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Xylem Sap Extraction Method from Hop Plants
蛇麻木质部汁液提取方法   

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本实验方案简略版
Molecular Plant Microbe Interactions
May 2016

Abstract

Verticillium wilt is one of the most important diseases on hop that significantly influence continuation of production on affected areas. It is caused by the soil borne vascular pathogen Verticillium nonalfalfae, which infects plants through the roots and then advances through the vascular (xylem) system. During infection, V. nonalfalfae secretes many different virulence factors. Xylem sap of infected plants is therefore a rich source for investigating the molecules that are involved in molecular interactions of Verticillium – hop plants. This protocol provides instructions on how to infect hop plants with V. nonalfalfae artificially and how to obtain xylem sap from hop plants.

Keywords: Verticillium nonalfalfae (非苜蓿轮枝菌), Vascular pathogen (维管病原体), Xylem sap (木质部汁液), Extraction method (提取方法), Molecular interactions (分子相互作用)

Background

Extraction of xylem sap from plants is mostly used for studies of xylem sap proteome and various methods have been used to extract sap from plant xylem tissues. Buhtz et al. (2004) used hand-held pipettes to collect xylem sap from cut plant stems for comparing xylem proteomes in different plants (broccoli, oilseed rape, pumpkin and cucumber). The same method was used for collecting xylem sap from Brassica napus (Kehr et al., 2005), Brassica oleracea (Ligat et al., 2011) and soybean (Subramanian et al., 2009). Alvarez et al. (2006) extracted the maize xylem sap proteome using ‘root pressure’, as described by Goodger et al. (2005). Dafoe and Constabel (2009) used a Tygon tube, which was fitted over the wood, to collect xylem sap from hybrid poplar and no additional pressure was applied. Information on the protein content of xylem sap is also available for apple, pear and peach (Biles and Abeles, 1991).

Because vascular plant fungal pathogens spread inside host plants through their xylem, this fluid is the most appropriate medium to search for in planta secreted virulence factors from the fungal pathogen. Rep et al. (2002) used a simple xylem sap extraction method by Satoh et al. (1992) whereby cut stems of Fusarium oxysporum f. sp. lycopersici-infected tomato were placed in a horizontal position and sap was dripped from the cut surface. A Scholander pressure chamber (Scholander et al., 1965) was used to obtain xylem sap from Verticillium longisporum-infected oilseed rape (Floerl et al., 2008).

It is hard to draw any conclusion as to which method works best for what kind of plant since no comparative studies have been performed on different methods on the same plant species. It appears that plant species with soft and juicy stems need no pressure added to obtain xylem sap and simple methods are efficient for xylem sap extraction. This may be related to the root pressure, which causes xylem sap to rise through a plant stem from the roots towards the leaves due to osmosis in the roots (Taiz and Zeiger, 2010). However, some species never generate any root pressure (Kramer and Boyer, 1995) so some external force (e.g., a pressure chamber) must be provided in order to extract xylem sap from such plants. So far, there is no specific extraction method available for collecting xylem sap from hop plant infected with Verticillium nonalfalfae. Hop plants have woody roots and exposure of roots to pressure causes no harm to root tissue and no contamination (e.g., with cytosol liquids, cell membranes and other parts). We therefore used a Scholander pressure chamber on hops. This is the first protocol for sampling xylem sap from hop plants.

Materials and Reagents

  1. Miracloth (EMD Millipore, catalog number: 475855-1R )
  2. Filter paper (LLG, catalog number: 9.045 840 )
  3. Tape
  4. Plastic bag
  5. Sterile pipet tips
  6. 2 ml microtubes (BRAND, catalog number: 780550 )
  7. Silicone tubing, plastic tubing, glass tubing, etc.
  8. Petri dish (Golias, catalog number: PE01K )
  9. Host plants (hop Humulus lupulus, susceptible cultivar ‘Celeia’ and resistant cultivar ‘Wye Target’)
  10. Fungal conidia (Verticillium nonalfalfae; lethal pathotype PV1 [isolate T2]) (Radisek et al., 2006)
  11. Fertilizer YaraKristalon yellow NPK 13-40-13 + ME [ME - trace elements: B - 0.025%; Cu * - 0.01%; Fe * - 0.07%; Mn * - 0.04%; Mo - 0.004%; Zn * - 0.025%; * - Chelate base] (Yara International ASA)
  12. Fertilizer YaraKristalon special NPK 18-18-18 + ME [ME - trace elements: B - 0.025%; Cu * - 0.01%; Fe * - 0.07%; Mn * - 0.04%; Mo - 0.004%; Zn * - 0.025%; * - Chelate base] (Yara International ASA)
  13. Growing medium:
    1. For fungus inoculum preparation: liquid GFM – general fungal medium (Kayser, 1992) (see Recipes)
    2. For plants: sterile soil substrate for growing plants
    3. For fungus re-isolation: potato dextrose agar + antibiotics (streptomycin sulphate, neomycin, chloramphenicol; each 100 mg/ml) = PDA + A plates (see Recipes)
  14. Streptomycin sulphate (Duchefa Biochemie, catalog number: S0148 )
  15. Chloramphenicol (Sigma-Aldrich, catalog number: C0378 )
  16. Sterile distilled water (IDT, catalog number: 231-791-5 )
  17. Protease inhibitor cocktail (Sigma-Aldrich, catalog number: P8340 )
  18. 96% ethanol
  19. Peptone (Sigma-Aldrich, catalog number: 73049-73-7 )
  20. Yeast extract (AMRESCO, catalog number: J850 )
  21. Glucose (Kemika, catalog number: 07051 )
  22. Potassium nitrate (KNO3) (EMD Millipore, catalog number: 105063 )
  23. Potato dextrose agar (Biolife, catalog number: 4019352 )
  24. Neomycin (Duchefa Biochemie, catalog number: M0135 )

Equipment

  1. Plastic pots (1 L, 2 L)
  2. 500 ml Erlenmeyer flask (BRAND, catalog number: 92824 )
  3. Rotary shaker (Infrost, catalog number: 29313 )
  4. 2 L plastic cup (BRAND, catalog number: 87822 )
  5. Growth chamber (Kambič Laboratory Equipment, model: RK-13300 )
  6. Fluorescent grow lamp (Idealo, model: Osram Fluora L 58 W/77 )
  7. Thoma counting chamber (BRAND, Wertheim, Germany)
  8. Wooden sticks for hop’s bine support
  9. Scalpel and tweezers
  10. Light microscope (Nikon Instruments)
  11. Scholander pressure chamber (Soilmoisture Equipment, model: 3005 )
  12. Polystyrene box with ice

Procedure

  1. Preparation of host plants
    1. Use one-year-old potted hop plants multiplied from softwood cuttings of the susceptible cultivar ‘Celeia’ and resistant ‘Wye Target’ and grown in 1 L plastic pots. It is important that plants have strong, thick stems, the diameter of which should be more than 5 mm (Figure 1). Use plants are produced by a professional grower. Grow plants in a greenhouse on mist benches. Water the plants every 2 days and fertilize once per week using foliar fertilizers that contain macro and micro elements (e.g., 0.1% solution of YaraKristalon yellow NPK 13-40-13 + ME). To maintain the appropriate health status, spray the plants once per week against pests and foliar diseases using protective plant protection products.


      Figure 1. Example of stems of hop plants, cultivar Celeia. Left – strong, thick stem. Right – weak, thin stem.

  2. Inoculum preparation
    1. Place a small piece of fungal mycelium (of the size of a 2 mm ‘ball’) from stock PDA plates in each of four 500 ml Erlenmeyer flasks filled with 250 ml of liquid GFM, supplemented with 100 mg/ml streptomycin sulphate and 100 mg/ml chloramphenicol.
    2. Incubate for approximately 5 days at 100 rpm on a rotary shaker at room temperature and in the dark (place the shaker in a dark room) (Figure 2).


      Figure 2. A picture of fungal culture grown after 5 days. White fungal mycelium in a sphere shape can be seen. Medium with fungal isolates that produce a lot of spores is duller.

    3. Prepare the conidia inoculum (spore size is 5 to 7 μm) by filtration of mycelia and the spore through miracloth (typical pore size is 22-25 µm): place a non-autoclaved piece of Miracloth (20 x 20 cm) on top of a 2 L plastic cup, hand held, and filter each flask separately, one by one. Wash spores and suspend in sterile distilled water. If the concentration is low, all the filtration material is needed.
    4. Use a Thoma counting chamber to determine the spore concentration. Determine the spore concentration four times for one filtrated suspension and calculate an average.
    5. Adjust the spore concentration to 5 x 106 conidia/ml with sterile distilled water.
    6. The final volume of inoculum with a concentration of 5 x 106 conidia/ml for artificial infection should be 1 L. Use a maximum 12 plants per one volume of inoculum.

  3. Artificial infection
    1. Uproot one-year-old hop plants that have been grown in 1 L plastic pots. Remove as much soil substrate as possible from the roots by hand.
    2. Rinse the roots in sterile water (just gently dip for a few seconds).
    3. Then dip the roots for 10 min in 1 L of inoculum that has been poured into a 2 L plastic cup.
    4. Inoculate a minimum of 30 plants per treatment.
    5. Dispose of the remaining fungal inoculum after autoclaving.
    6. Treat the control plants similarly, but dip their roots in sterile distilled water.
    7. Pot the plants in new pots of size 2 L.
    8. Grow the plants as a single bine in a growth chamber (RK-13300, Kambič) under a 12-h photoperiod of fluorescent light (L 58 W/77; Fluora, Osram) at a temperature of 22 °C and relative humidity of 65% during the light period and 20 °C and 70% during the dark period. During growth in the chamber, water the plants twice a week and fertilize once per week using foliar fertilizer containing a higher quantity of nitrogen (e.g., 0.2% solution of YaraKristalon special NPK 18-18-18 + ME).

  4. Xylem sap extraction
    1. Sample the plants 30 days post inoculation (± 3 days).
    2. Uproot the hop plants. Carefully remove the soil from roots but do not wash them in water.
    3. Cut the plants 5-10 cm above ground with a scalpel or blade (Figure 3). Use the lower part of the plant (root side with 5-10 cm of stem) for xylem sap extraction and discard the upper part of the plant (containing the rest of the stem and leaves). Wash the cut surface with sterile distilled water (drip a few drops of water onto the surface using a 100 µl pipette and soak the water up with filter paper).


      Figure 3. Plants are cut 5-10 cm above ground with a scalpel or blade

    4. Place the root in a plastic bag (Figure 4), sealed with tape against the base of the stem. The plastic bag does not have to be sealed very well because it is used only to protect the Scholander pressure cylinder from getting dirty. Insert the stem through the metal cover of the Scholander pressure cylinder (Figure 5).


      Figure 4. The root is placed in a plastic bag


      Figure 5. Metal cover of Scholander pressure cylinder with cut plant stem. The plant stem is placed through the upper part of the Scholander pressure cylinder.

    5. Place the root side in the Scholander pressure chamber (Scholander et al., 1965) and make an installation from sterile pipette tips for leading the xylem sap to a microtube, which is placed on ice (Figure 6).  Any other installation (e.g., silicone tubing, plastic tubing, glass tubing, etc.) can be used for leading the xylem sap to the microtube.


      Figure 6. Xylem sap extraction with Scholander pressure chamber. Left: Installation from pipette tips for leading xylem sap to microtube; right: Extraction of xylem sap using Scholander pressure chamber. The microtube is placed on ice, applying a pressure of 2-2.5 bar.

    6. Apply a pressure of 2-2.5 bar to extract xylem fluid.
    7. Blot the first drops of exuding fluid with filter paper to avoid phloem contamination.
    8. Then collect the xylem sap (Figure 7) for up to 3 h in microtubes (they must be placed on ice!) containing up to 20 µl 1x protease inhibitor cocktail.
    9. Store samples of xylem sap at -80 °C until analysis.


      Figure 7. The stream of xylem sap. Xylem fluid is seen in the installation from pipette tips.

    10. Use the root side for mycological re-isolation of the pathogen to confirm the presence of the pathogen in the inoculated plants. Perform re-isolation in a laminar. Spray the stems with 96% ethanol and place them very briefly on an open flame in order to sterilise the surface. Harvest the xylem sections from inoculated plants and place them on PDA + A plates. The xylem section is actually the whole inner part of the stem, which should be cut into 1-2 cm long pieces. Place three to six pieces from each plant on PDA + A plates. Fungal outgrowth can be detected after the plates have been incubated for 3-5 days in the dark at room temperature (Figure 8). Examine the emerging mycelium (if any) by light microscopy.


      Figure 8. Fungal outgrowth from xylem sections after 3-5 days. White and fluffy mycelium is typical of V. nonalfalfae. Fungal mycelia prefer to grow on the plant material (xylem sections are completely overgrown by mycelium) and not on the PDA medium.

Data analysis

  1. Up to 2 ml of xylem sap is usually collected from a single plant in a 3 h extraction.
  2. A very low protein concentration in xylem is expected; samples of 3-10 individual plants (5-10 ml of xylem sap) are therefore pooled into one biological replicate. In our experience, pooled samples have an estimated concentration in the range 5-20 ng/µl.

Notes

  1. As mentioned in the section ‘Procedure’ (part ‘B. Inoculum preparation’, step 6), a maximum 12 plants per one volume of inoculum (which is 1 L with a concentration of 5 x 106 conidia/ml) should be used. During root dipping of these plants, the inoculum gets diluted. If there are more plants to be inoculated with the same inoculum, the concentration of used inoculum must therefore be recovered to 5 x 106 conidia/ml by adding fresh conidia. It is even better to prepare new inoculum with a concentration of 5 x 106 conidia/ml.
  2. We recommend that plants are watered one day before xylem sap sampling.
  3. Regarding xylem sap extraction
    1. The plants for sap extraction should be cut 5-10 cm above ground. We suggest not cutting higher because we have observed that we obtain a smaller amount of xylem sap with longer stems.
    2. For every new plant, use a sterile scalpel to cut the stem, new filter paper to blot phloem drops and a new pipette tips installation leading the xylem sap to the microtube.
    3. When the plant stem is placed through the metal cover of the Scholander pressure chamber, the rubber wrap around stem should not be tightened too much, in order to allow the xylem fluid to run out.
    4. We recommend collection of xylem sap for a maximum three hours, because prolonging the time does not help to increase the amount of xylem fluid.
    5. After three hours of collection, remove the installation from pipette tips that lead the xylem sap to the microtube from the cut stem and manually empty by hand-held pipette. Around 100 µl of xylem fluid is recovered with this step.
    6. The user of this protocol can expect that no xylem fluid will be obtained from some plants, for unknown reasons.
  4. Emerging mycelium on re-isolation plates must be subjected to morphological analysis using light microscopy in order to confirm the presence of V. nonalfalfae. Aerial mycelium of V. nonalfalfae is generally abundant, floccose to pruinose, hyphae are smooth-walled and 1.5-3 µm wide. Conidiophores are erect or slanted, generally determinate, branched or unbranched, formed disjointedly throughout the colonies and hyaline.

Recipes

  1. GFM
    2 g peptone
    2 g yeast extract
    20 g glucose
    1 g KNO3
    Mix in 1,000 ml distilled water and autoclave for 20 min
  2. PDA with 300 mg/ml antibiotics
    1. Add 35 g of potato dextrose agar in 1,000 ml distilled water
    2. Shake and mix the powder and autoclave it for 20 min
    3. Cool down the PDA broth to 55 °C
    4. Add 1 ml of 100 mg/ml streptomycin sulphate, 1 ml of 100 mg/ml neomycin and 1 ml of 100 mg/ml chloramphenicol and mix well
    5. Pour in 90 x 15 mm Petri dishes

Acknowledgments

This protocol is adapted from a previously published paper, Flajsman et al., 2016. We acknowledge the Slovenian Research Agency, research programs P4-0077, for funds. We thank Prof. Dr. Dominik Vodnik from the Chair of Applied Botany of the Biotechnical Faculty for the use of their Scholander pressure chamber.

References

  1. Alvarez, S., Goodger, J. Q., Marsh, E. L., Chen, S., Asirvatham, V. S. and Schachtman, D. P. (2006). Characterization of the maize xylem sap proteome. J Proteome Res 5(4): 963-972.
  2. Biles, C. L. and Abeles, F. B. (1991). Xylem sap proteins. Plant Physiol 96(2): 597-601.
  3. Buhtz, A., Kolasa, A., Arlt, K., Walz, C. and Kehr, J. (2004). Xylem sap protein composition is conserved among different plant species. Planta 219(4): 610-618.
  4. Dafoe, N. J. and Constabel, C. P. (2009). Proteomic analysis of hybrid poplar xylem sap. Phytochemistry 70(7): 856-863.
  5. Flajsman, M., Mandelc, S., Radisek, S., Stajner, N., Jakse, J., Kosmelj, K. and Javornik, B. (2016). Identification of novel virulence-associated proteins secreted to xylem by Verticillium nonalfalfae during colonization of hop plants. Mol Plant Microbe Interact 29(5): 362-373.
  6. Floerl, S., Druebert, C., Majcherczyk, A., Karlovsky, P., Kues, U. and Polle, A. (2008). Defence reactions in the apoplastic proteome of oilseed rape (Brassica napus var. napus) attenuate Verticillium longisporum growth but not disease symptoms. BMC Plant Biol 8: 129.
  7. Goodger, J. Q., Sharp, R. E., Marsh, E. L. and Schachtman, D. P. (2005). Relationships between xylem sap constituents and leaf conductance of well-watered and water-stressed maize across three xylem sap sampling techniques. J Exp Bot 56(419), 2389-2400.
  8. Kayser, T. (1992). Protoplasten fusion sowie elektrophoretische Chromosomentrennung und Genkartierung bei filamentösen Pilzen: Penicillium janthinellum, Absidia glauca und Cochliobolus heterostrophus. Dissertation.
  9. Kehr, J., Buhtz, A. and Giavalisco, P. (2005). Analysis of xylem sap proteins from Brassica napus. BMC Plant Biol 5: 11.
  10. Kramer, P. J. and Boyer, J. S. (1995). Water relations of plants and soils. Academic Press.
  11. Ligat, L., Lauber, E., Albenne, C., San Clemente, H., Valot, B., Zivy, M., Pont-Lezica, R., Arlat, M. and Jamet, E. (2011). Analysis of the xylem sap proteome of Brassica oleracea reveals a high content in secreted proteins. Proteomics 11(9): 1798-1813.
  12. Radišek, S., Jakše, J. and Javornik, B. (2006). Genetic variability and virulence among Verticillium albo-atrum isolates from hop. Eur J Plant Pathol 116(4): 301-314.
  13. Rep, M., Dekker, H. L., Vossen, J. H., de Boer, A. D., Houterman, P. M., Speijer, D., Back, J. W., de Koster, C. G. and Cornelissen, B. J. (2002). Mass spectrometric identification of isoforms of PR proteins in xylem sap of fungus-infected tomato. Plant Physiol 130(2): 904-917.
  14. Satoh, S., Iizuka, C., Kikuchi, A., Nakamura, N. and Fujii, T. (1992). Proteins and carbohydrates in xylem sap from squash root. Plant Cell Physiol 33(7): 841-847.
  15. Scholander, P. F., Bradstreet, E. D., Hemmingsen, E. A. and Hammel, H. T. (1965). Sap pressure in vascular plants: Negative hydrostatic pressure can be measured in plants. Science 148(3668): 339-346.
  16. Subramanian, S., Cho, U. H., Keyes, C. and Yu, O. (2009). Distinct changes in soybean xylem sap proteome in response to pathogenic and symbiotic microbe interactions. BMC Plant Biol 9: 119.
  17. Taiz, L. and Zeiger, E. (2010). Plant physiology 5th Ed. Sinauer Associates.

简介

黄萎病是最重要的疾病之一,对受影响地区的生产继续产生重大影响。它是由土壤传播的血管病原体疣黄萎病引起的,其通过根感染植物,然后通过血管(木质部)系统前进。感染期间,非苜蓿科分泌许多不同的毒力因子。因此感染植物的木质部汁液因此是调查涉及轮枝孢属植物的分子相互作用的分子的丰富来源。该协议提供有关如何用V感染啤酒花植物的说明。人工制备非苜蓿,如何从啤酒花植物获得木质部汁液。

背景 从植物中提取木质部汁液主要用于木质部汁液蛋白质组学的研究,并且已经使用各种方法从植物木质部组织中提取汁液。 Buhtz等人。 (2004)使用手持式移液器从切割的植物茎收集木质部汁液,用于比较不同植物(西兰花,油菜,南瓜和黄瓜)中的木质部蛋白质组。采用相同的方法从欧洲油菜中收集木质部汁液(Kehr等,2005),芸苔属(Ligat et al。,et al。 al。,2011)和大豆(Subramanian等人,2009)。 Alvarez等人。 (2006)使用“根压力”提取玉米木质部汁液蛋白质组,如Goodger等人所述。 (2005)。 Dafoe和Constabel(2009)使用安装在木材上的Tygon管,从混合杨树收集木质部汁液,并且不施加额外的压力。关于木质部汁液的蛋白质含量的信息也适用于苹果,梨和桃子(Biles and Abeles,1991)。
 因为维管植物真菌病原体通过其木质部分传播到宿主植物内,所以这种液体是在真菌病原体分泌的毒力因子的植物中寻找植物的最合适的培养基。 Rep 等人。 (2002)使用Satoh等人的简单的木质部汁液提取方法。 (1992),其中切割尖孢镰孢 f。 sp。将感染番茄的感染番茄置于水平位置,并从切割表面滴下汁液。使用Scholander压力室(Scholander等人,1965)从长尾um霉感染的油籽油菜中获得木质部汁液(Floerl等人 ,2008)。
 很难得出任何结论,哪种方法对于什么样的植物最有效,因为没有对同一植物物种的不同方法进行比较研究。似乎具有软和多汁茎的植物物种不需要添加压力来获得木质部汁液,并且简单的方法对于木质部汁液提取是有效的。这可能与根部压力有关,由于根部渗透导致木质部汁液从根部向叶子上升,从而导致木质部汁液的上升(Taiz和Zeiger,2010)。然而,一些物种从未产生任何根部压力(Kramer和Boyer,1995),因此必须提供一些外力(例如压力室),以从这些植物中提取木质部汁液。迄今为止,没有具体的提取方法可用于从非苜蓿轮枝孢霉感染的啤酒花植物中收集木质部汁液。禾本科植物具有木质根部和根部暴露于压力,不会对根组织造成伤害,并且没有污染(例如细胞质液体,细胞膜和其他部分)。因此,我们在啤酒花上使用了一个Scholander压力室。这是从啤酒花植物中采集木质部汁液的第一个方案。

关键字:非苜蓿轮枝菌, 维管病原体, 木质部汁液, 提取方法, 分子相互作用

材料和试剂

  1. Miracloth(EMD Millipore,目录号:475855-1R)
  2. 滤纸(LLG,目录号:9.045 840)
  3. 磁带
  4. 塑料袋
  5. 无菌吸管技巧
  6. 2 ml微量管(BRAND,目录号:780550)
  7. 硅胶管,塑料管,玻璃管等
  8. 培养皿(Golias,目录号:PE01K)
  9. 寄主植物(hop ulus ulus ulus ulus ulus ulus,,ia ia ia ia)))))))))))))))))))))))
  10. 真菌分生孢子(非苜蓿轮枝孢霉属);致死性病原型PV1 [分离株T2])(Radisek等人,2006)
  11. 肥料YaraKristalon黄色NPK 13-40-13 + ME [微量元素:B - 0.025%; Cu * - 0.01%; Fe * - 0.07%; Mn * -0.04%; Mo - 0.004%; Zn * -0.025%; * - 螯合基地(Yara International ASA)
  12. 肥料YaraKristalon特殊NPK 18-18-18 + ME [ME - 微量元素:B - - 0.025%; Cu * - 0.01%; Fe * - 0.07%; Mn * -0.04%; Mo - 0.004%; Zn * -0.025%; * - 螯合基地(Yara International ASA)
  13. 生长媒介:
    1. 对于真菌接种物制剂:液体GFM-通用真菌培养基(Kayser,1992)(参见食谱)
    2. 对于植物:生长植物的无菌土壤底物
    3. 对于真菌重新分离:马铃薯葡萄糖琼脂+抗生素(硫酸链霉素,新霉素,氯霉素;每100毫克/毫升)= PDA + A板(参见食谱)
  14. 硫酸链霉素(Duchefa Biochemie,目录号:S0148)
  15. 氯霉素(Sigma-Aldrich,目录号:C0378)
  16. 无菌蒸馏水(IDT,目录号:231-791-5)
  17. 蛋白酶抑制剂混合物(Sigma-Aldrich,目录号:P8340)
  18. 96%乙醇
  19. 蛋白胨(Sigma-Aldrich,目录号:73049-73-7)
  20. 酵母提取物(AMRESCO,目录号:J850)
  21. 葡萄糖(Kemika,目录号:07051)
  22. 硝酸钾(KNO 3)(EMD Millipore,目录号:105063)
  23. 马铃薯葡萄糖琼脂(Biolife,目录号:4019352)
  24. 新霉素(Duchefa Biochemie,目录号:M0135)

设备

  1. 塑料盆(1升,2升)
  2. 500毫升锥形瓶(BRAND,目录号:92824)
  3. 旋转振动筛(Infrost,目录号:29313)
  4. 2升塑料杯(BRAND,目录号:87822)
  5. 生长室(Kambič实验室设备,型号:RK-13300)
  6. 荧光灯(Idealo,型号:Osram Fluora L 58 W/77)
  7. Thoma计数室(BRAND,Wertheim,Germany)
  8. 木棍用于跳跳的支持
  9. 手镯和镊子
  10. 光学显微镜(Nikon Instruments)
  11. Scholander压力室(土壤水分设备,型号:3005)
  12. 聚苯乙烯箱与冰

程序

  1. 宿主植物的制备
    1. 使用一年龄的盆栽啤酒花植物从敏感栽培品种"Celeia"的软木切片和抗性"Wye Target"中繁殖并生长在1L塑料罐中。重要的是,植物具有坚实的,较厚的茎,其直径应大于5毫米(图1)。使用植物由专业种植者生产。在温室里种植植物在雾台上。每2天浇灌一次植物,每周施肥一次,使用含有微量和微量元素(例如,0.1%YaraKristalon黄NPK 13-40-13 + ME的溶液)的叶面肥料。为了保持适当的健康状况,每周喷洒一次植物以防止害虫和叶面病害使用防护植物保护产品。


      图1.啤酒花茎茎的实例,品种Celeia。左侧坚强,较厚的茎。右弱,细茎。

  2. 接种物准备
    1. 在装有250毫升补充有100mg/ml硫酸链霉素和100mg的液体GFM的四个500毫升锥形瓶中的每一个中,将一小块真菌菌丝体(大小为2mm球的)放置在储备的PDA板上/ml氯霉素。
    2. 在室温下和黑暗中将旋转振荡器以100rpm的速度孵育约5天(将振荡器置于暗室中)(图2)。


      图2. 5天后生长的真菌培养物的图片。可以看到球形的白色真菌菌丝体。具有产生大量孢子的真菌分离物的培养基是钝的。

    3. 通过过滤菌丝体和孢子通过miracloth(典型孔径为22-25μm)准备分生孢子接种物(孢子大小为5至7μm):将未经高压灭菌的Miracloth(20×20cm)片放在一个2升塑料杯,手持,并逐个过滤每个烧瓶。清洗孢子并悬浮于无菌蒸馏水中。如果浓度低,则需要所有的过滤材料。
    4. 使用Thoma计数室确定孢子浓度。确定一个过滤悬浮液的四次孢子浓度,并计算平均值
    5. 用无菌蒸馏水将孢子浓度调至5×10 6 /分钟/ml。
    6. 用于人造感染的浓度为5×10 6分配孢子/ml的接种物的最终体积应为1 L.每1次接种量最多使用12株植物。

  3. 人工感染
    1. 在1L塑料盆中种植一年的啤酒花植物。用手从根部移除尽可能多的土壤基底。
    2. 用无菌水冲洗根部(只需轻轻浸泡几秒钟)。
    3. 然后将根浸入1L已倒入2L塑料杯的接种物中10分钟。
    4. 每次处理至少接种30株植物。
    5. 高压灭菌后处理剩余的真菌接种物。
    6. 对照对照植物类似,但将其根部浸于无菌蒸馏水中
    7. 将植物放在2L尺寸的新盆中
    8. 在生长室(RK-13300,Kambič)中,在温度为22℃,相对湿度为65℃的12小时荧光光照(L 58 W/77; Fluora,Osram)下生长植物作为单一生物光照期间为20%,暗期为70%。在室内生长期间,每周给植物两次施肥,并且每周施用一次含有较高氮含量的叶面肥(例如,0.2%的YaraKristalon特殊NPK 18-18-18 + ME)
  4. 木质部汁液提取
    1. 接种后30天取样植株(±3天)
    2. 拔掉啤酒花植物。仔细地从根部除去土壤,但不要将其在水中洗涤。
    3. 用手术刀或刀片切割地面5-10厘米的植物(图3)。使用植物的下部(根侧有5-10厘米的茎)用于木质部汁液提取,并丢弃植物的上部(含有茎和叶的其余部分)。用无菌蒸馏水清洗切面(使用100μl移液管将数滴水滴至表面,并用滤纸浸泡水)。


      图3.使用手术刀或刀片将地板切成5-10厘米的植物

    4. 将根放在塑料袋中(图4),用胶带密封在杆的底部。塑料袋不需要很好地密封,因为它只用于保护Scholander压力缸不会变脏。将杆插入Scholander压力缸的金属盖(图5)

      图4.根部放在塑料袋中


      图5.具有切割植物茎的Scholander压力缸的金属盖。植物茎被放置在Scholander压力缸的上部。

    5. 将根侧放在Scholander压力室(Scholander等人,1965)中,并从无菌移液管尖端进行安装,将木质部汁液引导到放置在冰上的微管(图6) 。  任何其他安装(例如,硅胶管,塑料管,玻璃管,等)可用于将木质部汁液引导到微管。 >

      图6.用Scholander压力室进行木质部汁液提取。左:从移液管安装,用于引导木质部汁液到微管;右:使用Scholander压力室提取木质部汁液。将微管置于冰上,施加2-2.5巴的压力
    6. 施加2-2.5巴的压力来提取木质部液体。
    7. 用滤纸涂抹第一滴渗出液以避免韧皮部污染。
    8. 然后将含有高达20μl1x蛋白酶抑制剂混合物的微管(它们必须放在冰上)收集木质部汁液(图7)达3小时。
    9. 将木质部汁液的样品在-80°C储存,直到分析

      图7.从移液器技巧安装中可以看到木质部汁液流。
    10. 使用根部进行真菌学重新分离病原体,以确认接种植物中病原体的存在。在层流中进行重新隔离。用96%乙醇喷洒茎,并将其放置在明火上,以便对表面进行灭菌。从接种的植物收获木质部,并将它们放在PDA + A板上。木质部实际上是茎的整个内部,它应该被切成1-2厘米长的碎片。在PDA + A板上放置三至六块植物。在室温下在黑暗中孵育3-5天后,可以检测到真菌生长物(图8)。通过光学显微镜检查出现的菌丝体(如果有的话)。


      图8. 3-5天后木质部切片的真菌生长。白色和蓬松的菌丝体是非洲苜蓿的典型。真菌菌丝体优选在植物材料上生长(木质部切片由菌丝体完全长满),而不是在PDA培养基上生长。

数据分析

  1. 通常在3小时提取中从单一植物中收集高达2ml的木质部汁液。
  2. 预期木质部中的蛋白质浓度非常低;因此将3-10个单株植物(5-10ml木质部汁液)的样品合并成一个生物复制品。根据我们的经验,合并样本的估计浓度范围在5-20 ng /μl。

笔记

  1. 如"程序"一节(部分"B.接种物制备",步骤6)中所述,每一个体积的接种物(其浓度为5×10 6)的最大12株植物分生孢子/ml)。在这些植物的根浸时,接种物被稀释。如果有更多的植物接种相同的接种物,则必须通过加入新鲜的分生孢子将所用接种物的浓度回收至5×10 6分配孢子/ml。更好地准备浓度为5×10 6 /分钟/ml的新接种物。
  2. 我们建议植物在木质部汁液取样之前一天浇水。
  3. 关于木质部汁液提取
    1. 提取汁液的植物应在地上5-10厘米处切割。我们建议不要削减更高,因为我们已经观察到我们获得较少量的具有较长茎的木质部汁液。
    2. 对于每个新植物,使用无菌手术刀切割茎,新的滤纸以吸取韧皮部滴和新的移液管尖端安装,将木质部汁液引导到微管。
    3. 当植物茎放置在Scholander压力室的金属盖上时,茎周围的橡胶圈不应该被拧紧太多,以使木质部液体流出。
    4. 我们建议收集木质部汁液最多三个小时,因为延长时间无助于增加木质部液体的量。
    5. 经过三个小时的收集,从移液管尖端移除安装,将木质部汁液从切割杆导向微管,并用手持移液器手动清空。本步骤回收约100μl木质部液体。
    6. 该协议的用户可以预期,由于未知的原因,不能从一些植物获得木质部流体。
  4. 必须使用光学显微镜对再隔离板上的新生菌丝进行形态学分析,以确认V的存在。 nonalfalfae 。 V的空气菌丝体非苜蓿通常是丰富的,絮状的,细菌的,菌丝是光滑的,宽1.5-3μm。分生孢子梗是直立或倾斜的,通常是确定的,分枝的或不分枝的,在整个殖民地和透明质素中不连贯地形成。

食谱

  1. GFM
    2克蛋白胨
    2克酵母提取物
    20 g葡萄糖 1 g KNO <3>
    在1000毫升蒸馏水中混合,高压灭菌20分钟
  2. PDA用300毫克/毫升抗生素
    1. 在1000 ml蒸馏水中加入35 g马铃薯葡萄糖琼脂
    2. 摇动并混合粉末,高压灭菌20分钟
    3. 将PDA肉汤冷却至55°C
    4. 加入1 ml的100 mg/ml硫酸链霉素,1 ml的100mg/ml新霉素和1 ml的100mg/ml氯霉素,并混匀
    5. 倒入90 x 15毫米培养皿

致谢

该协议是从以前发表的论文"Flajsman等",2016年改编而来的。我们承认斯洛文尼亚研究机构P4-0077研究计划的资金。我们感谢生物技术学院应用植物学主席Dominik Vodnik博士使用他们的Scholander压力室。

参考文献

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Copyright: © 2017 The Authors; exclusive licensee Bio-protocol LLC.
引用:Flajšman, M., Mandelc, S., Radišek, S. and Javornik, B. (2017). Xylem Sap Extraction Method from Hop Plants. Bio-protocol 7(6): e2172. DOI: 10.21769/BioProtoc.2172.
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