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Antifungal and Zearalenone Inhibitory Activity of Ocimum sanctum L. Essential Oil on Fusarium graminearum Determined by UHPLC and RT-qPCR
采用UHPLC和RT-qPCR技术在禾谷镰刀菌上测定罗勒属精油的抗真菌和玉米烯酮抑制活性   

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Abstract

Fusarium graminearum has been given special attention in the context of agricultural commodities due to its ability to grow in diverse climatic conditions, and to produce different mycotoxins including zearalenone (ZEA) and type-B trichothecenes, which cause ill health effects on humans, animals and plants. The application of synthetic antifungal agents for the control of F. graminearum result in negative health impacts in livestock and humans and the upsurge of resistant organisms as well. Therefore, there is a need to propose proper food grain management practices, including the application of herbal antifungal and mycotoxin controlling agents, to reduce the growth of toxigenic F. graminearum as well as the production of ZEA in agricultural commodities. Ocimum sanctum also known as Holy Basil or Tulsi is widely used as a medicinal plant in Ayurveda. The current protocol demonstrates to quantify the antifungal activity of O. sanctum L. essential oil (OSEO) as reflected by the decreased F. graminearum growth and ZEA production. Antifungal activities of OSEO are carried out by micro well dilution method and further validated quantitatively by scanning electron microscopic methods. Effects of OSEO on ZEA production is analysed by Quantitative reverse transcription PCR (RT-qPCR) and Ultra high performance liquid chromatography (UHPLC) methods from a broth culture of F. graminearum. Anti-mycotoxic efficacy of OSEO is assessed directly on F. graminearum inoculated maize grains. The protocol efficiently assessed the activity of OSEO as an herbal antagonistic agent against fungal infestation and ZEA production by F. graminearum. The protocol can be used to test a wide variety of herbal compounds for antifungal activity against F. graminearum or with modifications on other mycotoxigenic fungi, an important intervention in food safety and processing industries where the fungal infestation is a major concern.

Keywords: Zearalenone(玉米赤霉烯酮), Fusarium(镰刀菌), Ocimum sanctum oil(圣罗勒油), UHPLC(超高效液相色谱), Q-RTPCR(Q-RT-PCR)

Materials and Reagents

  1. 0.22 µm Millex-GP syringe filter unit (Sigma-Aldrich, catalog number: Z359904 )
  2. 96-well microtiter plates (Eppendorf, catalog number: 0030602200 )
  3. Column C18, 5 µm, 250 x 4.6 mm (Phenomenex, catalog number: 00G-4041-E0 )
  4. Carbon Conductive Tape (Ted Pella, Inc., catalog number: 16084-7 )
  5. Glass slides (HiMedia, catalog number: CG081 )
  6. Whatman No.1 paper (Sigma-Aldrich, catalog number: Z274852 )
  7. Zearalenone producing F. graminearum [The Microbial Type Culture Collection and Gene Bank, (MTCC), catalog number: 1893 ]
  8. Maize grains (Local agricultural market, Mysore, India)
  9. Zearalenone standard (Sigma-Aldrich, catalog number: Z2125 )
  10. Dimethyl sulfoxide (Merck Millipore, catalog number: 317275 )
  11. Distilled water
  12. Sodium chloride (NaCl) (Merck Millipore, catalog number: 1064040500 )
  13. Potassium chloride (KCl) (Merck Millipore, catalog number: 1049360250 )
  14. Sodium phosphate dibasic (Na2HPO4) (Merck Millipore, catalog number: 567550-1KG )
  15. Potassium phosphate monobasic (KH2PO4) (Merck Millipore, catalog number: 1048730250 )
  16. 0.1 M sodium cacodylate buffer, pH 6.5 (Sigma-Aldrich, catalog number: 70114 )
  17. 25% glutaraldehyde (Merck Millipore, catalog number: 354400 )
  18. Acetonitrile (Merck Millipore, catalog number: 100030 )
  19. Ethanol (Merck Millipore, catalog number: 100983 )
  20. Gold foil (Sigma-Aldrich, catalog number: 265829 )
  21. Immunoaffinity column of ZEA (Vicam, catalog number: G1026 )
  22. iScript One-Step RT-PCR Kit with SYBR Green (Bio-Rad Laboratories, catalog number: 1708892 )
  23. Liquid nitrogen (Local suppliers, Mysore, India)
  24. Nuclease-free water (Qiagen, catalog number: 129114 )
  25. Nystatin (Sigma-Aldrich, catalog number: N6261 )
  26. Ocimum sanctum L. essential oil (OSEO) (prepared as describe in Procedure step 2)
  27. Peptone (HiMedia, catalog number: RM001-500G )
  28. Porcelain mortar (Sigma-Aldrich, catalog number: Z529508 )
  29. RNA easy plant Mini kit (Qiagen, catalog number: 74903 )
  30. Sabouraud dextrose agar (HiMedia, catalog number: M063-500G )
  31. Sabouraud dextrose broth (HiMedia, catalog number: M033-500G )
  32. Synthesized primer sequences (Sigma-Aldrich, Bangalore, India)
  33. Tween 80 (Merck Millipore, catalog number: 822187 )
  34. Lactophenol-cotton blue (HiMedia, catalog number: S016-500ML )
  35. Phosphate-buffered saline (pH 7.4) (see Recipes)
  36. Lactophenol-cotton blue staining solution (see Recipes)

Equipment

  1. Milli-Q integral water purification system (Merck Millipore, catalog number: ZRXQ005WW )
  2. Aluminum stubs (Ted Pella, Inc., catalog number: 16111N )
  3. Autoclave (Medica Instrument Manufacturing Company, model: 7431PAD )
  4. Microcentrifuge (Sigma-Aldrich, Eppendorf, model: 5415 R )
  5. Centrifuge (Eppendorf, model: 5430 R )
  6. Hemocytometer (Sigma-Aldrich, catalog number: Z359629 )
  7. Hot-air oven (Memmert, model: UFP800DW )
  8. Incubator (Bio-age, model: BSR-R2 )
  9. Real-Time PCR system (Roche Diagnostics, Light cycler®, model: 480 )
  10. 0.5-10 µl micropipette (Eppendorf Research plus, catalog number: 3120000020 )
  11. 20-200 µl micropipette (Eppendorf Research plus, catalog number: 3120000054 )
  12. 100-1,000 µl micropipette (Eppendorf Research plus, catalog number: 3120000062 )
  13. Microscope (Leica Microsystems, model: Leica DM 1000 LED )
  14. 250 ml Erlenmeyer flasks (Duran Group, catalog number: 21 216 36 )
  15. 500 ml Erlenmeyer flasks (Duran Group, catalog number: 21 216 44 )
  16. NanoDrop 8000 spectrophotometer (Thermo Fisher Scientific, catalog number: ND-8000-GL )
  17. Nexera UHPLC system (Shimadzu Corporation, model: Nexera X2 )
  18. Scanning electron microscope (FEI, model: Quanta 200 )
    Note: This product has been discontinued by the manufacturer [Replaceable items (FEI, model: Quanta 250/450/650 )].
  19. Shaker Incubator (Bio-age, model: BSR-R1 )
  20. Sputter coater (Quorum Technologies, model: SC7620 )
  21. Water bath (NUVE, model: NB 5 )
  22. Weighing balance (Denver instruments, model: TB-215D )

Software

  1. GeneRunner software version 5.0.47 Beta

Procedure

  1. Grow the zearalenone (ZEA) producing F. graminearum (MTCC, 1893) on Sabouraud dextrose agar (SDA) for 7 days at 28 °C and collect the spores in 10 ml of peptone water containing 0.001% Tween 80 with a soft scrape. Determine the number of spores using a hemocytometer and adjust the spore suspension to 1 x 106 per ml.
  2. Collect the Ocimum sanctum L. plant and identify its botanical nomenclature, and dry the plant at room temperature under dark condition. Extract the essential oil from dried aerial parts following the technique of European Pharmacopoeia (Council of Europe, 1997). Prepare a stock solution of 0.05% OSEO in DMSO.
    Note: In the present study, Ocimum sanctum L. was collected from Mysore, Karnataka state, India and identification was done by Botanical Survey of India (Coimbatore, India).
  3. Determine the minimum inhibitory (MIC) and minimum fungicidal concentrations (MFC) of OSEO on F. graminearum by micro-well dilution technique in 96-well microtiter plate implementing the methodology of Clinical and Laboratory Standards Institute (2008) and Vieira et al. (2014) with following minor modifications.
    1. Add 10 µl of spore suspension (1 x 106 spores per ml) to the different concentrations of OSEO and adjust the total volume to100 µl per well with Sabouraud dextrose broth (SDB).
    2. Consider the wells without OSEO as control and incubate the microplates for 3 days at 28 °C in the dark.
    3. Observe the minimum concentration of OSEO without detectable fungal growth and determine as minimum inhibitory concentration (MIC).
    4. Spread plate 10 µl from each well on SDA plates and incubate at 28 °C for 3 days.
    5. Identify the minimum concentration of OSEO with no detectable fungal growth and determine as the MFC, specifying 99.5% killing of the original inoculum in comparison to nystatin (positive control).


    Figure 1. Determination of minimum inhibitory (MIC) and minimum fungicidal concentrations (MFC) of O. sanctum essential oil (OSEO) on F. graminearum by micro-well dilution method. Fungal growth is observed in control (OSEO untreated) and no detectable growth is observed at MFC value of OSEO.

  4. Analyse the effect of OSEO on spore germination of F. graminearum by the method of Rana et al. (1997) with minor modifications.
    1. Inoculate 10 µl of fungal spore suspension (1 x 106 spores per ml) on SDA slides containing different concentrations of OSEO (100-1,800 µg/ml) and incubate at 28 °C for 24 h.
    2. Consider SDA slide alone with fungal spores and without OSEO as control.
    3. Following the incubation period, stain each slide with lactophenol-cotton blue (100 µl) by dropping method at 28 °C for 15 min and observe for germ tubes under microscope.
    4. Examine at least 200 spores from each slide and calculate the percentage of spore germination using the formula,
      % Spore germination = ST/SC x 100
      Where, SC is number of spores germinated in control and ST is number of spores germinated in test.
  5. Determine the effect of OSEO on mycelial and spore structure of F. graminearum by scanning electron microscope (SEM) observation according to the method of Yamamoto-Ribeiro et al. (2013) with minor modifications.
    1. Collect the mycelia disk of 1 cm2 from a seven-day culture of F. graminearum and inoculate aseptically at the middle of SDA dishes that contained different concentrations of OSEO and incubate at 28 °C for 7 days in the dark.
    2. Consider the SDA medium without OSEO as control.
    3. After the incubation period, collect mycelial disk of 1 cm2 and rinse in phosphate-buffered saline (pH 7.4) and fix with 2.5% glutaraldehyde in 0.1 M sodium cacodylate buffer, pH 6.5 and dehydrate with gradient ethanol (20, 40, 70, 90 and 100%, keeping the mycelia for a longer duration in 100%).
    4. Paste the sample on dual side glue carbon conductive tape and fix to the surface of aluminum stubs.
    5. Further, expose the stubs towards critical-point dry out in CO2 and sputter-coat with gold to increase its conductivity.
    6. Observe the morphological quality of mycelia under scanning electron microscope at 20.0 KV in environmental mode.


    Figure 2. Determination of the antifungal activity of O. sanctum L. essential oil (OSEO) on F. graminearum by scanning electron microscopic observation (Kalagatur et al., 2015). The control (OSEO untreated) hyphae (a) is smooth, turgid and homogenous. Whereas hyphae treated with MIC (b) and MFC (c) values of OSEO exhibited craters, protuberances and collapsed. The control (OSEO untreated) spores (d) are round and smooth, and on other hand spores treated with MIC (e) and MFC (f) values of OSEO are disrupted and wrinkled.

  6. Determine the anti-mycotoxic activity of OSEO in liquid cultures. Add different concentrations of OSEO including, 250, 500, 1,000, 1,500 and 2,000 μg/ml to each 250 ml Erlenmeyer flask that contain 100 ml of SDB. Inoculate 10 μl of fungal suspension (1 x 106 spores/ml) of 7-day-old culture into these flasks under aseptic conditions. Consider the flask without OSEO as control and incubate the flasks under shaking condition (140-160 rpm) at 28 °C for 14 days in the dark.
  7. Following the incubation period, separate the culture media from fungal biomass by filtering through Whatman No.1 paper and use the broth for determination of ZEA. Wash the fungal mycelia twice with sterile distilled water and use 10 mg of mycelia for RNA extraction, and pack the leftover mycelia in pre-weighed Whatman no.1 filter paper and dry out at 60 °C for 24 h and determine the mycelial biomass by weighing.
  8. Detection and quantification of ZEA by UHPLC by the method of Ibáñez-Vea et al. (2011) with slight modifications.
    1. Blend the culture broth with an equivalent quantity of acetonitrile at 140 rpm under shaker incubator for 30 min.
    2. Subsequently, collect the supernatant of the sample by centrifugation at 4226.04 x g for 12 min and transfer 15 ml of the supernatant through an immunoaffinity column of ZEA, which is pre-conditioned under 10 ml of phosphate-buffered saline (pH 7.4).
    3. Next, Wash the column with 5 ml of PBS and 10 ml of distilled water.
    4. Finally, air-dry the column and elute the ZEA with 5 ml of acetonitrile. Maintain contact between acetonitrile and column antibodies at least for 5 min.
    5. Dry out the eluate completely over a water bath at 60 °C and redissolved the final residue in 1 ml of acetonitrile and filter through 0.22 µm of syringe filter.
    6. Use the filtrate for UHPLC determination and quantification of ZEA.
      1. Employ the Nexera UHPLC system attached with the column C18, 5 µm, 250 x 4.6 mm for detection and quantification of ZEA in reverse-phase with a fluorescence detector, and set excitation and emission wavelength at 334 and 450 nm, respectively.
      2. The mobile phase is acetonitrile-water (50:50, v/v) with a flow rate of 1 ml/min.
      3. Construct a five-point calibration curve for standard ZEA (100 ng-500 µg/ml) with peak area versus concentration of ZEA.
      4. The injection volume is 25 µl for both the standard solution and test sample.
      5. The sensing limitation of the technique is 100 ng/ml.
  9. Determination of the effect of OSEO on gene expression of PKS4 and PKS13, which are involved in ZEA biosynthesis of F. graminearum (Gaffoor and Trail, 2006; Kim et al., 2005) by RT-qPCR evaluation using GAPDH as an endogenous reference gene.
    1. Design the primers for target genes using the GeneRunner software version 5.0.47 Beta (Table 1).
    2. Briefly, flash-frozen the mycelia in liquid nitrogen and ground into a fine powder with a porcelain mortar. Extract the total RNA using RNEASY PLANT MINI KIT following manufacturer’s guidelines (Qiagen, Hilden, Germany).
    3. Quantify the total RNA by NanoDrop 8000 spectrophotometer.
    4. Carry out the RT-qPCR analysis of PKS4 and PKS13 in the Light Cycler 480 using iScript One-Step RT-PCR Kit with SYBR Green (Bio-Rad Laboratories, USA).
      1. Briefly, make 50 µl volume of reaction mixture consisting 25 µl of 2x SYBR Green RT-PCR reaction mix, 1 µl of iScript reverse transcriptase for one-step RT-PCR, 1 µl of primer (450 nM), 1 µl of template RNA (100 ng) and 22 µl of nuclease-free water (PCR grade).
      2. The thermal conditions for the reaction include 10 min of cDNA synthesis at 50 °C for 1 cycle, 5 min of polymerase activation at 95 °C and following by 35 cycles of PCR at 95 °C for 10 sec, 60 °C for 30 sec for combined annealing and extension.
      3. Attain individual narrow peak through melting curve analysis at distinct temperatures for each and every PCR product.
      4. Quantify the relative quantification levels of gene expression making use of second derivative maximum analysis with the determination of the crossing points for every single transcript.
      5. Normalize the crossing point values for each gene to the particular crossing point values with the reference gene GAPDH.


    Figure 3. Determination of the anti-mycotoxic activity of O. sanctum L. essential oil (OSEO) on F. graminearum by UHPLC and RT-qPCR analysis. The quantification of ZEA present in broth culture is determined by UHPLC and concentration of ZEA is declined with increasing the concentration of OSEO. The relative fold expression of PKS4 and PKS13 are determined by RT-qPCR analysis and it is down-regulated with increasing the concentration of OSEO.

  10. Assessment of anti-mycotoxic efficacy of OSEO on F. graminearum in maize grains.
    1. Sterilize the seeds by autoclave and dry in a hot-air oven at 60 °C for 2 h. 
    2. Treat 100 g of sterilized maize grains with various concentrations (250, 500, 1,000, 1,500 and 2,000 μg/g) of OSEO in 500 ml conical flask and inoculate 10 μl of fungal spore suspension (1 x 106 spores/ml) of 7-day-old culture into each conical flask and incubate for 14 days at 28 °C in the dark.
    3. Consider the grains not treated with OSEO as control and incubated for a period of 14 days at 28 °C in the dark condition.
    4. Following the incubation period, extract total RNA from fungal mycelia and carry out RT-qPCR evaluation for PKS4 and PKS13 genes as mentioned earlier.
    5. Further, ground the maize grains into a fine powder and dissolve in 500 ml of acetonitrile and centrifuge at 4226.04 x g for 30 min.
    6. Transfer 15 ml of supernatant through ZEA specific immunoaffinity column and quantify the ZEA by UHPLC as mentioned earlier.

    Table 1. Primers used for RT-qPCR analysis of zearalenone production

Recipes

  1. Phosphate-buffered saline (pH 7.4)
    8.0 g NaCl
    0.2 g KCl
    1.44 g Na2HPO4
    0.24 g KH2PO4
    Dissolve all these chemicals in 800 ml of distilled water and adjust pH to 7.4 with 1 N HCl, and make up the volume to 1,000 ml with distilled water and sterilize by autoclaving.
  2. Lactophenol-cotton blue staining solution
    20 ml lactic acid
    20 g phenol crystals
    0.05 g cotton blue
    20 ml glycerol
    20 ml distilled water

Notes

Zearalenone is toxic and classified as group 3 carcinogen by International Agency for Research on Cancer (IARC, 1999) and care should be taken.

Acknowledgments

Authors are thankful to the Director, DFRL and DRDO-BU-CLS for their support to carry out the study, and also acknowledge Rana et al. (1997), Clinical and Laboratory Standards Institute, (2008), Ibáñez-Vea et al. (2011), Yamamoto-Ribeiro et al. (2013) and Vieira et al. (2014). This protocol is invited from the report of Kalagatur et al. (2015) and Kumar et al. (2016).

References

  1. Council of Europe. (1997). “Methods of pharmacognosy,” in European Pharmacopoeia, 3rd Ed. European Department for the Quality of Medicines: 121-122.
  2. Gaffoor, I. and Trail, F. (2006). Characterization of two polyketide synthase genes involved in zearalenone biosynthesis in Gibberella zeae. Appl Environ Microbiol 72(3): 1793-1799.
  3. Ibáñez-Vea, M., Corcuera, L. A., Remiro, R., Murillo-Arbizu, M. T., González-Peñas, E., and Lizarraga, E. (2011). Validation of a UHPLC-FLD method for the simultaneous quantification of aflatoxins, ochratoxin A and zearalenone in barley. Food Chemistry, 127(1), 351-358.
  4. John, H. and Mahmoud, A. (eds) (2008). Reference method for broth dilution antifungal susceptibility testing of yeasts; Approved Standard-Third Edition, M27-A3. CLSI.
  5. Kalagatur, N. K., Mudili, V., Siddaiah, C., Gupta, V. K., Natarajan, G., Sreepathi, M. H., Vardhan, B. H. and Putcha, V. L. (2015). Antagonistic activity of Ocimum sanctum L. essential oil on growth and zearalenone production by Fusarium graminearum in maize grains. Front Microbiol 6: 892.
  6. Kim, Y. T., Lee, Y. R., Jin, J., Han, K. H., Kim, H., Kim, J. C., Lee, T., Yun, S. H. and Lee, Y. W. (2005). Two different polyketide synthase genes are required for synthesis of zearalenone in Gibberella zeae. Mol Microbiol 58(4): 1102-1113.
  7. Kumar, K. N., Venkataramana, M., Allen, J. A., Chandranayaka, S., Murali, H. S. and Batra, H. V. (2016). Role of Curcuma longa L. essential oil in controlling the growth and zearalenone production of Fusarium graminearum. LWT-Food Science and Technology 69, 522-528.
  8. Rana, B. K., Singh, U. P. and Taneja, V. (1997). Antifungal activity and kinetics of inhibition by essential oil isolated from leaves of Aegle marmelos. J Ethnopharmacol 57(1): 29-34.
  9. Vieira, P. R. N., de Morais, S. M., Bezerra, F. H. Q., Ferreira, P. A. T., Oliveira, I. R. and Silva, M. G. V. (2014). Chemical composition and antifungal activity of essential oils from Ocimum species. Industrial Crops and Products 55: 267-271.
  10. Yamamoto-Ribeiro, M. M., Grespan, R., Kohiyama, C. Y., Ferreira, F. D., Mossini, S. A., Silva, E. L., Filho, B. A., Mikcha, J. M. and Machinski, M., Jr. (2013). Effect of Zingiber officinale essential oil on Fusarium verticillioides and fumonisin production. Food Chem 141(3): 3147-3152.

简介

由于其在多种气候条件下生长的能力,并且产生不同的真菌毒素,包括玉米赤霉烯酮(ZEA)和B型单端孢霉烯,因此在农业商品的背景下已经特别关注禾谷镰刀菌对人类,动物和植物的健康影响。合成抗真菌剂用于控制F的应用。禾谷镰菌对家畜和人类的健康造成负面影响,以及抗性生物体的高涨。因此,需要提出适当的食物谷物管理实践,包括应用草本抗真菌剂和真菌毒素控制剂以减少产毒的F的生长。禾谷镰菌以及在农产品中生产ZEA。 罗勒属圣地也被称为圣罗勒或图尔西被广泛用作阿育吠陀的药用植物。目前的协议表明量化em的抗真菌活性。圣地牙本香精油(OSEO),如由降低的F所反映的。禾谷镰刀菌生长和ZEA生产。 OSEO的抗真菌活性通过微孔稀释法进行,并通过扫描电子显微镜方法定量进一步验证。通过定量逆转录PCR(RT-qPCR)和超高效液相色谱(UHPLC)方法从F的肉汤培养物分析OSEO对ZEA产生的影响。禾本科。 OSEO的抗真菌毒性功效直接在F上评估。禾谷镰菌接种的玉米谷物。该方案有效地评估OSEO作为抗真菌侵袭和ZE生产的草药拮抗剂的活性。禾本科。该方案可用于测试多种草药化合物对抗F的抗真菌活性。禾谷镰刀菌或对其他霉菌毒素真菌进行修饰,这是对食品安全和加工工业的重要干预,其中真菌侵染是主要关注的问题。

关键字:玉米赤霉烯酮, 镰刀菌, 圣罗勒油, 超高效液相色谱, Q-RT-PCR

材料和试剂

  1. 0.22μmMillex-GP注射器过滤装置(Sigma-Aldrich,目录号:Z359904)
  2. 96孔微量滴定板(Eppendorf,目录号:0030602200)
  3. 柱C18,5μm,250×4.6mm(Phenomenex,目录号:00G-4041-E0)
  4. 碳导电胶带(Ted Pella,Inc.,目录号:16084-7)
  5. 玻璃载玻片(HiMedia,目录号:CG081)
  6. Whatman 1号纸(Sigma-Aldrich,目录号:Z274852)
  7. 玉米赤霉烯酮生产。禾本科 [The Microbial Type Culture Collection and Gene Bank,(MTCC),目录号:1893]
  8. 玉米谷物(当地农业市场,印度迈索尔)
  9. 玉米赤霉烯酮标准品(Sigma-Aldrich,目录号:Z2125)
  10. 二甲基亚砜(Merck Millipore,目录号:317275)
  11. 蒸馏水
  12. 氯化钠(NaCl)(Merck Millipore,目录号:1064040500)
  13. 氯化钾(KCl)(Merck Millipore,目录号:1049360250)
  14. 磷酸氢二钠(Na 2 HPO 4)(Merck Millipore,目录号:567550-1KG)
  15. 磷酸二氢钾(KH 2 PO 4)(Merck Millipore,目录号:1048730250)
  16. 0.1M二甲胂酸钠缓冲液(pH6.5)(Sigma-Aldrich,目录号:70114)
  17. 25%戊二醛(Merck Millipore,目录号:354400)
  18. 乙腈(Merck Millipore,目录号:100030)
  19. 乙醇(Merck Millipore,目录号:100983)
  20. 金箔(Sigma-Aldrich,产品编号:265829)
  21. ZEA(Vicam,目录号:G1026)的免疫亲和柱
  22. 使用SYBR Green(Bio-Rad Laboratories,目录号:1708892)的iScript One-Step RT-PCR试剂盒
  23. 液氮(本地供应商,印度迈索尔)
  24. 无核酸酶水(Qiagen,目录号:129114)
  25. 制霉菌素(Sigma-Aldrich,目录号:N6261)
  26. 洋葱香精(OSEO)(按程序步骤2中所述制备)
  27. 蛋白胨(HiMedia,目录号:RM001-500G)
  28. 瓷研钵(Sigma-Aldrich,目录号:Z529508)
  29. RNA easy plant Mini kit(Qiagen,目录号:74903)
  30. Sabouraud葡萄糖琼脂(HiMedia,目录号:M063-500G)
  31. Sabouraud葡萄糖肉汤(HiMedia,目录号:M033-500G)
  32. 合成的引物序列(Sigma-Aldrich,Bangalore,India)
  33. 吐温80(Merck Millipore,目录号:822187)
  34. 乳糖苯酚蓝(HiMedia,目录号:S016-500ML)
  35. 磷酸盐缓冲盐水(pH 7.4)(参见配方)
  36. 乳糖 - 棉蓝染色溶液(见配方)

设备

  1. Milli-Q一体式水净化系统(Merck Millipore,目录号:ZRXQ005WW)
  2. 铝棒(Ted Pella,Inc.,目录号:16111N)
  3. 高压灭菌器(Medica Instrument Manufacturing Company,型号:7431PAD)
  4. 微量离心机(Sigma-Aldrich,Eppendorf,型号:5415R)
  5. 离心机(Eppendorf,型号:5430R)
  6. 血细胞计数器(Sigma-Aldrich,目录号:Z359629)
  7. 热风炉(Memmert,型号:UFP800DW)
  8. 孵化器(Bio-age,型号:BSR-R2)
  9. 实时PCR系统(Roche Diagnostics,Light cycler ,型号:480)
  10. 0.5-10μl微量移液管(Eppendorf Research plus,目录号:3120000020)
  11. 20-200μl微量移液管(Eppendorf Research plus,目录号:3120000054)
  12. 100-1000μl微量移液管(Eppendorf Research plus,目录号:3120000062)
  13. 显微镜(Leica Microsystems,型号:Leica DM 1000LED)
  14. 250ml锥形瓶(Duran Group,目录号:21-23636)
  15. 500ml锥形瓶(Duran Group,目录号:21 216 44)
  16. NanoDrop 8000分光光度计(Thermo Fisher Scientific,目录号:ND-8000-GL)
  17. Nexera UHPLC系统(Shimadzu Corporation,型号:Nexera X2)
  18. 扫描电子显微镜(FEI,型号:Quanta 200)
    注意:此产品已由制造商[可更换项目(FEI,型号:Quanta 250/450/650)]停产。
  19. 摇床培养箱(Bio-age,型号:BSR-R1)
  20. 溅射涂布机(Quorum Technologies,型号:SC7620)
  21. 水浴(NUVE,型号:NB 5)
  22. 称重天平(丹佛仪器,型号:TB-215D)

软件

  1. GeneRunner软件版本5.0.47 Beta

程序

  1. 生长玉米赤霉烯酮(ZEA)产生F。 (MTCC,1893)在Sabouraud葡萄糖琼脂(SDA)上在28℃下培养7天,并将孢子收集在含有0.001%Tween 80的蛋白胨水中,并用软刮刀收集。使用血细胞计数器确定孢子的数量,并将孢子悬浮液调节至1×10 6/ml。
  2. 收集罗勒属密苏里植物并鉴定其植物学命名,并在室温下在黑暗条件下干燥植物。按照欧洲药典(欧洲委员会,1997年)的技术从干燥的地面部分提取精油。制备0.05%OSEO的DMSO溶液 注意:在本研究中,从印度卡纳塔克邦的迈索尔收集了圣罗勒(Rosimum sanctum L.),并通过印度植物调查局(印度哥印拜陀)进行了鉴定。
  3. 确定OSEO在F上的最小抑制(MIC)和最小杀真菌浓度(MFC)。 (2008)和Vieira等人的方法,通过微孔稀释技术在96孔微量滴定板中对禾谷镰刀菌进行培养。 (2014年)进行了以下小改动。
    1. 向不同浓度的OSEO中加入10μl孢子悬浮液(每ml 1×10 6个孢子),并用Sabouraud葡萄糖肉汤(SDB)将每个孔的总体积调整到100μl。
    2. 考虑没有OSEO作为对照的孔,并在28℃下在黑暗中孵育微板3天。
    3. 观察OSEO的最小浓度,没有可检测的真菌生长,并确定为最小抑制浓度(MIC)。
    4. 将平板从SDA板上的每个孔中10μl铺在板上,并在28℃下孵育3天
    5. 确定没有可检测到的真菌生长的OSEO的最小浓度并确定为MFC,与制霉菌素(阳性对照)相比,指定99.5%的原始接种物的杀灭。


    图1.O的最小抑制(MIC)和最小杀真菌浓度(MFC)的测定。圣地牙本精油(OSEO)。 通过微孔稀释法。在对照(未处理的OSEO)中观察到真菌生长,并且在OSEO的MFC值没有观察到可检测到的生长。

  4. 分析OSEO对F的孢子萌发的影响。禾谷镰菌 通过Rana 的方法。 (1997)略有修改。
    1. 在含有不同浓度的OSEO(100-1,800μg/ml)的SDA载玻片上接种10μl真菌孢子悬浮液(每ml 1×10 6个孢子),并在28℃下孵育24小时。
    2. 考虑单独的SDA载玻片与真菌孢子和没有OSEO作为对照。
    3. 孵育后,通过滴落法在28℃下用乳糖醇 - 棉蓝(100μl)对每个载玻片染色15分钟,并在显微镜下观察胚芽管。
    4. 从每个幻灯片检查至少200个孢子,并使用公式计算孢子萌发的百分比,
      %孢子萌发= ST/SC×100
      其中,SC是对照中发芽的孢子数,ST是试验中发芽的孢子数
  5. 确定OSEO对EM的菌丝体和孢子结构的影响。根据Yamamoto-Ribeiro等人的方法通过扫描电子显微镜(SEM)观察来检测禾谷镰刀菌。 (2013)略有修改。
    1. 从7天的F培养物中收集1cm 2的菌丝盘。禾谷镰刀菌,并在含有不同浓度的OSEO的SDA培养皿的中间无菌接种,并在28℃在黑暗中孵育7天。
    2. 考虑没有OSEO的SDA介质作为控制。
    3. 孵育后,收集1cm 2的菌丝盘并在磷酸盐缓冲盐水(pH 7.4)中漂洗并用2.5M戊二醛在0.1M二甲胂酸钠缓冲液(pH 6.5)中固定,并用梯度乙醇脱水(20,40,70,90和100%,保持菌丝体在100%中更长的持续时间)。
    4. 将样品粘贴在双面胶碳导电胶带上,并固定到铝棒的表面。
    5. 此外,在CO 2中暴露出临界点的短棒,并用金溅射涂层以增加其导电性。
    6. 在环境模式下,在20.0 KV下,在扫描电子显微镜下观察菌丝体的形态学质量。


    图2.确定O的抗真菌活性。圣地牙本香精油(OSEO)。禾谷镰刀菌通过扫描电子显微镜观察(Kalagatur等人,2015)。对照(OSEO未处理的)菌丝(a)是平滑的,湍流和同质的。而用MIC(b)和OSEO的MFC(c)值处理的菌丝表现出凹坑,突起和塌陷。对照(OSEO未处理的)孢子(d)是圆形和光滑的,另一方面,用MIC(e)和MFC(f)处理的孢子被OSEO破坏和起皱。
  6. 确定液体培养物中OSEO的抗真菌毒性活性。向每个含有100ml SDB的250ml锥形瓶中加入不同浓度的OSEO,包括250,500,1,000,1,500和2,000μg/ml。在无菌条件下将10μl7天龄培养物的10μl真菌悬浮液(1×10 6个孢子/ml)接种到这些烧瓶中。考虑没有OSEO的烧瓶作为对照,在摇动条件下(140-160rpm)在28℃下在黑暗中孵育烧瓶14天。
  7. 在孵育期后,通过用Whatman no.1纸过滤将培养基与真菌生物质分离,并使用肉汤用于测定ZEA。用无菌蒸馏水洗涤真菌菌丝体两次,并使用10毫克菌丝体进行RNA提取,并将剩余的菌丝体包装在预先称重的Whatman no.1滤纸中,并在60℃干燥24小时,并通过以下步骤测定菌丝体生物量:称重。
  8. 通过UHPLC通过Ibá?ez-Vea等人的方法检测和定量ZEA。 (2011)略有修改。
    1. 将培养液与等量的乙腈在140rpm下在摇床培养箱中混合30分钟。
    2. 随后,通过在4226.04×g离心12分钟收集样品的上清液,并将15ml上清液通过ZEA的免疫亲和柱,其在10ml磷酸盐缓冲盐水(pH7.4)。
    3. 接下来,用5ml PBS和10ml蒸馏水洗涤柱子。
    4. 最后,空气干燥柱并用5ml乙腈洗脱ZEA。保持乙腈和柱抗体之间的接触至少5分钟。
    5. 将洗脱液在60℃的水浴中完全干燥,并将最终残余物再溶解于1ml乙腈中,并通过0.22μm注射器过滤器过滤。
    6. 使用滤液进行UHPLC测定和ZEA的定量。
      1. 使用与C18,5μm,250 x 4.6 mm色谱柱相连的Nexera UHPLC系统,用于用荧光检测器反相检测和定量ZEA,并分别在334和450 nm处设置激发和发射波长。
      2. 流动相为乙腈 - 水(50:50,v/v),流速为1ml/min。
      3. 构建具有峰面积对ZEA浓度的标准ZEA(100ng-500μg/ml)的五点校准曲线。
      4. 标准溶液和测试样品的注射体积为25μl。
      5. 该技术的感测限制为100ng/ml。
  9. 确定OSEO对PKS4和PKS1 3的基因表达的影响,其参与F的ZEA生物合成。禾谷镰刀菌(Graminearum)(Gaffoor和Trail,2006; Kim等人,2005)通过RT-qPCR评价,使用GAPDH作为内源参照基因。
    1. 使用GeneRunner软件版本5.0.47 Beta设计靶基因的引物(表1)。
    2. 简言之,在液氮中快速冷冻菌丝体并用瓷研钵研磨成细粉末。使用RNEASY PLANT MINI KIT根据制造商的指南(Qiagen,Hilden,Germany)提取总RNA。
    3. 通过NanoDrop 8000分光光度计定量总RNA。
    4. 使用具有SYBR Green(Bio-Rad Laboratories,USA)的iScript One-Step RT-PCR试剂盒在Light Cycler 480中进行PKS4和PKS13的RT-qPCR分析。 。
      1. 简言之,制备50μl体积的反应混合物,其包含25μl2x SYBR Green RT-PCR反应混合物,1μl用于一步RT-PCR的iScript逆转录酶,1μl引物(450nM),1μl模板RNA (100ng)和22μl不含核酸酶的水(PCR级)。
      2. 反应的热条件包括在50℃进行1个循环的cDNA合成10分钟,在95℃进行聚合酶活化5分钟,然后进行35个循环的PCR,95℃10秒,60℃30秒用于联合退火和延伸。
      3. 对于每个PCR产物,通过在不同温度下的解链曲线分析获得单个窄峰。
      4. 量化基因表达的相对定量水平,利用二阶导数最大值分析,确定每个单个转录物的交叉点。
      5. 将每个基因的交叉点值与参考基因 GAPDH 标准化为特定的交叉点值。


    图3.O的抗真菌毒性活性的测定。圣地牙本香精油(OSEO)。通过UHPLC和RT-qPCR分析测定禾谷镰菌。通过UHPLC测定肉汤培养物中存在的ZEA的定量,ZEA的浓度随着OSEO浓度的增加而下降。通过RT-qPCR分析测定PKS4和PKS13的相对折叠表达,并且随着OSEO浓度的增加而下调。

  10. OSEO对抗真菌毒性效力的评价。禾谷镰菌。
    1. 通过高压灭菌器灭菌种子,在60℃的热风烘箱中干燥2小时。
    2. 在500ml锥形瓶中处理100g各种浓度(250,500,1,000,1,500和2,000μg/g)的OSEO的灭菌的玉米粒,并接种10μl的真菌孢子悬浮液(1×10 6/孢子/ml)的7天龄培养物,并在28℃在黑暗中孵育14天。
    3. 考虑未用OSEO处理的谷物作为对照,并在28℃在黑暗条件下孵育14天的时间。
    4. 在孵育期后,从真菌菌丝体提取总RNA,并如前所述对PKS4和PKS13基因进行RT-qPCR评价。
    5. 此外,将玉米粒磨成细粉并溶解在500ml乙腈中,并在4226.04×g离心30分钟。
    6. 通过ZEA特异性免疫亲和柱转移15ml上清液,并如前所述通过UHPLC定量ZEA。

    表1.用于玉米赤霉烯酮生产的RT-qPCR分析的引物

食谱

  1. 磷酸盐缓冲盐水(pH 7.4)
    8.0克NaCl 0.2克KCl
    1.44g Na 2 HPO 4
    0.24g KH 2 PO 4 sub/
  2. 乳糖 - 棉兰染色溶液
    20ml乳酸 20克苯酚晶体
    0.05克棉蓝
    20ml甘油 20ml蒸馏水

笔记

玉米赤霉烯酮是有毒的,并且被国际癌症研究机构(IARC,1999)分类为第3组致癌物,并且应该注意。

致谢

作者感谢DFRL和DRDO-BU-CLS主任对本研究的支持,并感谢Rana等人。 (1997),Clinical and Laboratory Standards Institute,(2008),Ibá?ez-Vea等人。 (2011),Yamamoto-Ribeiro等人。 (2013)和Vieira等人。 (2014年)。该协议从Kalagatur等人的报告中被邀请。 (2015)和Kumar等人。 (2016年)。

参考文献

  1. 欧洲理事会。 (1997)。 "Methods in pharmacognosy",载于 European Pharmacopoeia,3 sup 欧洲药品质量部:121-122。
  2. Gaffoor,I。和Trail,F。(2006)。 表征参与玉米赤霉烯酮生物合成的两种聚酮化合物合酶基因赤霉病。  Appl Environ Microbiol 72(3):1793-1799。
  3. (a):a class ="ke-insertfile"href(a),a,b,c,e,e,e,e, ="http://www.sciencedirect.com/science/article/pii/S0308814611000720"target ="_ blank"验证UHPLC-FLD方法,用于同时定量大麦中的黄曲霉毒素,赭曲霉毒素A和玉米赤霉烯酮。 食品化学,127(1),351-358
  4. John,H。和Mahmoud,A.(eds)(2008)。  酵母菌液稀释抗真菌药敏试验的参考方法;批准的标准 - 第三版,M27-A3。 CLSI 。
  5. Kalagatur,NK,Mudili,V.,Siddaiah,C.,Gupta,VK,Natarajan,G.,Sreepathi,MH,Vardhan,BH和Putcha,VL(2015)。罗勒属密室的拮抗活性 L.精油对生长和玉米赤霉烯酮生产的影响禾本科镰刀菌。 6:892。
  6. Kim,YT,Lee,YR,Jin,J.,Han,KH,Kim,H.,Kim,JC,Lee,T.,Yun,SHand Lee,YW(2005)。  在玉米赤霉中合成玉米赤霉烯酮需要两种不同的聚酮化合物合酶基因。 Mol Microbiol 58(4):1102-1113。
  7. Kumar,KN,Venkataramana,M.,Allen,JA,Chandranayaka,S.,Murali,HS和Batra,HV(2016)。  L.精油在控制禾本科禾谷镰刀菌生长和玉米赤霉烯酮产生中的作用。 LWT-Food Science and Technology 69,522-528。
  8. Rana,BK,Singh,UP和Taneja,V。(1997)。  抗真菌活性和由从Aegle marmelos的叶分离的精油的抑制动力学。 57 Ethnopharmacol 57(1):29-34。
  9. Vieira,PRN,de Morais,SM,Bezerra,FHQ,Ferreira,PAT,Oliveira,IR和Silva,MGV(2014)。 
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Copyright: © 2016 The Authors; exclusive licensee Bio-protocol LLC.
引用:Kalagatur, N. K., Dhamodaran, N., Siddaiah, C., Mudili, V. and Sreepathi, M. H. (2016). Antifungal and Zearalenone Inhibitory Activity of Ocimum sanctum L. Essential Oil on Fusarium graminearum Determined by UHPLC and RT-qPCR. Bio-protocol 6(15): e1893. DOI: 10.21769/BioProtoc.1893.
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