Influence of cultivation conditions on pyrene degradation by the fungus Pleurotus Ostreatus D1

For the first time the dependence of completeness of pyrene degradation by the white-rot fungus Pleurotus ostreatus D1 on cultivation conditions was found. In Kirk’s medium about 65.6 ± 0.9% of the initial pyrene was metabolized after 3 weeks, with pyrene-4,5-dihydrodiol accumulating. This process was accompanied by laccase production only. In basidiomycetes rich medium, P. ostreatus D1 metabolized up to 89.8 ± 2.3% of pyrene within 3 weeks without pyrene-4,5-dihydrodiol accumulation throughout the time of cultivation. Phenanthrene and phthalic acid were identified as the metabolites produced from pyrene degradation under these conditions. Accumulation of phenanthrene with its subsequent disappearance was observed. One more metabolite probably was the product of phenanthrene degradation. Pyrene metabolism in basidiomycetes rich medium was accompanied first by laccase and tyrosinase production and later by versatile peroxidase production. The cell-associated activities of laccase, tyrosinase, and versatile peroxidase were found. The data obtained indicate that both enzymes (laccase and versatile peroxidase) are necessary for complete degradation of pyrene. Furthermore, both cell-associated and extracellular laccases can catalyse the first stages of pyrene degradation, and versatile peroxidase can be necessary for oxidation of the resulting metabolites.     

Degradation and transformation of anthracene by white-rot fungus Armillaria sp. F022

Characterization of anthracene metabolites produced by Armillaria sp. F022 was performed in the enzymatic system. The fungal culture was conducted in 100-mL Erlenmeyer flask containing mineral salt broth medium (20 mL) and incubated at 120 rpm for 5–30 days. The culture broth was then centrifuged at 10,000 rpm for 45 min to obtain the extract. Additionally, the effect of glucose consumption, laccase activity, and biomass production in degradation of anthracene were also investigated. Approximately, 92 % of the initial concentration of anthracene was degraded within 30 days of incubation. Dynamic pattern of the biomass production was affected the laccase activity during the experiment. The biomass of the fungus increased with the increasing of laccase activity. The isolation and characterization of four metabolites indicated that the structure of anthracene was transformed by Armillaria sp. F022 in two routes. First, anthracene was oxidized to form anthraquinone, benzoic acid, and second, converted into other products, 2-hydroxy-3-naphthoic acid and coumarin. Gas chromatography–mass spectrometry analysis also revealed that the molecular structure of anthracene was transformed by the action of the enzyme, generating a series of intermediate compounds such as anthraquinone by ring-cleavage reactions. The ligninolytic enzymes expecially free extracellular laccase played an important role in the transformation of anthracene during degradation period.     

Effect of different cultivation conditions and inducers on the production of laccase by the litter-dwelling fungal isolate Fusarium incarnatum LD-3 under solid substrate fermentation

The litter-dwelling fungus Fusarium incarnatum LD-3 has been identified as a novel producer of laccase. The present work was oriented towards the optimization of various cultivation conditions for maximizing laccase production under solid substrate fermentation. The process parameters were optimized by the “one factor at a time” approach. Maximum laccsase production was obtained at pH 5.0 and at a temperature of 28 °C with 60 % moisture content using rice bran as a substrate. The laccase production was enhanced in the presence of aromatic inducer, i.e. ortho-dianisidine at a concentration of 0.5 mM. Laccase production was further increased by 52.56 % when the medium was supplemented with 2 % (v/v) alcohol. Among the various amino acids tested as a growth factor and nitrogen source, D-Serine and DL-2 Amino n-butyric acid, DL-Alanine and L-Glycine were found to be the most suitable for laccase production. The highest laccase production (1,352.64 U/g) was achieved under optimized conditions, and was 2.1-fold higher than the unoptimized conditions. Thus, the novel litter-dwelling fungal isolate Fusarium incarnatum LD-3 seems to be an efficient producer of laccase and can be further exploited for biotechnological applications. This is the first report on the optimization of cultivation conditions and inducers for laccase production from Fusarium incarnatum LD-3.     

Molecular cloning and expression of a novel laccase showing thermo- and acid-stability from the endophytic fungus Phomopsis liquidambari and its potential for growth promotion of plants

A novel laccase (LACB3) from the endophytic fungus, Phomopsis liquidambari, was cloned and its potential to promote peanut growth was evaluated. The full-length cDNA is 1,731 bp, encoding a mature protein of 556 amino acids with a molecular mass of 60.1 kDa. Using 2,2′-azino-bis(3-ethylbenzthiazoline-6-sulfonate), LACB3 exhibited a K _m and k _cat of 85 μM and 92.7 s^?1, respectively. The enzyme had maximal activity at pH 2.5 and 50 °C and retained 50 % of its activity after 20 h at 50 °C. When LACB3 was applied to soil, the peanut biomass was increased by 12 %, and the content of vanillic acid, coumaric acid and 4-hydroxybenzoic acid in soil were decreased by 21, 27 and 40 %, respectively. These results suggest substantial potential for the use of P. liquidambari or its laccase in agriculture.     

Production of laccase and manganese peroxidase by Pleurotus pulmonarius in solid-state cultures and application in dye decolorization

The production of ligninolytic enzymes (laccase and Mn-dependent peroxidase) by the white-rot fungus Pleurotus pulmonarius (FR.) Quélet was studied in solid-state cultures using agricultural and food wastes as substrate. The highest activities of laccase were found in wheat bran (2,860?±?250 U/L), pineapple peel (2,450?±?230 U/L), and orange bagasse (2,100?±?270 U/L) cultures, all of them at an initial moisture level of 85 %. The highest activities of Mn peroxidase were obtained in pineapple peel cultures (2,200?±?205 U/L) at an initial moisture level of 75 %. In general, the condition of high initial moisture level (80–90 %) was the best condition for laccase activity, while the best condition for Mn peroxidase activity was cultivation at low initial moisture (50–70 %). Cultures containing high Mn peroxidase activities were more efficient in the decolorization of the industrial dyes remazol brilliant blue R (RBBR), Congo red, methylene blue, and ethyl violet than those containing high laccase activity. Also, crude enzymatic extracts with high Mn peroxidase activity were more efficient in the in vitro decolorization of methylene blue, ethyl violet, and Congo red. The dye RBBR was efficiently decolorized by both crude extracts, rich in Mn peroxidase activity or rich in laccase activity.     

Effect of Different Carbon Sources on Decolourisation of an Industrial Textile Dye Under Alkaline–Saline Conditions

White-rot fungal strains of Trametes versicolor and Phanerochaete chrysosporium were selected to study the decolourisation of the textile dye, Reactive Black 5, under alkaline–saline conditions. Free and immobilised T. versicolor cells showed 100 % decolourisation in the growth medium supplemented with 15 g l^?1 NaCl, pH 9.5 at 30 °C in liquid batch culture. Continuous culture experiments were performed in a fixed-bed reactor using free and immobilised T. versicolor cells and allowed 85–100 % dye decolourisation. The immobilisation conditions for the biomass and the additional supply of carbon sources improved the decolourisation performance during a long-term trial of 40 days. Lignin peroxidase, laccase and glyoxal oxidase activities were detected during the experiments. The laccase activity varied depending on carbon source utilized and glycerol-enhanced laccase activity compared to sucrose during extended growth.     

A Dual Role of Se on Cd Toxicity: Evidences from the Uptake of Cd and Some Essential Elements and the Growth Responses in Paddy Rice

This study was carried out to investigate the effects of selenium (Se) on the uptake and translocation of cadmium (Cd) and essential elements in paddy rice (Oryza sativa L., Shuangyou 998). Selenium could alleviate/aggravate Cd toxicity in paddy rice, which depended on the dosages of Se and/or Cd. When Cd treatment level was as low as 35.6 μM, ≤12.7 μM Se could inhibit the uptake of Cd in paddy rice and increase the biomass of paddy rice; however, with Cd levels reaching 89–178 μM, the addition of Se resulted in increases in Cd uptake and exacerbated the growth of paddy rice. Cd always inhibited the uptake of Se. Cd alone suppressed the uptake of Ca, Mg, Mn, Cu, and Zn; however, Se reversed the decreases in the concentrations of the said elements, suggesting an element regulation mechanism to relieve Cd toxicity. Without Cd in the solution, low doses of Se increased the biomasses of shoots and roots at the expense of the more or less decreases in the concentrations of Ca, Mg, K, Fe, Mn, Cu, and shoot Zn, indicating an antagonistic effect of Se on these cations. The presence of Cd could also reverse these decreases especially at the highest treatment levels for both Se and Cd, also suggesting an element regulation mechanism responsible for the detoxification of high dosages of Se. Consequently, when Se is used to alleviate Cd toxicity, attention must be paid to the Cd pollution extent and doses of Se supplement.     

Selenium Modifies the Effect of Short-Term Chilling Stress on Cucumber Plants

The objective of this study was to investigate the effect of selenium (Se) supply (0, control; 2.5, 5, 10, or 20 μM) on cucumber (Cucumis sativus L.) cv. Polan F1 plants grown under short-term low temperature stress. About 14–16 day-old seedlings, grown at an optimal temperature (25/20°C; day/night), were exposed to short-term chilling stress with a day/night temperature of 10°C/5°C for 24 h, for a further 24 h at 20°C/15°C, and then transferred to 25/20°C (re-warming) for 7 days. Se did not affect the fresh weight (FW) of plants at a concentration of 2.5–10 μM, but in the presence of 20 μM Se, the biomass of shoots significantly decreased. The contents of chlorophylls and carotenoids witnessed no significant change after Se supplementation. Compared with the control, the Se-treated plants showed an increase of proline content in leaves, once after chilling and again after 7 days of re-warming. However, proline levels were much higher immediately after chilling than after re-warming. The malondialdehyde (MDA) content in the root of plants treated with 2.5–10 μM Se decreased directly after stress. This was in comparison with the plants grown without Se, whereas it increased in roots and leaves of plants exposed to 20 μM Se. Seven days later, the MDA level in the root of plants grown in the presence of Se was still lower than those of plants not treated with Se and generally witnessed no significant change in leaves. Although Se at concentrations of 2.5–10 μM modified the physiological response of cucumber to short-term chilling stress, causing an increase in proline content in leaves and diminishing lipid peroxidation in roots, the resistance of plants to low temperature was not clearly enhanced, as concluded on the basis of FW and photosynthetic pigments accumulation.     

Glaucocalyxin A and B Regulate Growth and Induce Oxidative Stress in Lettuce (Lactuca sativa L.) Roots

Glaucocalyxin (Gla) A–C are major ent-kaurane diterpenoids isolated from Isodon japonicus var. glaucocalyx (Maxim.) H. W. Li. This study investigated the possible interference of these diterpenoids with root growth and its mechanism of action in lettuce (Lactuca sativa L.) seedlings. Results indicated the dual stimulatory and inhibitory effects of Gla A and B on root growth and their phytotoxic effects on root hair development. The promotion of root growth by lower levels of Gla A and B (20–40 μM) resulted from enhanced cell length and increased mitotic activity. However, higher concentrations (80–200 μM) of Gla A and B had inhibitory effects. In addition, Gla A and B inhibited root hair development of lettuce seedlings in a dose-dependent manner at concentrations between 20 and 200 μM. Exposure of lettuce roots to Gla A and B at 200 μM increased levels of malondialdehyde and the generation of O ₂ ^·? , indicating lipid peroxidation and induction of oxidative stress. Activities of the antioxidant enzymes superoxide dismutase, catalase, and peroxidase were significantly elevated. Reactive oxygen species (ROS) scavengers dihydroxybenzene disulfonic acid (Tiron) and dimethylthiourea at 100 μM could efficiently alleviate the phytotoxicity induced by Gla A and B at 200 μM. These results demonstrated that the deleterious effect of Gla A and B at higher concentrations (80–200 μM) on roots may occur through the imposition of oxidative stress on cell growth and cell division. Due to the lack of an α,β-unsaturated ketone in α-methylenecyclopentanone moiety, Gla C could not induce ROS generation and exhibited no effect on the roots, even at the highest concentration (200 μM). Therefore, the α-methylenecyclopentanone moiety in the ent-kaurene diterpenoids was presented as an essential possible active center for the phytotoxicity.     

Symbiotic Fungi Produce Laccases Potentially Involved in Phenol Degradation in Fungus Combs of Fungus-Growing Termites in Thailand

Fungus-growing termites efficiently decompose plant litter through their symbiotic relationship with basidiomycete fungi of the genus Termitomyces. Here, we investigated phenol-oxidizing enzymes in symbiotic fungi and fungus combs (a substrate used to cultivate symbiotic fungi) from termites belonging to the genera Macrotermes, Odontotermes, and Microtermes in Thailand, because these enzymes are potentially involved in the degradation of phenolic compounds during fungus comb aging. Laccase activity was detected in all the fungus combs examined as well as in the culture supernatants of isolated symbiotic fungi. Conversely, no peroxidase activity was detected in any of the fungus combs or the symbiotic fungal cultures. The laccase cDNA fragments were amplified directly from RNA extracted from fungus combs of five termite species and a fungal isolate using degenerate primers targeting conserved copper binding domains of basidiomycete laccases, resulting in a total of 13 putative laccase cDNA sequences being identified. The full-length sequences of the laccase cDNA and the corresponding gene, lcc1-2, were identified from the fungus comb of Macrotermes gilvus and a Termitomyces strain isolated from the same fungus comb, respectively. Partial purification of laccase from the fungus comb showed that the lcc1-2 gene product was a dominant laccase in the fungus comb. These findings indicate that the symbiotic fungus secretes laccase to the fungus comb. In addition to laccase, we report novel genes that showed a significant similarity with fungal laccases, but the gene product lacked laccase activity. Interestingly, these genes were highly expressed in symbiotic fungi of all the termite hosts examined.     

The effect of zinc on the growth and physiological and biochemical parameters in seedlings of Eruca sativa (L.) (Rocket)

Eruca sativa seedlings were treated with different Zn concentrations (0, 250, 500, 1,000, 2,000 μg g^?1 dried growth medium) under controlled conditions. The seedlings were harvested 20 days after Zn treatment. Physiological parameters, such as root and shoot length, fresh and dry weight, were measured and Zn content of roots and shoots was determined. Furthermore, various biochemical parameters were studied on E. sativa leaves: enzymatic antioxidants, such as superoxide dismutase (SOD), catalase (CAT), guaiacol peroxidase (POD), ascorbate peroxidase (APX), and non-enzymatic antioxidants, such as ascorbate, non-protein thiols. Malondialdehyde, which is an index of lipid peroxidation, was assayed. Zn treatment did not have any significant effect on fresh and dry weights, whereas 500 μg g^?1 Zn increased root growth significantly (p < 0.05). Zn accumulated in roots 2–8 times more than it did in leaves. Lipid peroxidation increased in proportion with the increase in Zn. Although a decrease in SOD and CAT activities at increased Zn was found, a significant increase in APX and POD was observed at 500 and 1,000 μg g^?1 Zn, respectively. In addition, an increase in the amounts of non-protein thiols and total AsA (Ascorbate) was observed with the increase in Zn.     

Biodegradation of chestnut shell and lignin-modifying enzymes production by the white-rot fungi Dichomitus squalens,Phlebia radiata

As a discarded lignocellulosic biomass, chestnut shell is of great potential economic value, thus a sustainable strategy is needed and valuable for utilization of this resource. Herein, the feasibility of biological processes of chestnut shell with Dichomitus squalens, Phlebia radiata and their co-cultivation for lignin-modifying enzymes (LMEs) production and biodegradation of this lignocellulosic biomass was investigated under submerged cultivation. The treatment with D. squalens alone at 12 days gained the highest laccase activity (9.42 ± 0.73 U mg^?1). Combined with the data of laccase and manganese peroxidase, oxalate and H₂O₂ were found to participate in chestnut shell degradation, accompanied by a rapid consumption of reducing sugar. Furthermore, specific surface area of chestnut shell was increased by 77.6–114.1 % with the selected fungi, and total pore volume was improved by 90.2 % with D. squalens. Meanwhile, the surface morphology was observably modified by this fungus. Overall, D. squalens was considered as a suitable fungus for degradation of chestnut shell and laccase production. The presence of LMEs, H₂O₂ and oxalate provided more understanding for decomposition of chestnut shell by the white-rot fungi.     

Comparative effects of selenite and selenate on growth and selenium accumulation in lettuce plants under hydroponic conditions

The effects of different concentrations of selenite (2–30 μM) and selenate (2–60 μM) on biomass production, leaf area, and concentrations of photosynthetic pigments in lettuce plants were investigated. On the basis of the obtained results, the threshold of toxicity for the selenite and selenate has been designated. The toxicity thresholds for selenite and selenate were determined at concentrations of 15 and 20 μM, respectively. Next, four selenium (Se) concentrations (2, 4, 6 or 15 μM), below or near the toxicity boundary, have been selected for the lettuce biofortification experiment. In the biofortified plants, the oxidant status (levels of lipid peroxidation and H₂O₂ concentrations), as well as Se and sulphur (S) accumulation were analysed. In the edible parts of the lettuce, the Se concentration was higher for selenate presence compared to selenite; however, this difference was not as obvious as it was noted in the case of the roots, where selenite application caused the high accumulation of Se. An application of 15 μM Se as selenite caused a decline in the biomass and an intensification of prooxidative processes in the plant’s tissues and as toxic should be excluded from further biofortification experiments. These results indicate that an application of either selenate or selenite to the nutrient solution at concentrations below 15 μM can be used for biofortification of lettuce with Se, evoking better plant growth and not inducing significant changes in the oxidant status, the concentration of assimilation pigments and S accumulation.     

Selenium (Se) Seed Priming Induced Growth and Biochemical Changes in Wheat Under Water Deficit Conditions

Insufficient stand establishment at early growth stages in wheat (Triticum aestivum L.) due to drought stress is a major problem that limits overall efficiency and yield of crop. Priming of seed is an effective method for raising seed performance and improving tolerance of crops to abiotic stresses especially drought. The seeds of two local wheat cultivars (Kohistan-97 and Pasban-90) were soaked in distilled water or sodium selenate solutions of 25, 50, 75, and 100 μM for 1/2 or 1 h at 25 °C and later re-dried to their original moisture levels before sowing. One-hour priming significantly increased root length stress tolerance index, dry matter stress tolerance index, and total biomass of seedlings; however, no significant effect of changing duration of Se seed priming was observed on plant height stress tolerance index and shoot/root ratio. Among cultivars, Kohistan-97 was found to be more responsive to Se seed treatment as 1 h priming at 100 μM significantly increased its total biomass by 43 % as compared to control treatment. Although biomass of seedlings was not affected with Se seed priming under normal conditions, but it increased significantly with increase in rates of Se under drought stress conditions. One-hour priming at 75 μM increased the total sugar content and total free amino acids in both wheat cultivars. A more significant decrease in soluble proteins of seedlings was observed by 1 h priming than 1/2 h priming under drought stress conditions.     

Production and in Vivo Antioxidant Activity of Zn,Ge, Se-enriched Mycelia by Cordyceps sinensis SU-01

Cordyceps sinensis, a traditionally edible and medicinal fungus in China, cannot be artificially solid-cultured. Zinc (Zn), germanium (Ge), and selenium (Se) are the essential trace elements for human body. In this work, C. sinensis SU-01 was cultivated in liquid medium simultaneously containing Zn, Ge, and Se. The bioactive ingredients and in vivo antioxidant activities of Zn, Ge, Se-enriched mycelia (ZGSM) of C. sinensis SU-01 were investigated. Under the determined conditions, the Zn, Ge, and Se contents of ZGSM were 2543.16 ± 158.92, 1873.85 ± 81.82, and 1260.16 ± 107.12 μg/g, respectively. The optimal concentrations of Zn, Ge, and Se had a positive effect on biosynthesis of protein, polysaccharide, cordycepic acid, and amino acids. The activities of glutathione peroxidase (GSH-Px) and superoxide dismutase (SOD) of mice blood were 3.72 ± 0.15 and 28.74 ± 2.53 % higher than that of control, respectively, and the content of malondialdehyde (MDA) was 41.01 ± 3.66 % lower than that of control.     

Induction of laccases in Trametes versicolor by aqueous wood extracts

The induction of laccase isoforms in Trametes versicolor HEMIM-9 by aqueous extracts (AE) from softwood and hardwood was studied. Samples of sawdust of Pinus sp., Cedrela sp., and Quercus sp. were boiled in water to obtain AE. Different volumes of each AE were added to fungal cultures to determine the amount of AE needed for the induction experiments. Laccase activity was assayed every 24 h for 15 days. The addition of each AE (50 to 150 μl) to the fungal cultures increased laccase production compared to the control (0.42 ± 0.01 U ml^?1). The highest laccase activities detected were 1.92 ± 0.15 U ml^?1 (pine), 1.87 ± 0.26 U ml^?1 (cedar), and 1.56 ± 0.34 U ml^?1 (oak); laccase productivities were also significantly increased. Larger volumes of any AE inhibited mycelial growth. Electrophoretic analysis revealed two laccase bands (lcc1 and lcc2) for all the treatments. However, when lcc2 was analyzed by isoelectric focusing, inducer-dependent isoform patterns composed of three (pine AE), four (oak AE), and six laccase bands (cedar AE) were observed. Thus, AE from softwood and hardwood had induction effects in T. versicolor HEMIM-9, as indicated by the increase in laccase activity and different isoform patterns. All of the enzymatic extracts were able to decolorize the dye Orange II. Dye decolorization was mainly influenced by pH. The optimum pH for decolorization was pH 5 (85 %), followed by pH 7 (50 %) and pH 3 (15 %). No significant differences in the dye decolorizing capacity were detected between the control and the differentially induced laccase extracts (oak, pine and cedar). This could be due to the catalytic activities of isoforms with pI 5.4 and 5.8, which were detected under all induction conditions.     

Brassinosteroids protect photosynthesis and antioxidant system of eggplant seedlings from high-temperature stress

The effects of 24-epibrassinolide under high temperature in eggplant (Solanum melongena L.) seedlings were studied by investigating the plant growth, chlorophyll content, photosynthesis and antioxidant systems. High temperature significantly inhibited the plant growth and markedly decreased the chlorophyll content, net photosynthetic rate, stomatal conductance and transpiration rate, while it increased intercellular CO₂ concentration. In a similar manner, high temperature also decreased significantly maximum quantum efficiency of PSII, potential photochemical efficiency, the quantum efficiency of PSII, photochemical quenching, the excitation capture efficiency of open centers, and increased non-photochemical quenching. Application of 0.05–0.2 μM EBR remarkably promoted the plant growth and alleviated high-temperature-induced inhibition of photosynthesis. Under high temperature, reactive oxygen species levels and lipid peroxidation were markedly increased, which were remarkably inhibited by application of 0.05–0.2 μM EBR. The activities of antioxidative enzymes such as superoxide dismutase, peroxidase, catalase and ascorbate peroxidase, and contents of ascorbic acid and reduced glutathione were significantly increased during high-temperature treatments, and these increases were more pronounced than those of EBR at 0.05–0.2 μM treatment. The EBR treatment also greatly enhanced contents of proline, soluble sugar and protein under high-temperature stress. Taken together, it can be concluded that 0.05–0.2 μM EBR could alleviate the detrimental effects of high temperatures on plant growth by increasing photosynthetic efficiency and enhancing antioxidant enzyme systems. Addition of 0.1 μM EBR had the best ameliorative effect against high temperature, while the addition of 0.4 μM EBR had no significant effects.     

Characterization of Lignocellulolytic Activities from a Moderate Halophile Strain of Aspergillus caesiellus Isolated from a Sugarcane Bagasse Fermentation

A moderate halophile and thermotolerant fungal strain was isolated from a sugarcane bagasse fermentation in the presence of 2 M NaCl that was set in the laboratory. This strain was identified by polyphasic criteria as Aspergillus caesiellus. The fungus showed an optimal growth rate in media containing 1 M NaCl at 28°C and could grow in media added with up to 2 M NaCl. This strain was able to grow at 37 and 42°C, with or without NaCl. A. caesiellus H1 produced cellulases, xylanases, manganese peroxidase (MnP) and esterases. No laccase activity was detected in the conditions we tested. The cellulase activity was thermostable, halostable, and no differential expression of cellulases was observed in media with different salt concentrations. However, differential band patterns for cellulase and xylanase activities were detected in zymograms when the fungus was grown in different lignocellulosic substrates such as wheat straw, maize stover, agave fibres, sugarcane bagasse and sawdust. Optimal temperature and pH were similar to other cellulases previously described. These results support the potential of this fungus to degrade lignocellulosic materials and its possible use in biotechnological applications.     

Biosynthesis and safety evaluation of ZnO nanoparticles

The secrets gleaned from nature have led to the development of biomimetic approaches for the growth of advanced nanomaterials. Biological methods for nanoparticle synthesis using microorganisms, enzymes, and plants or plant extracts have been suggested as possible ecofriendly alternatives to chemical and physical methods. Here, we report extracellular mycosynthesis of ZnO-NPs by Alternaria alternata (Fr.) Keissl (1912). On treating zinc sulfate solution with fungal culture filtrate, rapid reduction of ZnSO₄ was observed leading to the formation of highly stable ZnO-NPs in the solution and up-to-date literature survey showed this was the first report of biosynthesis of ZnO-NPs using this fungus. The particles thereby obtained were characterized by different analytical techniques. EDX-spectrum revealed the presence of zinc and oxygen in the nanoparticles. FTIR spectroscopy confirmed the presence of a protein shell outside the nanoparticles which in turn also support their stabilization. DLS and TEM analysis of the ZnO-NPs indicated that they ranged in size from 45 to 150 nm with average size of 75 ± 5 nm. But potential negative impacts of nanomaterials are sometimes overlooked during the discovery phase of research. Therefore, in the present study, bio-safety of mycosynthesized ZnO-NPs were evaluated by using cytotoxicity and genotoxicity assays in human lymphocyte cells, in vitro. Cytotoxicity studied as function of membrane integrity and mitochondrial dehydrogenase activity revealed significant (P < 0.05) toxicity at treatment concentration of 500 μg/ml and above. Additionally, DNA damaging potential was also studied using comet assay. The results revealed significant genotoxicity at the highest concentration (1,000 μg/ml).