Involvement of heme oxygenase as antioxidant defense in soybean nodules

Objective: We have previously demonstrated that the inducible form of heme oxygenase plays a critical role in protecting against oxidative stress in mammals. To gain further insight into the functions of this enzyme in plants, we have tested its activity and expression in soybean nodules subjected to cadmium (Cd) stress.

Materials and methods: Four-weeks-old soybean nodulated plants were treated with different cadmium chloride concentrations (0, 50 and 200 μM) during 48 h. Oxidative stress parameters such as TBARS content, GSH levels and antioxidant enzyme activities were measured as well as heme oxygenase activity and expression. Besides, the effect of biliverdin and Zn-protophorphyrin IX were analized.

Results: Treatment with 200 μM Cd during 48 h caused a 67% increase in TBARS content, whereas GSH decreased 44%, and total superoxide dismutase, gluthatione reductase and guaiacol peroxidase were also inhibited 54, 20 and 60%, respectively. A total of 200 μM Cd produced the overexpression of heme oxygenase-1, as well as a 10-fold enhancement of its activity. Co-administration of biliverdin (10 μM) completely prevented the effects caused by Cd. Treatment with Zn protoporphyrin IX, a strong inhibitor of heme oxygenase, expectedly decreased heme oxygenase-1 activity to half. When the inhibitor was given together with Cd, completely prevented the enzyme induction and oxidative stress parameters were significantly enhanced.

Conclusion: Taking together, these results are indicating that heme oxygenase plays a protective role against oxidative cell damage in soybean nodules.     

Dithioerythritol (DTE) prevents inhibitory effects of triphenyltin (TPT) on the key enzymes of the human sex steroid hormone metabolism

Organotins are known to induce imposex (pseudohermaphroditism) in marine neogastropods and are suggested to act as specific endocrine disruptors, inhibiting the enzyme-mediated conversion of steroid hormones. Therefore, we investigated the in vitro effects of triphenyltin (TPT) on human 5-reductase type 2 (5-Re 2), cytochrome P450 aromatase (P450arom), 17β-hydroxysteroid dehydrogenase type 3 (17β-HSD 3), 3β-HSD type 2 and 17β-HSD type 1 activity. First, the present study demonstrates that significant amounts of TPT occurred in the blood of eight human volunteers (0.17–0.67 μg organotin cation/l, i.e. 0.49–1.92 nmol cation/l). Second, TPT showed variable inhibitory effects on all the enzymes investigated. The mean IC₅₀ values were 0.95 μM for 5-Re 2 (mean of n=4 experiments), 1.5 μM for P450arom (n=5), 4.0 μM for 3β-HSD 2 (n=1), 4.2 μM for 17β-HSD 3 (n=3) and 10.5 μM for 17β-HSD 1 (n=3). To exclude the possibility that the impacts of TPT are mediated by oxidizing essential thiol residues of the enzymes, the putative compensatory effects of the reducing agent dithioerythritol (DTE) were investigated. Co-incubation with DTE (n=3) resulted in dose-response prevention of the inhibitory effects of 100 μM deleterious TPT concentrations on 17β-HSD 3 (EC₅₀ value of 12.9 mM; mean of n=3 experiments), 3β-HSD 2 (0.90 mM; n=3), P450arom (0.91 mM; n=3) and 17β-HSD 1 (0.21 mM; n=3) activity. With these enzymes, the use of 10 mM DTE resulted in an at least 80% antagonistic effect, whereas, the effect of TPT on 5-Re 2 was not compensated. In conclusion, the present study shows that TPT acts as an unspecific, but significant inhibitor of human sex steroid hormone metabolism and suggests that the inhibitory effects are mediated by the interaction of TPT with critical cysteine residues of the enzymes.     

Grafting of cucumber as a means to minimize copper toxicity

The aim of the current work was to determine whether grafting could improve copper (Cu) tolerance of cucumber, and to study the changes induced by the rootstock in the shoot growth at agronomical and physiological levels. A greenhouse experiment was carried out to determine yield, growth, fruit quality, leaf gas exchange, chlorophyll and carotenoids contents, electrolyte leakage, mineral composition and assimilate partitioning of cucumber plants (Cucumis sativus L. cv. ‘Akito’), either ungrafted or grafted onto the commercial rootstock ‘Shintoza’ (Cucurbita maxima Duchesne × Cucurbita moschata Duchesne) and cultured in nutrient film technique (NFT). Plants were supplied with nutrient solutions having three levels of Cu concentration [0.3 (control), 47, or 94 μM]. Significant depression of yield, shoot and root biomass production, and fruit quality (low fruit pH, and high Cu content) in Cu treated cucumber plants was observed, and this effect varied as a function of Cu concentration in NFT solution. The relative yield of cucumber decreased by ≈3.4% for each unit of increase in leaf tissue Cu concentrations above the threshold value (7.8 μg g⁻¹). At the two higher Cu concentrations (47 and 94 μM Cu), the percentages of yield, shoot and root biomass weight reductions were significantly lower in grafted plants in comparison to those of the ungrafted plants. Excessive Cu, especially at 94 μM Cu, inhibited photosynthesis, pigment synthesis, and membrane integrity. The Cu-related reductions in net assimilation, stomatal conductance, chlorophyll and carotenoid content were more severe in ungrafted plants in comparison with those grafted on 'Shintoza’. The percentage of electrolyte leakage was significantly higher in ungrafted plants especially those with severe Cu toxicity (94 μM Cu). The accumulation of Cu in leaf tissue at 47 and 94 μM Cu, with respect to control, were significantly lower in grafted plants (138 and 181%, respectively) in comparison to that of ungrafted plants (about 235 and 392%, respectively). Significant reduction of macro- (N, K, Ca and Mg) and micro-elements (Fe, Mn and Zn) in cucumber leaf tissue was found under moderate and severe Cu stresses especially on ungrafted plants. The improved crop performance of grafted plants was attributed to their strong capacity to inhibit Cu accumulation in the aerial parts and to maintain a better plant nutritional status.     

Fertile plant regeneration from protoplasts of a seed-propagated cultivar of Lilium × formolongi by utilizing meristematic nodular cell clumps

Plant regeneration from protoplasts of Lilium × formolongi cv. Azusa was achieved by utilizing suspension cultures of meristematic nodular cell clumps with a high plant regeneration ability. Creamy-white calli with embryogenic potential were initially induced from the seeds on Murashige and Skoog (MS) medium containing 4.1 μM picloram. The calli were then transferred into a liquid medium of the same composition, in which they turned into compact cell clumps which consisted of meristematic nodules. Protoplasts were readily isolated from these meristematic nodular cell clumps. Colonies were successfully formed from the protoplasts by embedding in 0.1% gellan gum-solidified MS medium containing 4.1 μM picloram and 0.5 M glucose. They regenerated shoots and roots on MS medium containing 2.2 μM benzylaminopurine (BAP). The plants thus obtained produced flowers with normal fertile pollen 8 months after successful transfer into soil. These plants had normal chromosome numbers (2n = 24) but had shorter leaves than original plants. They set seeds after as well as cross pollination.     

The presence of the potentially toxic genera Ostreopsis and Coolia (Dinophyceae) in the North Aegean Sea, Greece

The examination of macrophyte, water and sediment samples, collected at depths less than 1.5 m from 50 different sites along the North Aegean coasts, has revealed, for the first time in Greek coastal waters, the presence of two Ostreopsis species (O. ovata and O. cf. siamensis) and Coolia monotis in the majority of the sampling sites (94% and 100%, respectively). Other epiphytic dinoflagellates of the genera Prorocentrum and Amphidinium and diatoms were accompanying species in this epiphytic community. Morphometric features, plate formula and thecal ornamentation were used for species identification. O. ovata cells were smaller in dorsoventral (DV) diameter and width (W) (26.18–61.88 μm and 13.09–47.60 μm, respectively) in comparison with O. cf. siamensis (35.70–65.45 μm and 23.80–49.98 μm, respectively). In contrast, the anterioposterior (AP) diameter of O. cf. siamensis was smaller (14.28–26.18 μm) resulting in DV/AP ≈ 3, whereas the above ratio for O. ovata was less than 2 (AP ranging between 14.28–35.70 μm). Moreover, the theca of O. ovata cells was ornamented with scattered pores, which fluctuated in a wider range (0.07–0.32 μm) than those of O. cf. siamensis (0.23–0.29 μm). Coolia monotis cells were almost round with average DV diameter 26.88 μm, AP 25.66 μm and width 26.76 μm. Small and large cells were recorded in both field and culture populations of Ostreopsis spp. and C. monotis, while hyaline cysts were observed for O. ovata. The presence of O. ovata and O. cf. siamensis exhibited a clear seasonal pattern dominating (maximum abundance up to 4.05 × 10⁵ cells gr⁻¹ fwm) the period from midsummer to late autumn in years 2003 and 2004, while C. monotis was found also in winter and spring months.     

Efficient <Emphasis Type="Italic">Agrobacterium</Emphasis>-mediated transformation of <Emphasis Type="Italic">Pennisetum glaucum</Emphasis> (L.) R. Br. using shoot apices as explant source

A critical step in the development of a reproducible Agrobacterium tumefaciens mediated transformation system for a recalcitrant species, such as pearl millet, is the establishment of optimal conditions for efficient T-DNA delivery into target tissue from which plants can be regenerated. A multiple shoot regeneration system, without any intervening callus phase, was developed and used as a tissue culture system for Agrobacterium-mediated transformation. Agrobacterium super virulent strain EHA105 harboring the binary vector pCAMBIA 1301 which contains a T-DNA incorporating the hygromycin phosphotransferase (hpt II) and β-glucuronidase (GUS) genes was used to investigate and optimize T-DNA delivery into shoot apices of pearl millet. A number of factors produced significant differences in T-DNA delivery; these included optical density, inoculation duration, co-cultivation time, acetosyringone concentration in co-cultivation medium and vacuum infiltration assisted inoculation. The highest transformation frequency of 5.79% was obtained when the shoot apex explants were infected for 30 min with Agrobacterium O.D.₆₀₀ = 1.2 under a negative pressure of 0.5 × 10⁵ Pa and co-cultivated for 3 days in medium containing 400 μM acetosyringone. Histochemical GUS assay and polymerase chain reaction (PCR) analysis confirmed the presence of the GUS gene in putative transgenic plants, while stable integration of the GUS gene into the plant genome was confirmed by Southern analysis. This is the first report showing reproducible, rapid and efficient Agrobacterium-mediated transformation of shoot apices and the subsequent regeneration of transgenic plants in pearl millet. The developed protocol will facilitate the insertion of desirable genes of useful traits into pearl millet.     

Anti-allergic substances from the rhizomes of Dioscorea membranacea

Extracts of five species of Thai medicinal plants, locally known as Hua-Khao-Yen, were screened for anti-allergic activities using RBL-2H3 cells. Of the five species studied, the ethanolic extract of Dioscorea membranacea exhibited potent inhibitory activity against β-hexosaminidase release as a marker of degranulation in RBL-2H3 cells, with an IC₅₀ value of 37.5 μg/mL. Eight compounds were isolated from this crude ethanolic extract, [two naphthofuranoxepins (1, 2), one phenanthraquinone (3), three steroids (4–6), and two steroidal saponins (7, 8)], and tested for their anti-allergic activities. The results showed that dioscorealide B (2) possessed the highest activity with an IC₅₀ value of 5.7 μM, followed by dioscoreanone (3, IC₅₀ = 7.7 μM), dioscorealide A (1, IC₅₀ = 27.9 μM), and diosgenin (9, IC₅₀ = 29.9 μM). Structure–activity relationship studies of naphthofuranoxepins on anti-allergic activity revealed that the hydroxylation at position 8 conferred higher activity than methoxylation. For diosgenin derivatives, the aglycone was found to possess higher activity than the diglucosylated molecule; whereas substitution with rhamnoglucosides apparently results in loss of activity. Furthermore, effects of dioscorealide A, dioscorealide B, and dioscoreanone on antigen-induced release of TNF- and IL-4 in the late phase reaction were also examined.     

Generation and analyses of the transgenic potatoes expressing heterologous thermostable β-amylase

β-Amylase hydrolyzes the -1,4-glycosidic linkages of starch resulting in the release of maltose. This reaction is of industrial importance for maltose production and for the preparation process of fermented foods and alcoholic beverages. A demand for an acceleration of the rate of enzymatic cleavage of the starch macro-molecule is a prerequisite for large-scale and highly efficient production. Increasing the temperature up to the optimum of approximately 60 °C can significantly speed up the reaction. However, at higher temperatures, the effect on protein denaturation becomes dominant, and the conversion rate decreases. The primary objective of this study was to generate transgenic plants of the “Kennebec” potato variety for production of thermostable β-amylase using Agrobacterium-mediated transformation. Four chimeric genes encoding the β-amylase with or without signal peptide sequences for targeting expression in cytoplasm, amyloplasts, or vacuoles were constructed and driven by high tuber expression promoter from Sucrose synthetase gene Sus4. Forty-two transgenic lines were selected for this study. Transgenic lines with various β-amylase constructs were verified for the existence and expression of the transgenes by PCR approaches. The expression level of the introduced β-amylase protein was estimated by immunoblot analyses using polyclonal antibodies. Recombinant β-amylase was successfully expressed in Escherichia coli B21 (DE3), and temperature ranges of these inducible recombinant proteins were found to be between 40 and 90 °C. This enzymatic complex produced in the in vitro cultured microtubers and field-grown tubers from transgenic potatoes were proved to be stable and active at 60 °C. The relative activities of β-amylase in tubers of field-grown potatoes were compared, and the maximum increase was found with transgenic line #6A of the pSUS4-AMY construct which has an 11-fold greater increase than the untransformed “Kennebec”. Variations of the chemical compositions were found in the selected transgenic lines. Results of this study suggest the feasibility of utilizing thermostable β-amylase in transgenic potatoes for the starch-processing industries.     

Anti-proliferative lichen compounds with inhibitory activity on 12(S)-HETE production in human platelets

Several lichen compounds, i.e. lobaric acid (1), a β-orcinol depsidone from Stereocaulon alpinum L., (+)-protolichesterinic acid (2), an aliphatic -methylene-γ-lactone from Cetraria islandica Laur. (Parmeliaceae), (+)-usnic acid (3), a dibenzofuran from Cladonia arbuscula (Wallr.) Rabenh. (Cladoniaceae), parietin (4), an anthraquinone from Xanthoria elegans (Link) Th. Fr. (Calaplacaceae) and baeomycesic acid (5), a β-orcinol depside isolated from Thamnolia vermicularis (Sw.) Schaer. var. subuliformis (Ehrh.) Schaer. were tested for inhibitory activity on platelet-type 12(S)-lipoxygenase using a cell-based in vitro system in human platelets. Lobaric acid (1) and (+)-protolichesterinic acid (2) proved to be pronounced inhibitors of platelet-type 12(S)-lipoxygenase, whereas baeomycesic acid (5) showed only weak activity (inhibitory activity at a concentration of 100 μg/ml: 1 93.4±6.62%, 2 98,5±1.19%, 5 14.7±2.76%). Usnic acid (3) and parietin (4) were not active at this concentration. 1 and 2 showed a clear dose–response relationship in the range of 3.33–100 μg/ml. According to the calculated IC₅₀ values the highest inhibitory activity was observed for the depsidone 1 (IC₅₀=28.5 μM) followed by 2 (IC₅₀=77.0 μM). The activity of 1 was comparable to that of the flavone baicalein, which is known as a selective 12(S)-lipoxygenase inhibitor (IC₅₀=24.6 μM).     

Effect of oxygen transfer rate on β-carotene production from synthetic medium by Blakeslea trispora in shake flask culture

The effect of oxygen transfer rate (OTR) on β-carotene production by Blakelsea trispora in shake flask culture was investigated. The results indicated that the concentration of β-carotene (704.1 mg/l) was the highest in culture grown at maximum OTR of 20.5 mmol/(l h). In this case, the percentage of zygospores was over 50.0% of the biomass dry weight. On the other hand, OTR level higher than 20.5 mmol/(l h) was found to be detrimental to cell growth and pigment formation. To elucidate the effect of oxidative stress on β-carotene synthesis, the accumulation of hydrogen peroxide during fermentation under different OTRs was determined. A linear response of β-carotene synthesis to the level of H₂O₂ was observed, indicating that β-carotene synthesis is stimulated by H₂O₂. However, there was an optimal concentration of H₂O₂ (2400 μM) in enhancing β-carotene synthesis. At a higher concentration of H₂O₂, β-carotene decreased significantly due to its toxicity.     

Halophila ovalis (R. Br.) Hook. f. from a submarine hot spring in southern Japan

Colonies of the seagrass Halophila ovalis are found growing adjacent to coral Acropora sp. and Seriatopora hystrix in a submarine hot spring (at 15.7 m depth, 28.6°C) at the north coast of Taketomi Island, near the southern tip of Japan. Halophila plants grow in sea water containing sulphide 930 μg S ml⁻¹ and on the substratum with fine precipitates of the submarine hot spring which have sulphide content up to 5400 μg S g⁻¹ DW. The accumulated sulphide concentration reaches as high as 8400 μg S g⁻¹ DW in under ground tissues and 5700 μg S g⁻¹ DW in above-ground tissues, respectively. It is suggested that, not the sulphide concentration but light and possibly water temperature are the limiting factors for the Halophila colonization in the submarine hot spring.     

A robust genetic transformation protocol to obtain transgenic shoots of Solanum tuberosum L. cultivar ‘Kufri Chipsona 1’

The genetic transformation of plants is an important biotechnological tool used for crop improvement for many decades. The present study was focussed to investigate various factors affecting genetic transformation of potato cultivar ‘Kufri Chipsona 1’. It was observed that explants pre-cultured for 2 days on MS2 medium (MS medium containing 10 µM silver nitrate, 10 µM BA, 15 µM GA3), injured with a surgical blade and co-cultivated with Agrobacterium tumefaciens strain EHA105 [O.D600 (0.6)] for 2 days results in maximum transient β-glucuronidase (GUS) expression. The addition of 100 µM acetosyringone in MS2 medium also increased rate of transient GUS expression in both the explants. Clumps of putative transgenic shoots were regenerated using the optimised culture conditions from leaf and internodal explants. The stable integration of T-DNA was established using histochemical staining for GUS and amplification of DNA fragment specific to nptII and uidA genes. Within the clumps, around 67.85% of shoots showed uniform GUS expression in all the tissues and about 32.15% shoots show intermittent GUS expression establishing chimeric nature. Uniform GUS staining of the tissue was used as initial marker of non-chimeric transgenic shoots. Quantitative expression of nptII transgene was found to be directly proportional to uniformity of GUS staining in transgenic shoots. The present investigation indicated that manipulation of culture conditions and the medium composition may help to get transgenic shoots with uniform expression of transgene in all the tissues of potato cultivar ‘Kufri Chipsona 1’.     

New series of potent delta-opioid antagonists containing the H-Dmt-Tic-NH-hexyl-NH-R motif

Heterodimeric compounds H-Dmt-Tic-NH-hexyl-NH-R (R = Dmt, Tic, and Phe) exhibited high affinity to δ- (K_iδ = 0.13–0.89 nM) and μ-opioid receptors (K_iμ = 0.38–2.81 nM) with extraordinary potent δ antagonism (pA₂ = 10.2–10.4). These compounds represent the prototype for a new class of structural homologues lacking μ-opioid receptor-associated agonism (IC₅₀ = 1.6–5.8 μM) based on the framework of bis-[H-Dmt-NH]-alkyl (Okada, Y.; Tsuda, Y.; Fujita, Y.; Yokoi, T.; Sasaki, Y.; Ambo, A.; Konishi, R.; Nagata, M.; Salvadori, S.; Jinsmaa, Y.; Bryant, S. D.; Lazarus, L. H. J. Med. Chem. 2003, 46, 3201), which exhibited both high μ affinity and bioactivity.     

Accumulation of osmolytes and osmotic adjustment in water-stressed wheat (Triticum aestivum) and maize (Zea mays) as affected by calcium and its antagonists

Maize (Zea mays) and wheat (Triticum aestivum) were water stressed for 4 days at early vegetative growth (15-day-old) using PEG-6000 (−1.0 MPa), in the presence of 1 mM CaSO₄, 50 μM Verapamil (VP; calcium channel blocker); 50 μM Trifluoperazine (TFP; calmodulin antagonist) and then put to recovery in order to investigate the changes in osmoregulation in plants having C₃ and C₄ metabolism. Accumulation of proline (Pro) and quaternary ammonium compounds (QAC's), activities of pyrroline-5-carboxylate reductase (P5CR), proline dehydrogenase (PDH), water potential (Ψ_w), osmotic adjustment (OA), relative elongation rate (RER) and electrolyte leakage (EL) were examined during stress and recovery. Maize had significantly higher accumulation of Pro while wheat showed relatively more accumulation of QAC's. The activities of P5CR and PO were also significantly higher in maize than wheat. Maize shoots under stress showed higher Ψ_w, OA, RER and less EL than wheat shoots. Upon recovery from stress, maize regained its growth and water potential faster than wheat. Ca²⁺ elevated the accumulation of osmolytes in both the plants but OA was less sensitive to it. In the presence of Ca²⁺, wheat showed significantly more accumulation of osmolytes, higher Ψ_w, RER than maize. Ca²⁺ inhibitors partially reversed the effects of calcium indicating its involvement in governing solute accumulation. The differential sensitivity of maize and wheat towards water stress may be related to variation in endogenous calcium expression and its function.     

Synthesis and in vitro anti-hepatitis B and C virus activities of ring-expanded ('fat') nucleobase analogues containing the imidazo[4,5-e][1,3]diazepine-4,8-dione ring system

As part of our structure–activity relationship studies, we report here the synthesis and in vitro anti-HBV and anti-HCV activities of a number of ring-expanded (‘fat’) nucleobases containing the imidazo[4,5-e][1,3]diazepine-4,8-dione ring system. One of the compounds, ZP-88, exhibited a good activity/toxicity profile against HBV by inhibition of the synthesis of extracellular virion release (EC₅₀ = 1.7 μM, CC₅₀ = 286 μM, SI = 168) and intracellular HBV replication intermediates (EC₅₀ = 8.4 μM, CC₅₀ = 286 μM, SI = 34) in cultured human hepatoblastoma 2.2.15 cells. By contrast, most of the compounds tested against HCV had only marginal activity/toxicity profile, although that was still better than that of the reference compound ribavirin.     

Antibacterial activity from Penicillium corylophilum Dierckx

A strain of Penicillium corylophilum isolated from Brazilian soil sample was submitted to different culture conditions to investigate the production of secondary metabolites with antimicrobial activity. The largest number of conidia was obtained after 5 days of incubation in oat medium and the highest level of antimicrobial activity was produced when the fungus culture was developed in the Czapek medium. The activity against Staphylococcus aureus was found only in the chloroform extract from Czapek culture broth, which also showed activity against Micrococcus luteus. Fumiquinozoline F was isolated from the active chloroform extract by using chromatographic methods. The minimal inhibitory concentration (MIC) values for M. luteus and S. aureus were 99 μg/mL and 137 μg/mL, respectively.     

Isolation and characterization of a novel thermostable non-specific lipid transfer protein-like antimicrobial protein from motherwort (Leonurus japonicus Houtt) seeds

In screening for potent antimicrobial proteins from plant seeds, a novel heat-stable antimicrobial protein, designated LJAMP2, was purified from seeds of the motherwort (Leonurus japonicus Houtt), a medicine herb, with a procedure involving cation exchange chromatography on a CM FF column, and reverse phase HPLCs on C8 column and C18 column. LJAMP2 exhibited a molecular mass of 6.2 kDa determined. Automated Edman degradation determined the partial N-terminal sequence of LJAMP2 to be NH2-AIGCNTVASKMAPCLPYVTGKGPLGGCCGGVKGLIDAARTTPDRQAVCNCLKTLAKSYSG, which displays homology with plant non-specific lipid transfer proteins (nsLTPs). In vitro bioassays showed that LJAMP2 inhibits the growth of a variety of microbes, including filamentous fungi, bacteria and yeast. The growth of three phytopathogenic fungi, Alternaria brassicae, Botrytis maydis, and Rhizoctonia cerealis, are inhibited at 7.5 μM of LJAMP2, whereas Bacillus subtilis is about 15 μM. The IC₅₀ of LJAMP2 for Aspergillus niger, B. maydis, Fusarium oxysporum, Penicillium digitatum and Saccharomyces cerevisiae are 5.5, 6.1, 9.3, 40.0, and 76.0 μM, respectively.     

Purification, characterization and synergistic activity of β-1,3-glucanase and antibiotic extract from an antagonistic Bacillus subtilis NSRS 89-24 against rice blast and sheath blight

Antifungal compounds in the culture filtrate from Bacillus subtilis NSRS 89-24 that inhibited the growth of Pyricularia grisea and Rhizoctonia solani were mainly heat stable as the filter sterilized culture filtrate showed higher activity than an autoclaved one. The heat stable and labile components were due to an antibiotic and a β-1,3-glucanase, respectively. This β-1,3-glucanase was purified and characterized. Glucanase activity in the culture medium of B. subtilis NSRS 89-24 was inducible in the presence of 0.3% chitin, reaching a maximum on day 5. After purification, activity was associated with a protein of molecular mass of approximately 95.5 kDa by both gel filtration and native PAGE. Two major bands of M_r 64.6 and 32.4 kDa were revealed by SDS–PAGE. The enzyme had a K_m of 0.9 mg/ml, and V_max of 0.11 U, the optimal pH was 6.5–9.5 and was stable up to 50 °C. Both the pure enzyme and the antibiotic extract from the culture filtrate of the B. subtilis separately inhibited R. solani and P. grisea with MIC values of 12.5 and 6.25 mU/ml and 3.13 and 1.56 μg/ml, respectively. The glucanase enzyme in combination with the antibiotic showed a strong synergistic inhibitory effect on the hyphal growth of both fungi.