hot-water and hot aqueous KOH (2% w/v) followed by ethanol precipitation. Following their purification by gel permeation chromatography on Sepharose CL-4B, the structural characteristics of the D-glucans were determined by FT-IR and 13C NMR spectroscopy and, after methylation, by GC-MS. The hot-water extract produced a fraction designated Q1A that was a beta-(1-->6)-D-glucan with the following structure: [Formula: see text] The alkaline extract, when subjected to repeated freeze-thawing, yielded two fractions: K1P (insoluble) that comprised a beta-(1-->3)-D-glucan with beta-D-glucose branches at C-6 with the structure: [Formula: see text] and K1SA (soluble) consisting of a backbone chain of alpha-(1-->4)-linked D-glucopyranosyl residues substituted at O-6 with alpha-D-glucopyranosyl residues: [Formula: see text]

Anticlastogenic activity exhibited by botryosphaeran, a new exopolysaccharide produced by Botryosphaeria rhodina MAMB-05.

Biopolymers such as exopolysaccharides (EPS) are produced by microbial species and possess unusual properties known to modify biological responses, among them are antimutagenicity and immunomodulation. Botryosphaeran, a newly described fungal (1-->3; 1-->6)-beta-d-glucan produced by Botryosphaeria rhodina MAMB-05, was administered by gavage to mice at three doses (7.5, 15 and 30mg/kgb.w.per day) over 15 days, and found to be non-genotoxic by the micronucleus test in peripheral blood and bone marrow. Botryosphaeran administered at doses of 15 and 30mg EPS/kgb.w. decreased significantly (p<0.001) the clastogenic effect of cyclophosphamide-induced micronucleus formation resulting in a reduction of the frequency of micronucleated cells of 78 and 82% in polychromatic erythrocytes of bone marrow, and reticulocytes in peripheral blood, respectively. The protective effect was dose-dependent, and strong anticlastogenic activity was exerted at low EPS doses. Variance analysis (ANOVA) showed no significant differences (p<0.05) among the median body weights of the groups of mice treated with botryosphaeran during experiments evaluating genotoxic and protective activities of botryosphaeran. This is the first report on the biological activity attributed to botryosphaeran.

Influence of nutrients on enhancing laccase production by Botryosphaeria rhodina MAMB-05.

The physiological requirements needed to enhance the production of laccases by the ascomycete Botryosphaeria rhodina MAMB-05 in submerged cultivation were examined under non-induced and induced (veratryl alcohol, VA) conditions. Under non-induced conditions (-VA), the initial pH, C:N ratio, and inorganic N source did not influence laccase production, in contrast to Tween 80, soybean oil, and copper, which significantly increased laccase production, and proline and urea, which suppressed laccase formation. In addition, Tween 60 could serve as the sole carbon source for the production of these enzymes. Under VA-induced conditions of fungal growth, factors such as inoculum type, time-point of addition of inducer, initial pH, C:N ratio, and type of N source, influenced the production of laccases; however, unlike the non-induced conditions, proline and urea did not act as suppressors. Each of these physiological conditions exerted different effects on biomass production. The nutritional conditions examined for B. rhodina MAMB-05 are discussed in relation to their influence on fungal growth and laccase production.

Genetic diversity among Botryosphaeria isolates and their correlation with cell wall-lytic enzyme production

Nine isolates of Botryosphaeria spp. were evaluated for their growth and the production of cell wall-lytic enzymes (laccase, pectinase and beta-1,3-glucanase) when grown on basal medium in the absence and presence of the laccase inducer, veratryl alcohol (VA). The genetic relationship among the nine isolates collected from different host plants was determined by RAPD analyses. ITS sequence analysis showed eight closely related isolates classified as Botryosphaeria rhodina, and one isolate classified as Botryosphaeria ribis. RAPD analysis resolved the isolates into three main clusters based upon levels of laccase and beta-1,3-glucanase activity. There appears to be no correlation between pectinase production and genetic diversity among the nine isolates. However, the strain characterized as B. ribis, positioned out of the main cluster, was found to be the highest producer of pectinases in the presence of VA.

Nove isolados de Botryosphaeria spp foram avaliados quanto ao crescimento e produção de enzimas líticas da parede celular (lacase, pectinase e beta-1,3-glucanase) quando cultivados em meio basal na ausência e presença do indutor de lacase álcool veratrílico (VA). As relações genéticas entre os nove isolados coletados de diferentes plantas hospedeiras foram determinadas por RAPD. A análise das seqüências de nucleotídeos da região ITS mostrou oito isolados estreitamente relacionados, os quais foram classificados como Botryosphaeria rhodina e um isolado como Botryosphaeria ribis. A análise por RAPD agrupou os isolados em três grupos principais condizentes com os níveis de atividades de lacase e beta-1,3-glucanase. Nenhuma correlação foi detectada entre a produção de pectinase e a diversidade genética nos nove isolados. Entretanto, a linhagem caracterizada como B. ribis, posicionada fora dos grupos principais, se mostrou maior produtora de pectinase na presença de álcool veratrílico.