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Soybean Cyst Nematode, Heterodera glycines, Infection Assay Using Soybean Roots
大豆异皮线虫和大豆胞囊线虫感染大豆根部的实验分析   

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参见作者原研究论文

本实验方案简略版
Molecular Plant Pathology
Dec 2014

Abstract

Soybean cyst nematode (SCN; Heterodera glycines), an obligate parasite of plants, is the most damaging pathogen of soybean, causing $469 to $818 million in soybean yield losses annually in the United States. However, there are no soybean cultivars available that are resistant to all SCN populations. Therefore, much research is being conducted to develop soybean cultivars resistant to SCN (Matthews et al., 2013; Matthews et al., 2014; Youssef et al., 2013). Here we describe the rearing and harvesting of SCN, as well as how SCN can be assayed by determining the Female Index.

Materials and Reagents

  1. Plastic Pasteur pipettes (Corning, Falcon®, catalog number: 357575 )
  2. Nylon cloth at 30 micron pore
  3. 90 mm Whatman circle filter size
  4. White filter paper (GE Healthcare, catalog number: 10347009 )
  5. Fleaker filtration unit (Cole Parmer fluid handling and analysis Item) (Cole-Parmer Instrument Compan, catalog number: EW-08917-50 )
  6. Disposable Petri Dishes (Kord-Valmark Labware Products, catalog number: 2900 )
  7. Compost soil
  8. Soybean seeds (William 82 or Essex) (Glycine max) (ARS-USDA)
  9. Sodium hypochlorite (Commercial Bleach-Clorox contains 8.3% Na-hyperchlorite)
  10. Sucrose (Baker Analyzed Reagent, catalog number: 4072-1 )
  11. Table sugar (Commercial sugar)
  12. Ethyl alcohol (The Warner-Graham Company, catalog number: 64-17-5 )
    Note: Currently, it is “Sigma-Aldrich, catalog number: 64-17-5 ”.
  13. Zinc sulfate heptahydrate (Sigma-Aldrich, catalog number: 91f-0135 )
  14. Sucrose solution (see Recipes)
  15. Sodium hypochlorite solution (see Recipes)
  16. 3 mm Zinc sulfate heptahydrate (ZnSO4) solution (see Recipes)

Equipment

  1. Greenhouse with a sink to divert drain water to a chlorine holding tank to kill nematodes that go down the drain
  2. 8-10 inch round Pots (Myers Lawn&Garden)
  3. Sieves (Sieve sizes: #20 = 841 micron; #60 = 250 micron; #80 = 177 micron; #100 = 149 micron; #250 = 58 micron; #500 = 25 micron) (U.S Standard Sieve Series)
  4. 1 liter glass cylinder (Pyrex USA)
  5. Dissecting microscope (Spenser)
  6. Gyrotory water bath Shaker (LabX, New Brunswick Scientific, model: G76 )
  7. 1 liter glass beaker (Pyrex)
  8. Platform Shaker (New Brunswick Scientific)
  9. 10 ml pipette (Thermo Fisher Scientific, Fisherbrand)

Procedure

Note: All procedures must be in compliance with APHIS requirements to prevent nematodes from escaping the laboratory and greenhouse.

  1. Culture pots containing soybean plants inoculated with SCN were maintained for three or more months. To collect cysts from stock pots, cut the stem at the soil line of a soybean plant from a stock pot inoculated with SCN for three months. The ball of roots and soil were placed into a two gallon bucket containing water to loosen the root system from the soil ball (Figure 1). How to make a soybean cyst nematodes culture pots was described in Matthews et al. (2013).


    Figure 1. Culture pots containing soybean plants inoculated with soybean cyst nematodes


    Figure 2. A. Soybean roots containing SCN. (Arrow indicates nematode cyst)

  2. The roots were placed on nested sieves, #20 on top and #100 on the bottom, as shown in Figure 3. The roots were gently massaged with the fingers and rinsed under running water to collect the cysts and the females on the #100 sieve.


    Figure 3. Nested sieves, #20 on top and #100 on the bottom

  3. The cysts were collected at the edge of the screen by gently washing the inside of the screen with running water. Water containing cysts were gathered at the edge of the screen. The cysts were rapidly poured into a sterile 500 ml plastic beaker.
  4. Cysts remaining in the soil-water mixture in the bucket were separated from the soil by stirring with a stick. The water was poured into the nested sieves, #20 on top and #100 on the bottom, and the cysts and females were collected on the #100 sieve. This step was repeated for 4 to 5 times.
    Note: Autoclave the soil and the roots waste. Treat the bucket with 10% bleach solution to kill any remaining nematodes. We have a valve in the sink drain pipe to divert water containing nematodes into a holding tank for treating the effluent with chlorine.
  5. The water containing the cysts and females was poured into a 1 L glass cylinder. After 20 to 30 min, the females and cysts sank to the bottom of the cylinder.
  6. The females and soil were allowed to settle for 5-10 min, and as much water as possible was poured off. The sucrose solution was added to a final volume of 500 ml. A piece of parafilm was placed over the top of the cylinder and turned upside down twice to mix the solution. In 15 min the females and cysts floated to the top of the solution separating them from most of the soil particles and root debris as shown in Figure 4.


    Figure 4. The females and cysts floated to the top of the sucrose solution separating them from most of the soil particles and root debris (the arrow points to the females and cysts)

    Note: The solution of sucrose (454 grams/L) was made while waiting for the females to settle, so it is fresh to avoid fungal growth. Table sugar works fine.
  7. To clean the females and cysts further, they were poured into a 1 L glass beaker. The cylinder was rotated while pouring to recover the females stuck on the walls of the cylinder. The solution from the beaker was poured into a 3 inch diameter #100 sieve and rinsed well to remove the sucrose solution. The sieve was rinsed and contents collected in a 250 ml beaker.
  8. To clean the females from any remaining root debris, the beaker was rotated to resuspend the females and cysts and allowed to settle a few seconds. The females and cysts were allowed to sink to the bottom of the beaker, because they are heavier than most of the root debris. The root debris was discarded by pouring the water into a glass beaker, leaving the females and cysts in the beaker bottom. This step is repeated several times until most of the root debris was removed.
  9. To release nematodes and eggs from the cysts, place a 100 sieve containing females on top of an autoclaved 4 cm deep plastic plate (lid of pipet tip box). Add sterile water to just above the level of the sieve. Gently crush the females and cysts with a clean rubber stopper. Occasionally lift the sieve to allow the eggs to fall through to the lid below. Crush the females and cysts for 5 to 10 min. When the water is cloudy, switch sieve to a new autoclaved 4 cm deep plastic plate (lid of pipet tip box) with fresh sterile ddH2O. Crush again. Check the progress of crushing of the females under the microscope. When finished crushing, pour the solution through a 3 inch diameter #250 nested with a #500 sieve. The #250 sieve catches the female and cyst shells. The #500 sieve contains the purified eggs, while the small debris flows through the #500 sieve.


    Figure 5. The females and cysts placed on top of an autoclaved 350 ml plate to release the eggs

  10. Eggs were sterilized by placing the #500 sieve with eggs in an autoclaved 4 cm deep plastic plate (lid of pipet tip box). Bleach solution was added for 1.5 min to the eggs, and the sieve was rotated. The eggs were drained and rinsed with 1 L sterile water. A little sterile water was added to the eggs and the eggs were poured into a fresh sterile 4 cm deep plastic plate (lid of pipet tip box). The volume of the egg and sterile water mixture was brought to 120 ml with sterile water. Then, 1.2 ml sterile 300 mM ZnSO4.7H2O (zinc sulfate heptahydrate) was added to make a final concentration of 3 mM ZnSO4. The ZnSO4 enhances hatching and controls fungal growth. The plate was covered with plastic wrap and placed on a heated shaker at 28 °C at 50-75 rpm for aeration for at least three days. Eggs continued to hatch each day thereafter for over a week. Progress of hatching was monitored using a dissecting microscope. Hatching nematodes are shown in Figure 6.


    Figure 6. Hached J2 juvenile nematodes and eggs

  11. J2 stage juveniles were collected by placing a clean nylon cloth of 30 micron pore size into a 1 L glass beaker containing 200 ml reverse osmosis water. The egg/juvenile solution was poured into the nylon cloth and collected. The cloth was dunked like a tea bag into the water for several minutes. This process was repeated with a fresh beaker and water if the juveniles were very concentrated. The volume was increased to no more than 200 ml in a 1 L beaker, and then placed on a shaker at 50-75 rpm to concentrate the juveniles to the center of the beaker. After roughly a half hour, the J2 juveniles were removed with a Pasteur pipette and placed in a 400 ml beaker. Sterile water was added to the desired volume for the experiment and the concentration was checked by pipetting 5 μl of the J2 solution on a drop of water on a microscopic slide to count the nematodes. This was repeated three times and the average was calculated. To get 1,000 J2 per ml, you want 5 J2 per 5 μl solution.
  12. To inoculate the roots of soybean plants, the plants were grown in sterile sand for 4 weeks. Immediately before inoculation, the plants were watered well then allowed to drain to keep them hydrated. Two holes about 1.5 to 2 cm deep, and 0.5 to 1 cm from the stem were made in the sand on either side of the plant. The solution of nematodes was gently swirled during the inoculation period to keep the nematodes suspended evenly and aerated. A 10 ml pipette was used to measure and dispense the solution of J2 nematodes. To each hole 1 ml of a 1,000 J2/ml solution was added for a total of 2,000 J2/plant. The hole was gently filled with sand. The plants were not watered for two days to give the juveniles a chance to find and migrate into the roots.
  13. After 30-35 days the females were harvested from each plant, as described in steps 2 and 3. The sand ball containing the soybean roots with nematodes was gently placed in a 1 L plastic beaker. Much of the sand was removed by gently massaging the roots over a beaker. The beaker containing the water and sand were retained for collection of cysts and females. The cell packs or pots were rinsed, and the solution was collected to obtain females that may have adhered to the walls. The solution containing the cysts and females were poured into another and labeled. The roots were gently rubbed under running water to dislodge the cysts and females onto a pair of nested sieves. The #20 sieve allowed the cysts to pass through while keeping out larger debris. The cysts and females were retained on the #100 sieve. The cysts and females were poured into a clean labeled beaker. Cysts and females that were in the sand and in water used to rinse the walls of the pot were collected by passing the water through the nested sieves. More water was added to the beaker and swirled to release cysts and females trapped in the sand. The water was passed through the sieve. This process of rinsing the sand to obtain cysts and females was repeated three times or until the water was clear after swirling. The roots were saved in labeled 100 ml plastic beakers for dry weight measurements.
  14. The cysts were collected on a lined 90 mm Whatman filter circle using a Fleaker filtration unit (Figure 7). Water was added to wet the filter. The collected nematode solution was added under vacuum. The filter was removed from the unit and a lid was placed on a disposable standard Petri plate. The cysts were counted under a dissecting microscope. The dry weight of each root was recorded in a spreadsheet.


    Figure 7. Cysts of SCN collected on lined Whatman filter paper

  15. Female index calculation. Remove the outliers in the female count data using Grubbs’ test (Grubbs, 1969). A free online version can be found at the GraphPad QuickCalcs Web site (http://graphpad.com/quickcalcs/grubbs1/). Check the normality of the data using the Shapiro-Wilk test (Shapiro and Wilk, 1965). A free, online version implemented by S. Dittamin can be found at http://dittami.gmxhome.de/shapiro/). The female index can be calculated as follows: Female index = (Ng/Nc) X 100, where Ng = mean number of females on roots of experimental plants. Nc = mean number of females present on roots of control plants. Means can be compared using Welch’s unpaired t test for unequal variance (Welch, 1947). A free version is available online at the GraphPad QuickCalcs Web site (http://graphpad.com/quickcalcs/ttest1/). Number of cysts per dry weight of the root can be calculated to standardize the experiment, especially if root size varies greatly.

Recipes

  1. 1 L of sucrose solution
    454 g sucrose
    1 L reverse osmosis water
  2. Sodium hypochlorite solution
    Make a 0.4% sodium hypochlorite solution by adding 14.8 ml standard bleach (8.3% sodium hypochlorite) to 285.2 ml sterile water
  3. 3 mm Zinc sulfate heptahydrate (ZnSO4) solution
    1.2 ml sterile 300 mM ZnSO4.7H2O (zinc sulfate heptahydrate) to make a final concentration of 3 mm ZnSO4

Acknowledgments

This protocol is adapted from our previous work, including, Matthews et al. (2013) and Youssef et al. (2013). We thank Peggy MacDonald for technical support. Mention of trade name, proprietary product or vendor does not constitute a guarantee or warranty of the product by the U. S. Department of Agriculture or imply its approval to the exclusion of other products or vendors that also may be suitable. The authors have no conflict of interest.

References

  1. Grubbs, F. (1969). Procedures for detecting outlying observations in samples. Technometrics 11: 1-21.
  2. Matthews, B. F., Beard, H., MacDonald, M. H., Kabir, S., Youssef, R. M., Hosseini, P. and Brewer, E. (2013). Engineered resistance and hypersusceptibility through functional metabolic studies of 100 genes in soybean to its major pathogen, the soybean cyst nematode. Planta 237(5): 1337-1357.
  3. Matthews, B. F., Beard, H., Brewer, E., Kabir, S., MacDonald, M. H. and Youssef, R. M. (2014). Arabidopsis genes, AtNPR1, AtTGA2 and AtPR-5, confer partial resistance to soybean cyst nematode (Heterodera glycines) when overexpressed in transgenic soybean roots. BMC Plant Biol 14: 96.
  4. Shapiro, S. S. and Wilk, M. B. (1965). Analysis of variance test for normality (complete samples). Biometrika 52: 591-611.
  5. Youssef, R. M., MacDonald, M. H., Brewer, E. P., Bauchan, G. R., Kim, K. H. and Matthews, B. F. (2013). Ectopic expression of AtPAD4 broadens resistance of soybean to soybean cyst and root-knot nematodes. BMC Plant Biol 13: 67.

简介

作为植物的专性寄生虫的大豆胞囊线虫(SCN; Heterodera glycines)是大豆最具破坏性的病原体,在美国每年导致大豆产量损失469至8.18亿美元。 然而,没有可用的对所有SCN群体有抗性的大豆栽培品种。 因此,正在进行大量研究以开发对SCN有抗性的大豆栽培种(Matthews等人,2013; Matthews等人,2014; Youssef et al。 ,2013)。 在这里我们描述饲养和收获的SCN,以及如何SCN可以通过确定女性指数测定。

材料和试剂

  1. 塑料巴斯德移液管(Corning,Falcon ,目录号:357575)
  2. 30微米孔的尼龙布
  3. 90毫米Whatman圆形滤镜尺寸
  4. 白色滤纸(GE Healthcare,目录号:10347009)
  5. Fleler过滤单元(Cole Parmer流体处理和分析项目)(Cole-Parmer Instrument Compan,目录号:EW-08917-50)
  6. 一次性培养皿(Kord-Valmark Labware Products,目录号:2900)
  7. 堆肥土
  8. 大豆种子(William 82或Essex)(大豆)(ARS-USDA)
  9. 次氯酸钠(商业Bleach-Clorox含有8.3%高氯酸钠)
  10. 蔗糖(Baker Analyzed Reagent,目录号:4072-1)
  11. 表糖(商业糖)
  12. 乙醇(Warner-Graham公司,目录号:64-17-5)
    注意:目前,它是"Sigma-Aldrich,目录号:64-17-5"。
  13. 硫酸锌七水合物(Sigma-Aldrich,目录号:91f-0135)
  14. 蔗糖溶液(见配方)
  15. 次氯酸钠溶液(见配方)
  16. 3毫米的硫酸锌七水合物(ZnSO 4)溶液(参见配方)

设备

  1. 温室有一个水槽,将排水转移到一个氯容器,以杀死线虫下来的线虫
  2. 8-10英寸圆盆(梅尔斯草坪花园)
  3. 筛(筛尺寸:#20 = 841微米;#60 = 250微米;#80 = 177微米;#100 = 149微米;#250 = 58微米;#500 = 25微米)(美国标准筛系列) >
  4. 1升玻璃圆筒(Pyrex USA)
  5. 解剖显微镜(Spenser)
  6. Gyrotory水浴摇床(LabX,New Brunswick Scientific,型号:G76)
  7. 1升玻璃烧杯(Pyrex)
  8. 平台振动器(New Brunswick Scientific)
  9. 10ml的移液管(Thermo Fisher Scientific,Fisherbrand)

程序

注意:所有程序必须符合APHIS要求,以防止线虫逃离实验室和温室。

  1. 将含有用SCN接种的大豆植物的培养罐保持三个月或更长时间。为了从储存罐收集孢囊,在来自接种SCN的储存罐的大豆植物的土壤线处切断茎3个月。将根和土壤的球放入含有水的两加仑桶中以从土壤球中松开根系统(图1)。如何制作大豆胞囊线虫培养罐在Matthews等人(2013)中有描述。


    图1.含有用大豆胞囊线虫接种的大豆植物的培养罐


    图2. A.含有SCN的大豆根。(箭头表示线虫胞囊)

  2. 将根置于嵌套的筛子上,在顶部#20和在底部#100,如图3所示。用手指轻轻按摩根并在流水下漂洗以收集在#100筛上的孢囊和雌性。


    图3.嵌套筛网,#20在顶部,#100在底部

  3. 通过用流水轻轻洗涤筛子的内部,在筛子的边缘收集囊肿。含水孢囊聚集在筛子的边缘。将孢囊迅速倒入无菌的500ml塑料烧杯中。
  4. 通过用棒搅拌,将保留在桶中的土壤 - 水混合物中的孢囊与土壤分离。将水倒入嵌套的筛中,在顶部#20和在底部#100,并且在#100筛上收集孢囊和雌性。将该步骤重复4?5次。
    注意:高压灭菌土壤和根系废物。用10%漂白溶液处理桶以杀死任何剩余的线虫。我们在水槽排水管中有一个阀门,用于将含有线虫的水转移到一个用氯处理废水的储水槽中。
  5. 将含有孢囊和雌性的水倒入1L玻璃圆筒中。 20到30分钟后,雌性和囊肿沉积到圆柱体的底部。
  6. 使女性和土壤沉降5-10分钟,并倒出尽可能多的水。加入蔗糖溶液至终体积为500ml。将一片石蜡膜放置在圆筒的顶部上并颠倒两次以混合溶液。在15分钟内,雌性和囊肿浮在溶液的顶部,将它们与大多数土壤颗粒和根碎片分离,如图4所示。


    图4.雌性和囊肿漂浮在蔗糖溶液的顶部,将它们与大多数土壤颗粒和根碎片分离(箭头指向雌性和囊肿)

    注意:在等待雌性沉降的同时制备蔗糖溶液(454克/L),因此其是新鲜的以避免真菌生长。表糖工作正常。
  7. 为了进一步清洁雌性和囊肿,将它们倒入1L玻璃烧杯中。在倾倒的同时旋转圆柱体以回收粘在圆柱体壁上的雌性。将来自烧杯的溶液倒入3英寸直径的#100筛中并充分漂洗以除去蔗糖溶液。冲洗筛并将内容物收集在250ml烧杯中。
  8. 为了从任何剩余的根碎片清洁女性,旋转烧杯以重悬雌性和囊肿并允许沉降几秒钟。女性和囊肿被允许下沉到烧杯的底部,因为它们比大多数根碎片更重。通过将水倒入玻璃烧杯中弃去根碎片,在烧杯底部留下雌性和囊肿。该步骤重复几次,直到大部分根碎片被除去。
  9. 为了从囊肿释放线虫和卵,在高压灭菌4cm深的塑料板(移液管吸头盒的盖子)的顶部放置含有雌性的100筛。添加无菌水到刚好高于筛子的水平。轻轻地粉碎女性和囊肿用干净的橡胶塞。偶尔提起筛子,让鸡蛋掉落到下面的盖子。粉碎女性和囊肿5至10分钟。当水混浊时,将筛子转移到新的灭菌的ddH 2 O的高压灭菌的4cm深的塑料板(移液管尖端盒的盖子)上。再次粉碎。检查在显微镜下女性的压碎的进展。当完成粉碎时,将溶液倾倒通过用#500筛网嵌套的3英寸直径#250。 #250筛子捕获女性和囊肿壳。 #500筛包含纯化的蛋,而小碎片流过#500筛。


    图5.将雌性和囊肿放置在高压灭菌的350ml板的顶部以释放蛋

  10. 通过将具有蛋的#500筛子置于高压灭菌的4cm深的塑料板(移液管吸头盒的盖子)中来灭菌鸡蛋。向蛋中加入漂白溶液1.5分钟,旋转筛子。将蛋排干并用1L无菌水冲洗。将少量无菌水加入蛋中,并将蛋倒入新鲜无菌的4cm深的塑料板(移液管吸头盒的盖子)中。用无菌水使蛋和无菌水混合物的体积达到120ml。然后,加入1.2ml无菌的300mM ZnSO 4·7H 2 O·7H 2 O(硫酸锌七水合物),使最终浓度为3mM ZnSO 4 4 。 ZnSO 4增强孵化并控制真菌生长。用塑料包膜覆盖板,并置于28℃,50-75rpm的加热振荡器上通气至少三天。其后每天继续孵蛋超过一周。使用解剖显微镜监测孵化的进展。孵化线虫如图6所示。


    图6. Hached J2幼虫线虫和卵

  11. 通过将30微米孔径的干净的尼龙布置于含有200ml反渗透水的1L玻璃烧杯中收集J2阶段幼体。将蛋/幼体溶液倒入尼龙布中并收集。布像一个茶包浸入水中几分钟。如果幼鱼非常浓缩,用新鲜的烧杯和水重复该过程。在1L烧杯中将体积增加至不超过200ml,然后以50-75rpm置于振荡器上以将幼体集中到烧杯的中心。大约半小时后,用巴斯德吸管取出J2幼体,并置于400ml烧杯中。加入无菌水至实验所需的体积,并通过在显微镜载玻片上的一滴水上吸取5μlJ2溶液以计数线虫来检查浓度。将其重复三次,并计算平均值。为了每毫升1000个J2,你需要5个每5μl溶液的J2。
  12. 为了接种大豆植物的根,将植物在无菌砂中生长4周。在接种之前,将植物浇水,然后排出以保持它们水合。在植物两侧的沙子上制出两个深约1.5至2厘米深,距离茎0.5至1厘米的孔。在接种期间使线虫溶液轻轻地涡旋以保持线虫均匀悬浮并充气。使用10ml移液管测量和分配J2线虫的溶液。向每个孔中加入1ml的1,000J2/ml溶液,总共为2,000J2 /植物。孔里轻轻地充满了沙子。植物不浇水两天,以使幼体有机会发现并迁移到根中。
  13. 在30-35天后,如步骤2和3中所述从每个植物收获雌性。将含有具有线虫的大豆根的沙球轻轻地放置在1L塑料烧杯中。通过在烧杯上轻轻按摩根部来除去大部分沙。保留含有水和砂的烧杯用于收集孢囊和雌性。冲洗细胞堆叠或盆,并收集溶液以获得可能粘附到壁的雌性。将含有囊肿和雌性的溶液倒入另一个中并进行标记。在流水下轻轻擦拭根部以将囊肿和雌性移到一对嵌套的筛子上。 #20筛允许囊肿通过,同时保持更大的碎片。囊肿和雌性保留在#100筛上。将囊肿和雌性倒入干净的标记烧杯中。通过使水通过嵌套的筛子来收集在沙子和用于冲洗罐壁的水中的囊和雌。将更多的水加入到烧杯中并旋动以释放陷入沙子中的囊肿和雌性。使水通过筛子。冲洗砂以获得囊肿和雌性的这个过程重复三次或直到旋转后水清澈。将根保存在标记的100ml塑料烧杯中用于干重测量
  14. 使用Fleaker过滤单元(图7)在衬有90mm Whatman过滤器圆上收集孢囊。加入水以润湿过滤器。在真空下加入收集的线虫溶液。从单元中取出过滤器,并将盖放置在一次性标准Petri板上。在解剖显微镜下计数囊肿。每个根的干重记录在电子表格中。


    图7.在内衬的Whatman滤纸上收集的SCN囊肿

  15. 女性指数计算。使用Grubbs检验去除雌性计数数据中的异常值(Grubbs,1969)。可以在GraphPad QuickCalcs网站上找到免费在线版本( http://graphpad.com/quickcalcs/grubbs1/)。使用Shapiro-Wilk检验检查数据的正态性(Shapiro和Wilk,1965)。由S. Ditamin执行的免费在线版本可在 http://dittami.gmxhome.de/shapiro/)。雌性指数可以如下计算:雌性指数=(N g g/N c)×100,其中N g g =平均雌性对实验植物的根。 N c c =存在于对照植物的根上的雌性的平均数。可以使用Welch的不成对的 t 测试比较不平等方差的平均值(Welch,1947)。可以在GraphPad QuickCalcs网站上免费获得版本( http://graphpad.com/quickcalcs/ttest1/)。可以计算每根根干重的孢囊数,以使实验标准化,特别是如果根大小变化很大。

食谱

  1. 1L蔗糖溶液 454克蔗糖 1 L反渗透水
  2. 次氯酸钠溶液
    通过向285.2ml无菌水中加入14.8ml标准漂白剂(8.3%次氯酸钠)制备0.4%次氯酸钠溶液。
  3. 3mm的硫酸锌七水合物(ZnSO 4)溶液
    1.2ml无菌的300mM ZnSO 4·7H 2 O(硫酸锌七水合物),使最终浓度为3mm ZnSO 4

致谢

该协议改编自我们以前的工作,包括Matthews等人(2013)和Youssef等人(2013)。我们感谢Peggy MacDonald的技术支持。提及商业名称,专利产品或供应商不构成美国农业部对产品的担保或保证,也不意味着批准其他可能适用的其他产品或供应商。作者没有利益冲突。

参考文献

  1. Grubbs,F。(1969)。 在样本中检测异常观察的程序。 Technometrics 11:1-21。
  2. Matthews,B.F.,Beard,H.,MacDonald,M.H.,Kabir,S.,Youssef,R.M.,Hosseini,P.and Brewer,E。 通过对大豆中100种基因对其主要病原体,大豆胞囊的功能代谢研究的工程化抗性和过敏性线虫。 237(5):1337-1357。
  3. Matthews,B.F.,Beard,H.,Brewer,E.,Kabir,S.,MacDonald,M.H.and Youssef,R.M。(2014)。 拟南芥基因, AtNPR1 , > AtTGA2和 AtPR-5 ,当在转基因大豆根中过表达时赋予对大豆胞囊线虫( Heterodera glycines )的部分抗性。 BMC Plant Biol 14:96.
  4. Shapiro,S.S。和Wilk,M.B。(1965)。 正常模型的方差测试分析(完整示例)。 Biometrika 52:591-611。
  5. Youssef,R.M.,MacDonald,M.H.,Brewer,E.P.,Bauchan,G.R.,Kim,K.H。和Matthews,B.F。(2013)。 AtPAD4的异位表达拓宽了大豆对大豆胞囊和根结线虫的抗性。 BMC Plant Biol 13:67.
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Copyright: © 2016 The Authors; exclusive licensee Bio-protocol LLC.
引用:Matthews, B. F. and Youssef, R. M. (2016). Soybean Cyst Nematode, Heterodera glycines, Infection Assay Using Soybean Roots. Bio-protocol 6(2): e1707. DOI: 10.21769/BioProtoc.1707.
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