Quercetin mitigates the deoxynivalenol mycotoxin induced apoptosis in SH-SY5Y cells by modulating the oxidative stress mediators.

Deoxynivalenol (DON) is Fusarium mycotoxin that is frequently found in many cereal-based foods, and its ingestion has a deleterious impact on human health. In this investigation, we studied the mechanism of DON-induced neurotoxicity and followed by cytoprotective efficacy of quercetin (QUE) in contradiction of DON-induced neurotoxicity through assessing the oxidative stress and apoptotic demise in the human neuronal model, i.e. SH-SY5Y cells. DON diminished the proliferation of cells in the manner of dose and time-dependent as revealed by cell viability investigations, i.e. MTT and lactate dehydrogenase assays. Additional studies, such as intracellular reactive oxygen species (ROS), lipid peroxidation (LPO), mitochondrial membrane potential (MMP), DNA damage, cell cycle, and neuronal biomarkers (amino acid decarboxylase, tyrosine hydroxylase, and brain-derived neurotrophic factor) demonstrated that DON induces apoptotic demise in neuronal cells through oxidative stress intermediaries. On another hand, pre-treatment of neuronal cells with 1 mM of quercetin (QUE) showed decent viability upon exposure to 100 µM of DON. In detailed studies demonstrated that QUE (1 mM) pre-treated cells show strong attenuation efficiency against DON-induced ROS generation, LPO, MMP loss, DNA impairment, cell cycle arrest, and down-regulation of neuronal biomarkers. The consequences of the investigation concluded that QUE mitigates the DON-induced stress viz., decreased ROS production and LPO generation, upholding MMP and DNA integrity and regulation of neuronal biomarker gene expression in SH-SY5Y cells.

Inhibitory effect of C. zeylanicum, C. longa, O. basilicum, Z. officinale, and C. martini essential oils on growth and ochratoxin A content of A. ochraceous and P. verrucosum in maize grains.

In the study, antifungal and ochratoxin A (OTA) production inhibitory activities of essential oils (EOs) of Cinnamomum zeylanicum, Curcuma longa, Ocimum basilicum, Zingiber officinale, and Cymbopogon martini were reported on Aspergillus ochraceus and Penicillium verrucosum. EOs were obtained by hydrodistillation and GC-MS technique was chosen to deduce their chemical profile. Major chemical compounds in EOs of C. zeylanicum, C. longa, O. basilicum, Z. officinale, and C. martini were (E)-cinnamaldehyde (35.81 %), ar-turmerone (46.13 %), eugenol (36.58 %), geranyl proprionate (18.93 %), and geranyl acetate (14.88 %), respectively. The EOs shown potent antioxidant activity by DPPH and ABTS assays. The EOs presented superlative antifungal activity against P. verrucosum related to A. ochraceus. The C. zeylanicum and C. martini EOs shown superlative antifungal activity related to other EOs. The C. zeylanicum and C. martini EOs completely inhibited the growth and OTA production of P. verrucosum and A. ochraceous at 1500 and 2500 µg/g in maize grains, respectively.

Single Nucleotide Polymorphisms in Starch Biosynthetic Genes Associated With Increased Resistant Starch Concentration in Rice Mutant.

Resistant Starch (RS), plays a crucial role in human health and nutrition by controlling glucose metabolism. RS or dietary fibre content in rice is low because it goes through a variety of process before it is ready for cooking and consumption. Hence, this study was carried out to develop a rice mutant with increased RS. The rice mutant (γ278) with increased RS was developed by utilizing gamma (γ) rays as a mutagen. Mutant γ278 was characterized for mutations in the starch biosynthetic genes viz., GBSSI, SSI, SSIIa, SSIIIa, SBEIa, and SBEIIb to reveal the functional mutations/variations led to high RS content in rice. A total of 31 sequence variants/mutations in six genes were identified. We report the discovery of three deleterious mutation/variants each in GBSSI, SSIIa, and SSIIIa with the potential to increase RS content in rice. Further, wild × mutant crosses were made to develop an F2 population to study the effect of combination of deleterious mutations. The SNP (GBSSI:ssIIa:ssIIIa) combination responsible for high RS content in F2 population was identified and recorded highest amylose content (AC) (26.18%) and RS (8.68%) content. In conclusion, this marker combination will be highly useful to develop a rice variety with increased RS.

Biofabrication of Zinc Oxide Nanoparticles With Syzygium aromaticum Flower Buds Extract and Finding Its Novel Application in Controlling the Growth and Mycotoxins of Fusarium graminearum.

Fusarium graminearum is a leading plant pathogen that causes Fusarium head blight, stalk rot, and Gibberella ear rot diseases in cereals and posing the immense threat to the microbiological safety of the food. Herein, we report the green synthesis of zinc oxide nanoparticles from Syzygium aromaticum (SaZnO NPs) flower bud extract by combustion method and investigated their application for controlling of growth and mycotoxins of F. graminearum. Formation of SaZnO NPs was confirmed by spectroscopic methods. The electron microscopic (SEM and TEM) analysis revealed the formation of triangular and hexagonal shaped SaZnO NPs with size range 30-40 nm. The synthesized SaZnO NPs reduced the growth and production of deoxynivalenol and zearalenone of F. graminearum in broth culture. Further analysis revealed that treatment of mycelia with SaZnO NPs resulted in the accumulation of ROS in the dose-dependent manner. Also, SaZnO NPs treatment enhanced lipid peroxidation, depleted ergosterol content, and caused detrimental damage to the membrane integrity of fungi. Moreover, SEM observations revealed that the presence of diverged micro-morphology (wrinkled, rough and shrank surface) in the macroconidia treated with SaZnO NPs. Taken together, SaZnO NPs may find a potential application in agriculture and food industries due to their potent antifungal activity.

Isolation and identification of enterotoxigenic Staphylococcus aureus isolates from Indian food samples: evaluation of in-house developed aptamer linked sandwich ELISA (ALISA) method.

Staphylococcus aureus is one of the major food contaminants worldwide, and its enterotoxins are documented as food poisoning and bioterrorism agents. In the present study, an attempt was made to account on the incidences of toxigenic S. aureus and its antibiotic resistance profiles in ready to eat bakery food products from different parts of Southern India (Andhra Pradesh, Karnataka, Kerala, Tamil Nadu, and Telangana). A total of 100 food samples, including milk, cake, cheese and chicken products were assessed for S. aureus and Staphylococcal Enterotoxin B (SEB) by PCR. Among the subjected food samples, a total of 51 isolates belong to genus Staphylococcus and out of that, 34 isolates were S. aureus. Among 34 S. aureus isolates, 14 isolates were found positive for SEB. The PCR results were further co-evaluated with in-house developed aptamer linked immunosorbent assay (ALISA) for the specific and sensitive detection of SEB. The obtained ALISA results were promising and found consistent with PCR analysis. Furthermore, 24%, 47%, 91%, 82%, 59%, and 47% of S. aureus isolates were found resistant to chloramphenicol, methicillin, penicillin, ampicillin, erythromycin, and oxacillin, respectively and concluded as a multidrug resistance (MDR). In conclusion, the present study revealed high presence of toxigenic and MDR resistant S. aureus species among the studied regions of Southern India. The present study cautions the need of stringent food safety regulations in India to control the toxigenic and MDR S. aureus from food sources and to minimize the risks associated with S. aureus.

Assessment of Detoxification Efficacy of Irradiation on Zearalenone Mycotoxin in Various Fruit Juices by Response Surface Methodology and Elucidation of Its in-vitro Toxicity.

Fruits are vital portion of healthy diet owed to rich source of vitamins, minerals, and dietary fibers, which are highly favorable in keeping individual fit. Unfortunately, these days, one-third of fruits were infested with fungi and their toxic metabolites called mycotoxins, which is most annoying and pose significant health risk. Therefore, there is a need to suggest appropriate mitigation strategies to overcome the mycotoxins contamination in fruits. In the present study, detoxification efficiency of irradiation on zearalenone (ZEA) mycotoxin was investigated in distilled water and fruit juices (orange, pineapple, and tomato) applying statistical program response surface methodology (RSM). The independent factors were distinct doses of irradiation and ZEA, and response factor was a percentage of ZEA reduction in content. A central composite design (CCD) consists of 13 experiments were planned applying software program Design expert with distinct doses of irradiation (up to 10 kGy) and ZEA (1-5 µg). The results revealed that independent factors had a positive significant effect on the response factor. The analysis of variance (ANOVA) was followed to fit a proper statistical model and suggested that quadratic model was appropriate. The optimized model concluded that doses of irradiation and ZEA were the determinant factors for detoxification of ZEA in fruit juices. Further, toxicological safety of irradiation mediated detoxified ZEA was assessed in the cell line model by determining the cell viability (MTT and live/dead cell assays), intracellular reactive oxygen species (ROS), mitochondrial membrane potential (MMP), nuclear damage, and caspase-3 activity. The higher level of live cells and MMP, lower extent of intracellular ROS molecules and caspase-3, and intact nuclear material were noticed in cells treated with irradiation mediated detoxified ZEA related to non-detoxified ZEA. The results confirmed that toxicity of ZEA was decreased with irradiation treatment and detoxification of ZEA by irradiation is safe. The study concluded that irradiation could be a potential post-harvest food processing technique for detoxification of ZEA mycotoxin in fruit juices. However, irradiation of fruit juices with high dose of 10 kGy has minimally altered the quality of fruit juices.

Combinational Inhibitory Action of Hedychium spicatum L. Essential Oil and γ-Radiation on Growth Rate and Mycotoxins Content of Fusarium graminearum in Maize: Response Surface Methodology.

Nowadays, contamination of agricultural commodities with fungi and their mycotoxins is one of the most annoying with regard to food safety and pose serious health risk. Therefore, there is a requisite to propose suitable mitigation strategies to reduce the contamination of fungi and mycotoxins in agricultural commodities. In the present study, combinational inhibitory effect of Hedychium spicatum L. essential oil (HSEO) and radiation was established on growth rate, production of deoxynivalenol (DON) and zearalenone (ZEA) by Fusarium graminearum in maize grains. The HSEO was obtained from rhizomes by hydrodistillation technique and chemical composition was revealed by GC-MS analysis. A total of 48 compounds were identified and major compounds were 1,8-cineole (23.15%), linalool (12.82%), and ß-pinene (10.06%). The discrete treatments of HSEO and radiation were effective in reducing the fungal growth rate and mycotoxins content, and the complete reduction was noticed at 3.15 mg/g of HSEO and 6 kGy of radiation. Response surface methodology (RSM) was applied to evaluate the combinational inhibitory effect of HSEO and radiation treatments on fungal growth rate and mycotoxins content. A total of 13 experiments were designed with distinct doses of HSEO and radiation by central composite design (CCD) of Stat-Ease Design-Expert software. In combinational approach, complete reductions of fungal growth, DON, and ZEA content were noticed at 1.89 mg/g of HSEO and 4.12 kGy of radiation treatments. The optimized design concluded that combinational treatments of HSEO and radiation were much more effective in reducing the fungal growth and mycotoxins content compared to their discrete treatments (p < 0.05). Responses of the design were assessed by second-order polynomial regression analysis and found that quadratic model was well fitted. The optimized design has larger F-value and adequate precision, smaller p-value, decent regression coefficients (R2 ) and found statistically significant (p < 0.05). In addition, correlation matrix, normal plot residuals, Box-Cox, and actual vs. predicted plots were endorsed that optimized design was accurate and appropriate. The proposed combinational decontamination technique could be highly applicable in agriculture and food industry to safeguard the food and feed products from fungi and mycotoxins.

Mycotoxin zearalenone induced gonadal impairment and altered gene expression in the hypothalamic-pituitary-gonadal axis of adult female zebrafish (Danio rerio).

In the present study, we aimed to assess the adverse effects of zearalenone (ZEA) at environmentally relevant concentrations (0.5, 1, 5 and 10 µg l-1 ) on hypothalamic-pituitary-gonadal axis associated reproductive function using zebrafish model. ZEA was exposed to female zebrafish for 21 days to assess growth indices such as condition factor, hepatosomatic index, gonadosomatic index and caspase 3 activity. Further, expression of estrogen receptor (ER) α and CYP19a1b genes in the brain, ERα and vitellogenin (Vtg) genes in the liver and follicle-stimulating hormone receptor, luteinizing hormone receptor, ERα, steroidogenic acute regulatory protein, 3ß-hydroxysteroid dehydrogenase (HSD), 17-ßHSD and CYP19a1 genes in the ovary were also investigated. Our results showed that there were no significant changes in the condition factor and hepatosomatic index, whereas a significant (P < .05) reduction in the gonadosomatic index, increase in caspase 3 activities and Vtg expression was observed at higher concentration. However, no significant changes were observed at lower treatment levels. Further, we also observed significant (P < .05) upregulation in ERα, Vtg, luteinizing hormone receptor, steroidogenic acute regulatory protein, 3ß-HSD, 17ß-HSD, CYP19a1 and CYP19a1b genes in treatment groups with higher levels of ZEA. Moreover, in histopathological examination, we observed oocyte atresia and oocyte membrane detachment in ovaries at the highest concentration. In conclusion, the present study revealed the negative impact of ZEA on zebrafish reproductive system by involvement of the hypothalamic-pituitary-gonadal axis-associated reproductive function.

Antifungal Activity of Chitosan Nanoparticles Encapsulated With Cymbopogon martinii Essential Oil on Plant Pathogenic Fungi Fusarium graminearum.

Application of synthetic fungicides in agricultural commodities has been restricted due to development of fungicide resistance fungi and deleterious impact on environment and health of farm animals and humans. Hence, there is an urge for development of mycobiocides, and the present study was undertaken to determine the antifungal activity of Cymbopogon martinii essential oil (CMEO) on post-harvest pathogen Fusarium graminearum. The CMEO was extracted by hydrodistillation and GC-MS chemical profile revealed the presence of 46 compounds and abundant was geraniol (19.06%). The minimum inhibitory concentration and minimum fungicidal concentration of CMEO were determined as 421.7 ± 27.14 and 618.3 ± 79.35 ppm, respectively. The scanning electron microscopic observation of CMEO exposed macroconidia was exhibited a detrimental morphology with vesicles, craters, protuberance, and rough surfaces related to control fungi. The CMEO induced the death of fungi through elevating intracellular reactive oxygen species and lipid peroxidation, and depleting ergosterol content. Regrettably, essential oils are highly volatile and become unstable and lose their biological features on exposure to light, heat, pH, moisture, and oxygen. To overcome these issues, chitosan encapsulated CMEO nanoparticles (Ce-CMEO-NPs) were prepared. The synthesized Ce-CMEO-NPs have spherical morphology with Zeta potential of 39.3-37.2 mV and their corresponding size was found in range of 455-480 nm. The Fourier transform infrared analysis confirmed that bio-active constituents of CMEO were well stabilized due to chitosan conjugation and successfully formed Ce-CMEO-NPs. The in vitro release assay observed that the release of CMEO is stabilized due to the complex formation with chitosan and thereby, increases the lifetime antifungal activity of CMEO by gradual release of antifungal constituents of Ce-CMEO-NPs. In conclusion, antifungal and antimycotoxin activities of CMEO and Ce-CMEO-NPs against F. graminearum were assessed in maize grains under laboratory conditions over a storage period of 28 days. Interestingly, Ce-CMEO-NPs were presented efficient and enhanced antifungal and antimycotoxin activities related to CMEO, and it could be due to perseverance of antifungal activity by controlled release of antifungal constituents from Ce-CMEO-NPs. The study concluded that Ce-CMEO-NPs could be highly appropriate as mycobiocides in safeguarding the agricultural commodities during storage period in agricultural and food industries.

Endophytic Fungi-Alternative Sources of Cytotoxic Compounds: A Review.

Cancer is a major cause of death worldwide, with an increasing number of cases being reported annually. The elevated rate of mortality necessitates a global challenge to explore newer sources of anticancer drugs. Recent advancements in cancer treatment involve the discovery and development of new and improved chemotherapeutics derived from natural or synthetic sources. Natural sources offer the potential of finding new structural classes with unique bioactivities for cancer therapy. Endophytic fungi represent a rich source of bioactive metabolites that can be manipulated to produce desirable novel analogs for chemotherapy. This review offers a current and integrative account of clinically used anticancer drugs such as taxol, podophyllotoxin, camptothecin, and vinca alkaloids in terms of their mechanism of action, isolation from endophytic fungi and their characterization, yield obtained, and fungal strain improvement strategies. It also covers recent literature on endophytic fungal metabolites from terrestrial, mangrove, and marine sources as potential anticancer agents and emphasizes the findings for cytotoxic bioactive compounds tested against specific cancer cell lines.

Chitosan nanoparticles having higher degree of acetylation induce resistance against pearl millet downy mildew through nitric oxide generation.

Downy mildew of pearl millet caused by the biotrophic oomycete Sclerospora graminicola is the most devastating disease which impairs pearl millet production causing huge yield and monetary losses. Chitosan nanoparticles (CNP) were synthesized from low molecular weight chitosan having higher degree of acetylation was evaluated for their efficacy against downy mildew disease of pearl millet caused by Sclerospora graminicola. Laboratory studies showed that CNP seed treatment significantly enhanced pearl millet seed germination percentage and seedling vigor compared to the control. Seed treatment with CNP induced systemic and durable resistance and showed significant downy mildew protection under greenhouse conditions in comparison to the untreated control. Seed treatment with CNP showed changes in gene expression profiles wherein expression of genes of phenylalanine ammonia lyase, peroxidase, polyphenoloxidase, catalase and superoxide dismutase were highly upregulated. CNP treatment resulted in earlier and higher expression of the pathogenesis related proteins PR1 and PR5. Downy mildew protective effect offered by CNP was found to be modulated by nitric oxide and treatment with CNP along with NO inhibitors cPTIO completely abolished the gene expression of defense enzymes and PR proteins. Further, comparative analysis of CNP with Chitosan revealed that the very small dosage of CNP performed at par with recommended dose of Chitosan for downy mildew management.

Development of a FRET-based fluorescence aptasensor for the detection of aflatoxin B1 in contaminated food grain samples.

The present study aimed to develop an aptamer-based FRET detection strategy for the specific and sensitive detection of AFB1 in contaminated food grains. The study comprises generation of ssDNA aptamers against AFB1 by whole-cell SELEX and their application in a FRET-based platform utilizing graphene oxide (GO) and quantum dots (QDs). The generated aptamers were characterized to determine their specificity and sensitivity using indirect ELISA where AFB1-OVA was used as a coating antigen. Among the aptamers generated, the ATB1 aptamer showed good reactivity and selectivity against AFB1. This aptamer was further characterized to determine its secondary structure and KD value, which was found to be 5.9 kcal mol-1. The characterized aptamers were conjugated onto Cd/Se quantum dots to develop a fluorimetric system for the detection of aflatoxin B1 using a graphene oxide platform. The presence of graphene oxide quenches the fluorescence ability of the quantum dots due to π-π stacking interactions between the aptamer and GO. Upon target addition, the aptamer forms a complex with aflatoxin B1 thereby restoring the fluorescence intensity. The developed assay shows a linear response from 0.002 µg µl-1 to 0.2 µg µl-1 with a detection limit of 0.004 µg µl-1 for the AFB1 standard toxin and showed no cross-reactivity with other closely related mycotoxins. To validate the reliability of the developed method, several field samples spiked with AFB1 were included in this study and the results obtained were cross verified using a standard commercial AFB1 kit. In conclusion, the developed method may find good utility in routine food testing laboratories for risk assessment of AFB1.

Application of Activated Carbon Derived from Seed Shells of Jatropha curcas for Decontamination of Zearalenone Mycotoxin.

In the present study, activated carbon (AC) was derived from seed shells of Jatropha curcas and applied to decontaminate the zearalenone (ZEA) mycotoxin. The AC of J. curcas (ACJC) was prepared by ZnCl2 chemical activation method and its crystalline structure was determined by X-ray diffraction analysis. The crystalline graphitic nature of ACJC was confirmed from the Raman spectroscopy. Scanning electron microscope showed the porous surface morphology of the ACJC surface with high pore density and presence of elemental carbon was identified from the energy dispersive X-ray analysis. From Brunauer-Emmett-Teller (BET) analysis, SBET, micropore area, and average pore diameter of ACJC were calculated as 822.78 (m2/g), 255.36 (m2/g), and 8.5980 (Å), respectively. The adsorption of ZEA by ACJC was accomplished with varying contact time, concentration of ZEA and ACJC, and pH of media. The ACJC has adsorbed the ZEA over a short period of time and adsorption of ZEA was dependent on the dose of ACJC. The effect of different pH on adsorption of ZEA by ACJC was not much effective. Desorption studies confirmed that adsorption of ZEA by ACJC was stable. The adsorption isotherm of ZEA by ACJC was well fitted with Langmuir model rather than Freundlich and concluded the homogeneous process of sorption. The maximum adsorption of ZEA by ACJC was detected as 23.14 µg/mg. Finally, adsorption property of ACJC was utilized to establish ACJC as an antidote against ZEA-induced toxicity under in vitro in neuro-2a cells. The percentage of live cells was high in cells treated together with a combination of ZEA and ACJC compared to ZEA treated cells. In a similar way, ΔΨM was not dropped in cells exposed to combination of ACJC and ZEA compared to ZEA treated cells. Furthermore, cells treated with a combination of ZEA and ACJC exhibited lower level of intracellular reactive oxygen species and caspase-3 compared to ZEA treated cells. These in vitro studies concluded that ACJC has successfully protected the cells from ZEA-induced toxicity by lowering the availability of ZEA in media as a result of adsorption of ZEA. The study concluded that ACJC was a potent decontaminating agent for ZEA and could be used as an antidote against ZEA-induced toxicity.

Elicitation of resistance and associated defense responses in Trichoderma hamatum induced protection against pearl millet downy mildew pathogen.

Endophytic Trichoderma hamatum UoM 13 isolated from pearl millet roots was evaluated for its efficiency to suppress downy mildew disease. Under laboratory conditions, T. hamatum seed treatment significantly enhanced pearl millet seed germination and seedling vigor. T. hamatum seed treatment resulted in systemic and durable immunity against pearl millet downy mildew disease under greenhouse and field conditions. T. hamatum treated seedlings responded to downy mildew infection with high lignification and callose deposition. Analysis of defense enzymes showed that T. hamatum treatment significantly enhanced the activities of glucanase, peroxidase, phenylalanine ammonia-lyase, and polyphenol oxidase in comparison to untreated control. RT-PCR analysis revealed differentially expressed transcripts of the defense enzymes and PR-proteins in treated, untreated, and checks, wherein PR-1, PR-5, and cell wall defense HRGPs were significantly over expressed in treated seedlings as against their lower expression in controls. T. hamatum treatment significantly stimulated endogenous salicylic acid (SA) levels and significantly upregulated important SA biosynthesis gene isochorismate synthase. The results indicated that T. hamatum UoM13 treatment induces resistance corresponding to significant over expression of endogenous SA, important defense enzymes, PR-proteins, and HRGPs, suggesting that SA biosynthetic pathway is involved in pearl millet for mounting systemic immunity against downy mildew pathogen.

Antifungal and Zearalenone Inhibitory Activity of Pediococcus pentosaceus Isolated from Dairy Products on Fusarium graminearum.

The present study was aimed to evaluate the bio-control efficacy of Pediococcus pentosaceus isolated from traditional fermented dairy products originated from India, against the growth and zearalenone (ZEA) production of Fusarium graminearum. The cell-free supernatants of P. pentosaceus (PPCS) were prepared and chemical profiling was carried out by GC-MS and MALDI-TOF analysis. Chemical profiling of PPCS evidenced that, the presence of phenolic antioxidants, which are responsible for the antifungal activity. Another hand, MALDI-TOF analysis also indicated the presence of antimicrobial peptides. To know the antioxidant potential of PPCS, DPPH free radical scavenging assay was carried out and IC50 value was determined as 32 ± 1.89 µL/mL. The antifungal activity of P. pentosaceus was determined by dual culture overlay technique and zone of inhibition was recorded as 47 ± 2.81%, and antifungal activity of PPCS on F. graminearum was determined by micro-well dilution and scanning electron microscopic techniques. The minimum inhibitory concentration (MIC) of PPCS was determined as 66 ± 2.18 µL/mL in the present study. Also a clear variation in the micromorphology of mycelia treated with MIC value of PPCS compared to untreated control was documented. Further, the mechanism of growth inhibition was revealed by ergosterol analysis and determination of reactive oxygen species (ROS) in PPCS treated samples. The effects of PPCS on mycelial biomass and ZEA production were observed in a dose-dependent manner. The mechanism behind the suppression of ZEA production was studied by reverse transcriptase qPCR analysis of ZEA metabolic pathway genes (PKS4 and PKS13), and results showed that there is a dose dependent down-regulation of target gene expression in PPCS treated samples. The results of the present study were collectively proved that, the antifungal and ZEA inhibitory activity of PPCS against F. graminearum and it may find a potential application in agriculture and food industry as a natural bio-controlling agent.

Molecular Diversity of Seed-borne Fusarium Species Associated with Maize in India.

A total of 106 maize seed samples were collected from different agro-climatic regions of India. Sixty-two Fusarium isolates were recovered, 90% of which were identified as Fusarium verticillioides based on morphological and molecular characters. Use of the tef-1α gene corrected/refined the morphological species identifications of 11 isolates, and confirmed those of the remaining isolates. Genetic diversity among the Fusarium isolates involved multilocus fingerprinting profiles by Inter Simple Sequence Repeats (ISSR) UPGMA and tef-1α gene phenetic analyses; for which, we observed no significant differences among the isolates based on geographic origin or fumonisin production; most of the subdivision related to species. Genotyping was performed on the F. verticillioides isolates, using 12 primer sets from the fumonisin pathway, to elucidate the molec-ular basis of fumonisin production or non-production. One fumonisin-negative isolate, UOMMF-16, was unable to amplify nine of the 12 fumonisin cluster genes tested. We also used the CD-ELISA method to confirm fumonisin production for our 62 Fusarium isolates. Only 15 isolates were found to be fumonisin-negative. Interestingly, genotypic characterization re-vealed six isolates with various gene deletion patterns that also tested positive for the production of fumonisins via CD-ELISA. Our findings confirm the importance of molecular studies for species delimitation, and for observing genetic and phenotypic diversity, among the Fusaria.

A novel IgY-Aptamer hybrid system for cost-effective detection of SEB and its evaluation on food and clinical samples.

In the present study, we introduce a novel hybrid sandwich-ALISA employing chicken IgY and ssDNA aptamers for the detection of staphylococcal enterotoxin B (SEB). Cloning, expression and purification of the full length recombinant SEB was carried out. Anti-SEB IgY antibodies generated by immunizing white leg-horn chickens with purified recombinant SEB protein and were purified from the immunized egg yolk. Simultaneously, ssDNA aptamers specific to the toxin were prepared by SELEX method on microtiter well plates. The sensitivity levels of both probe molecules i.e., IgY and ssDNA aptamers were evaluated. We observed that the aptamer at 250 ngmL(-1) concentration could detect the target antigen at 50 ngmL(-1) and the IgY antibodies at 250 ngmL(-1), could able to detect 100 ngmL(-1) antigen. We further combined both the probes to prepare a hybrid sandwich aptamer linked immune sorbent assay (ALISA) wherein the IgY as capturing molecule and biotinylated aptamer as revealing probe. Limit of detection (LOD) for the developed method was determined as 50 ngmL(-1). Further, developed method was evaluated with artificially SEB spiked milk and natural samples and obtained results were validated with PCR. In conclusion, developed ALISA method may provide cost-effective and robust detection of SEB from food and environmental samples.

Antagonistic activity of Ocimum sanctum L. essential oil on growth and zearalenone production by Fusarium graminearum in maize grains.

The present study was aimed to establish the antagonistic effects of Ocimum sanctum L. essential oil (OSEO) on growth and zearalenone (ZEA) production of Fusarium graminearum. GC-MS chemical profiling of OSEO revealed the existence of 43 compounds and the major compound was found to be eugenol (34.7%). DPPH free radical scavenging activity (IC50) of OSEO was determined to be 8.5 µg/mL. Minimum inhibitory concentration and minimum fungicidal concentration of OSEO on F. graminearum were recorded as 1250 and 1800 µg/mL, respectively. Scanning electron microscope observations showed significant micro morphological damage in OSEO exposed mycelia and spores compared to untreated control culture. Quantitative UHPLC studies revealed that OSEO negatively effected the production of ZEA; the concentration of toxin production was observed to be insignificant at 1500 µg/mL concentration of OSEO. On other hand ZEA concentration was quantified as 3.23 µg/mL in OSEO untreated control culture. Reverse transcriptase qPCR analysis of ZEA metabolic pathway genes (PKS4 and PKS13) revealed that increase in OSEO concentration (250-1500 µg/mL) significantly downregulated the expression of PKS4 and PKS13. These results were in agreement with the artificially contaminated maize grains as well. In conlusion, the antifungal and antimycotoxic effects of OSEO on F. graminearum in the present study reiterated that, the essential oil of O. sanctum could be a promising herbal fungicide in food processing industries as well as grain storage centers.

Mould incidence and mycotoxin contamination in freshly harvested maize kernels originated from India.

BACKGROUND: In this study, mould incidence and mycotoxin contamination were determined in freshly harvested maize samples collected from different agroclimatic regions of India. A total of 150 freshly harvested maize samples from major maize-growing areas of India (Karnataka, Andhra Pradesh and Tamilnadu) were collected during winter seasons 2010-2011 and 2011-2012 to determine their toxigenic fungal incidences, and mycotoxins were analyzed and quantified by high-perfomance liquid chromatography. A total of 288 fungal isolates comprising Fusarium, Aspergillus and Penicillium species were tested for aflatoxin B1 (AFB1), ochratoxin A (OTA), trichothecenes (deoxynivalenol (DON) and T-2 toxin) and fumonisin B1 (FB1). Chemotype determination of fungal isolates was carried out by molecular and chemical analysis through polymerase chain reaction (PCR) and high-performance thin layer chromatography respectively. The diversity and distribution of the mycoflora among the studied samples were recorded in terms of frequency, density, importance value index and diversity indices. RESULTS: A total of 288 fungal isolates were recovered from the 150 maize samples, of which 28 were positive for AFB1, 20 for OTA, 58 for FB1, 23 for DON and 11 for T-2 toxin chemotypes by PCR. Species-specific PCR assays were in line with morphological analysis. Toxigenic fungal incidences were found throughout the study region, and most of the toxins under study exceeded the maximum legal limits. The range of observed toxin concentrations were 48-58 µg AFB1, 76-123 µg FB1, 38-50 µg T-2, 72-94 µg DON and <5 µg OTA kg⁻¹ grain sample. CONCLUSION: Owing to the high incidences of toxigenic moulds and mycotoxins in the study area, there is a need for the creation of mycotoxin awareness among maize farmers of India to control the chronic adverse health effects on humans and livestock due to mycotoxins.