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Dec 2020

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Phytophthora infestans (Late blight) Infection Assay in a Detached Leaf of Potato
在离体叶片中的马铃薯疫霉(晚疫病)感染实验   

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Abstract

Phytophthora infestans is a hemibiotroph oomycete that primarily infects potato and tomato. It infects stems, leaves, and tubers and fruits of potato and tomato. High throughput and reproducible infection assays are prerequisites to find sources of resistance in any crop. In this protocol, we describe a detached leaf assay (DLA) for conducting the virulence assay of P. infestans in potato leaves. A late blight infection assay using a potato detached leaf is a semi-high throughput assay in which hundreds of plants can be screened in a laboratory setting.

Keywords: Phytophthora infestans (致病疫霉), Potato (马铃薯), Detached leaf assay (离体叶片分析), Late blight (晚疫病)

Background

Potato (Solanum tuberosum L.) is one of the most important non-cereal food crops in terms of food and nutritional value (Zhang et al., 2017). Late blight of potato caused by the oomycete pathogen P. infestans is one of the most devastating potato diseases in the world and is the most important yield-limiting factor in potato production (Haverkort et al., 2008 and 2016; Fisher et al., 2012). Breeding for late blight resistance is considered an important factor to fight against this disease. For this purpose, identification of novel sources of resistance in the available germplasm is a crucial step. Several testing methods such as field tests, whole plant assays, and detached leaf assays (DLA) have been developed. DLA provides increased infection and potato leaves showing more susceptibility to P. infestans than the field and whole plant assays (Stewart, 1990; Vleeshouwers et al., 1999; Vossen et al., 2016). Since the genotype of the host and pathogen are generally static in infection assays, observed differences in susceptibility among testing methods are likely due to variation in environmental conditions. DLA is suited for identification of qualitative resistance available in the germplasm which is typically a qualitative trait governed by a single or a few disease resistant (R) genes.

Materials and Reagents

  1. Conical tubes

  2. Combitips advanced 1.0 ml (Eppendorf, catalog number: 0030089430)

  3. Cheese cloth

  4. Petri Dish (100 mm × 15 mm) (Fisher Scientific, catalog number: FB0875713)

  5. Parafilm (PM-996)

  6. Nunc square standard height bioassay dishes (Thermo Fisher ScientificTM, catalog number: 240835)

  7. Heavy-duty paper towels (Uline, catalog number: S-13631BLU)

  8. Spreader (Universal Medical, catalog number: HS86655)

  9. P. infestans isolate, US-23

  10. Sterile water

  11. 70% ethanol

  12. Potato genotypes, obtained from U.S. Potato GenBank (Sturgeon Bay, WI)

  13. Fertilizer (Peters' Professional 20-10-20 Peat Lite special)

  14. Growing media

    1. For P. infestans Rye A media (Caten and Jinks, 1968) (60 g of Rye grain, 15 g of agar and 20 g of sucrose for 1 L of distilled water, for detail please see Reference)

    2. For plants soil mixtures (All-purpose mix BM1, Berger)

Equipment

  1. Eppendorf Repeater E3 (Eppendorf, catalog number: 4987000398)

  2. Biological safety cabinet

  3. Light microscope

  4. Scanner (Epson, model: Perfection V700 Photo)

  5. Scalpel

  6. Hemocytometer

  7. Lamp

  8. Cold room at 4 °C or refrigerator

  9. Incubator (Fisher Scientific, catalog number: 97-990E)

  10. 10 × 10 cm pots

  11. 15 × 15 cm pots

Software

  1. ImageJ

Procedure

  1. Growing conditions of plants and collection of leaves

    1. Germinate potato seeds or plant tubers, cuttings or tissue culture plantlets in a greenhouse in soil-less potting mix (BM1, Berger) in a 10 × 10 cm pot.

    2. Transplant the seedlings after two weeks into the 15 × 15 cm pots.

    3. Maintain a temperature of 22 °C during the day (a photoperiod of 17.5 h) and 20 °C during the night (dark) in the greenhouse.

    4. To maintain the health of plants, irrigate regularly however avoid excessive moisture, fertilize once a week and spray against insects and foliar disease, when necessary. However, avoid spraying with fungicides for one week before inoculations.


  2. Growing conditions of P. infestans

    1. Grow P. infestans isolate US-23 on a Rye A media plate (Figure 1) at 18 °C. This plate can be used as a master plate for next 3 months for the further sub-culture.



      Figure 1. P. infestans growth in Rye A plates after 12 days


    2. Cut three plugs (5 mm) of P. infestans isolate, US-23 from Rye A media.

    3. Place these three plugs in a new Rye A media plate in a triangular fashion (Figure 1).

    4. Seal the plates properly with parafilm and incubate in the dark at 18 °C.

    5. Keep the plates facing downward to avoid any moisture development on the plugs.


  3. Inoculum preparation

    1. Harvest the sporangia from a 10-14-day old Rye A cultured plate (Figure 1) by flooding the plate with 5 ml ice-cold sterilized water (to expedite the release of zoospores) and mix properly with a spreader.

    2. Keep the plate at 4 °C for 2 to 4 h to release zoospores.

    3. Harvest the zoospores by filtering the liquid from each plate through two layers of cheesecloth (to remove mycelia) and dilute in 20 ml of ice-cold sterilized water.

    4. Count the motile zoospores using a hemocytometer under a microscope and adjust the concentration to 50,000 zoospores per ml of water. Generally, we make 100 ml final solution (zoospores and water) with 50,00 zoospores from a 10-14-day old Rye cultured plate.


  4. Infection assay

    1. Collect healthy, full-grown compound leaves having at least three leaflets from 5-8-week-old plants if available. Care should be taken not to collect old (start showing sign of yellowing) or very young leaves (not-fully developed).

    2. Set up a bioassay plate lined with wet Uline heavy-duty paper towels (Figure 2).



      Figure 2. Late blight infection assay conducted on bioassay plate lined with wet paper towel. Top cover was removed to take the picture.


    3. Drop Inoculate the abaxial side of each leaflet with 10 μl droplets (4 to 6 droplets per leaflet) of inoculum (50,000 zoospores per ml) with an Eppendorf repeater.

    4. Keep the bioassay plates in a room with natural light at a temperature of 21 °C.


  5. Symptom monitoring

    1. The first symptoms can be observed 3 days after inoculation as black/brown lesions, sporulation, and a water-soaked area at the point of pathogen inoculation.

    2. Subsequently, symptoms enlarge and cover the whole leaf in the case of highly susceptible genotypes after 5 days.

    3. Assess the leaves visually for the appearance of symptoms using a 1-9 scale (Karki et al., 2020) (Figure 3) or quantified by using ImageJ software (Rueden et al., 2017), 5 days after inoculation.



      Figure 3. The 1-9 scale used to evaluate the late blight infection. The mean leaf area covered with blackish/brown lesions, sporulation, and water-soaking was calculated using ImageJ and assigned a value based on this area. 1 = ≥90%, 2 = 81-90%, 3 = 71-80%, 4 = 61-70%, 5 = 41-60%, 6 = 21-40%, 7 = 10-20% with cell death at the point of inoculation, 8 = ≤10% with cell death at the point of inoculation, and 9 = 0% infection, no visible symptoms, clean leaves.


  6. Quantification of diseased area

    1. Obtain a digital image of the leaf using a flatbed scanner or camera (Image type: 24-bit color, Resolution: 300 dpi).

    2. Start ImageJ and open the image (File > Open…).

    3. Select the ‘Straight Line’ tool.

    4. Draw a line to an object of known size (e.g., coin or ruler).

    5. Go to ‘Analyze > Set scale and enter the preferred unit of length (e.g., cm, mm, or inches) and the known distance of the line from Step 4. Click OK.

    6. Use the ‘Paintbrush tool’ to highlight the diseased area of the leaf. Double-click the paintbrush icon to set the brush options, including brush width. Care should be taken to select only the diseased area and not other injuries.

    7. Go to ‘Image > Type > 8 bit’.

    8. Go to ‘Image > Adjust >Threshold (to get red leaf images).

    9. Select the dark background checkbox, and slide both bars to the very right (255 value). The selected infected area should be red and the remainder of the leaf photo should be in greyscale.

    10. Close the threshold settings window.

    11. Use ‘Freehand selection’ tool and trace the outline of the whole leaf area.

    12. Go to ‘Analyze > Set Measurement’ and select only the ‘Area’ and ‘Area fraction’ checkboxes, deselect any other checkboxes, and click OK.

    13. Go to ‘Analyze > Measure’ or press Ctrl+M to measure the diseased leaf area. A separate ‘Results Window’ will pop up showing the whole area and the % area, which represent the surface area of the outlined leaf in square units defined in step 5 and the percent infected area of the leaf, respectively.

    14. When closing the ‘Results Window’, you will have the option to save the results.

Competing interests

The authors declare no conflicts of interest.

Acknowledgments

This protocol describes details of the late blight infection assay used in experiments previously published by Karki et al. (2020). Work was supported by the USDA NIFA/NSF Plant Biotic Interactions Program award number: 2018-67014-28488.

References

  1. Caten, C. E., and Jinks, J. L. (1968). Spontaneous Variability of Single Isolates of Phytophthora infestans. I. Cultural Variation. Can J Botany 46 (4): 329-48.
  2. Fisher, M. C., Henk, D. A., Briggs, C. J., Brownstein, J. S., Madoff, L. C., McCraw, S. L. and Gurr, S. J. (2012). Emerging fungal threats to animal, plant and ecosystem health. Nature 484(7393): 186-194.
  3. Haverkort, A. J., Boonekamp, P. M., Hutten, R., Jacobsen, E., Lotz, L. A. P., Kessel, G. J. T., Visser, R. G. F. and Van Der Vossen, E. A. G. (2008). Societal Costs of Late Blight in Potato and Prospects of Durable Resistance through Cisgenic Modification. Potato Res 51 (1): 47-57.
  4. Haverkort, A. J., Boonekamp, P. M., Hutten, R., Jacobsen, E., Lotz, L. A. P., Kessel, G. J. T., Vossen, J. H. and Visser, R. G. F. (2016). Durable Late Blight Resistance in Potato Through Dynamic Varieties Obtained by Cisgenesis: Scientific and Societal Advances in the DuRPh Project. Potato Res 59 (1): 35-66.
  5. Karki, H. S., Jansky, S. H. and Halterman, D. A. (2020). Screening of Wild Potatoes Identifies New Sources of Late Blight Resistance. Plant Dis: PDIS06201367RE. 
  6. Rueden, C. T., Schindelin, J., Hiner, M. C., DeZonia, B. E., Walter, A. E., Arena, E. T. and Eliceiri, K. W. (2017). ImageJ2: ImageJ for the next generation of scientific image data. BMC Bioinformatics 18(1): 529.
  7. Stewart, H. E. (1990). Effect of Plant Age and Inoculum Concentration on Expression of Major Gene Resistance to Phytophthora infestans in Detached Potato Leaflets. Mycol Res 94(6): 823-826.
  8. Vleeshouwers, V. G. A. A., van Dooijeweert, W., Paul Keizer, L. C., Sijpkes, L., Govers, F. and Colon, L. T. (1999). A Laboratory Assay for Phytophthora infestans Resistance in Various Solanum Species Reflects the Field Situation. Eur J Plant Pathol 105 (3): 241-50.
  9. Vossen, J. H., van Arkel, G., Bergervoet, M., Jo, K. R., Jacobsen, E. and Visser, R. G. (2016). The Solanum demissum R8 late blight resistance gene is an Sw-5 homologue that has been deployed worldwide in late blight resistant varieties. Theor Appl Genet 129(9): 1785-1796.
  10. Zhang, H., Xu, F., Wu, Y., Hu, H. and Dai, X. (2017). Progress of Potato Staple Food Research and Industry Development in China. J Integr Agric 16(12):2924-2932.

简介

[摘要]疫霉菌是一种主要感染马铃薯和番茄的半生营养菌卵菌。它感染马铃薯和番茄的茎,叶,块茎和果实。高通量和可重复的感染测定是寻找任何农作物抗药性来源的先决条件。在此协议中,我们描述了分离叶分析(DLA),用于进行马铃薯叶中致病疫霉的毒力测定。使用马铃薯离体叶的晚疫病感染测定是一种半高通量测定,其中可以在实验室环境中筛选数百种植物。

[背景]就食品和营养价值而言,马铃薯(Solanum tuberosum L.)是最重要的非谷物粮食作物之一(Zhang等人,2017)。晚疫病引起卵菌病原体马铃薯的晚疫病菌是世界上最具破坏性的马铃薯的疾病之一,是在马铃薯生产中最重要的产量限制因素(Haverkort等人,2008和2016 ;费舍尔等人, 2012 )。后期抗白叶枯病的育种被认为是抗击这种疾病的重要因素。为此,在可用种质中鉴定新的抗性来源是至关重要的一步。已经开发了几种测试方法,例如田间测试,整株植物测定和离体叶片测定(DLA)。DLA提供的感染增加,并且马铃薯叶表现出对疫病菌的敏感性高于田间和整个植物的检测方法(Stewart ,1990 ;Vleeshouwers等,1999 ;Vossen等,2016)。由于宿主和病原体的基因型在感染检测中通常是静态的,因此在检测方法之间观察到的敏感性差异可能是由于环境条件的变化所致。DLA适合于鉴定种质中可用的定性抗性,通常是由单个或几个抗病(R)基因控制的定性性状。

关键字:致病疫霉, 马铃薯, 离体叶片分析, 晚疫病

材料和试剂
锥形管
Combitips先进1.0毫升仪(Eppendorf,Ç atalog编号:0030089430)
芝士布
培养皿(100毫米 × 15毫米)(Fisher Scientific,目录号:FB0875713)
封口膜(PM-996)
Nunc公司平方标准高度生物测定皿(赛默飞世尔科技TM ,Ç atalog号:240835)
重型纸巾(Uline,目录号:S-13631BLU)
撒布机(Universal Medical ,目录号:HS86655)
P. infestans分离株,US-23
无菌水
70%乙醇
马铃薯基因型,获自美国马铃薯基因库(威斯康星州斯特金湾)
肥料(彼得斯的专业20-10-20泥炭精简版)
不断增长的媒体
对于致病疫霉黑麦A培养基(Caten和Jinks ,1968年)(60克黑麦谷物,15克琼脂和20克蔗糖与1升蒸馏水的混合情况,请参见参考资料)
用于植物土壤混合物(通用混合物BM1,Berger)

设备


Eppendorf Repeater E3(Eppendorf,目录号:4987000398)
生物安全柜
光学显微镜
扫描仪(Epson ,型号:Perfection V700 Photo)
解剖刀
血细胞计数器

4 °C的冷藏室或冰箱
孵化器(Fisher Scientific,目录号:97-990E)
10 × 10厘米的花盆
15 × 15厘米的花盆

小号的吨洁具


图像

程序


植物的生长条件和叶片的收集
在10 × 10厘米盆中的无土盆栽混合物(BM1,Berger)中,将温室中的马铃薯种子或块茎,插条或组织培养苗发芽。
两周后将幼苗移植到15 × 15厘米的盆中。
在温室中,白天应保持22 °C (17.5小时的光照周期)的温度,而在夜晚(黑暗)则保持20°C的温度。
为了保持植物的健康,应定期灌溉,但要避免过多的水分,每周施肥一次,并在必要时喷洒杀虫剂和叶面疾病。但是,接种前一周应避免喷洒杀菌剂。

致病疫霉的生长条件
生长致病疫霉分离物US-23上一个黑麦的介质板(图1)18 ℃下。该板可在接下来的3个月用作进一步的继代培养的母板。



图1. 12天后黑麦A板中的疫霉菌生长


从黑麦A培养基上切下3株美国疫病菌隔离株US-23的3个塞子(5毫米)。
将这三个插头以三角形方式放置在新的Rye A介质板上(图1)。
用石蜡膜正确密封板,并在黑暗中于18°C孵育。
保持板朝下,以免插头上产生水分。

接种制剂
通过用5 ml冰冷的灭菌水(以加速游动孢子的释放)注入10到14天的黑麦A培养板(图1)来收获孢子囊。
保持板在4°C 2至4小时以释放游动孢子。
通过用两层粗棉布过滤每块板中的液体(去除菌丝体)并在20 ml冰冷的无菌水中稀释来收集游动孢子。
使用血细胞计数器在显微镜下计数运动的游动孢子,并将浓度调整为每毫升水中50,000个游动孢子。通常,我们用10-14天的黑麦培养板制成的50 ml游动孢子制成100 ml最终溶液(游动孢子和水)。 

感染测定
如果可以的话,从5-8周龄的植物中收集至少有三张小叶的健康,成熟的复合叶子。注意不要收集旧的(开始显示出泛黄的迹象)或非常幼小的叶子(未完全发育)。
设置一个内衬湿Uline重型纸巾的生物测定板(图2)。



图2.在衬有湿纸巾的生物测定板上进行的晚疫病感染测定。取下顶盖拍照。


滴剂用Eppendorf重复器接种10μl接种物(每毫升50,000个游动孢子)的10μl液滴(每片小册子4至6滴)接种每个小叶的背面。
将生物测定板放置在温度为21°C的自然光室内。

症状监测
接种后3天可以观察到最初的症状,如黑色/棕色病变,孢子形成和病原体接种点的水浸区域。
随后,在高度敏感的基因型5天后,症状会扩大并覆盖整个叶片。
接种后5天,使用1-9量表(Karki等人,2020)(图3)或使用ImageJ软件(Rueden等人,2017)对叶片外观进行症状评估。



图3.用于评估晚疫病感染的1-9量表。使用ImageJ计算覆盖有黑色/棕色病斑,孢子形成和浸水的平均叶面积,并根据该面积指定一个值。1 =≥90%,2 = 81-90%,3 = 71-80%,4 = 61-70%,5 = 41-60%,6 = 21-40%,7 = 10-20%在接种时,8 =≤10%,在接种时死亡,9 = 0%,无明显症状,干净。


Q病变区域的uantification
使用平板扫描仪或照相机获取树叶的数字图像(图像类型:24位彩色,分辨率:300 dpi)。
启动ImageJ并打开图像(文件>打开…)。
选择“直线”工具。
在已知大小的物体(例如硬币或直尺)上画一条线。
转到“分析>设置比例,然后输入首选的长度单位(例如,厘米,毫米或英寸)和距步骤4的已知线距。单击确定。
使用“画笔工具”突出显示叶子的患病区域。双击画笔图标以设置画笔选项,包括画笔宽度。应注意仅选择患病区域,而不选择其他伤害。
转到“图像>类型> 8位”。
转到“图像>调整>阈值(以获取红色叶子图像)” 。
选中深色背景复选框,然后将两个条向右滑动(255个值)。所选感染区域应为红色,叶子照片的其余部分应为灰度。
关闭阈值设置窗口。
使用“徒手选择”工具并跟踪整个叶子区域的轮廓。
转到“分析>设置度量”,然后仅选择“面积”和“面积分数”复选框,取消选择任何其他复选框,然后单击“确定”。
转到“分析>测量”或按Ctrl + M来测量患病的叶子面积。将会弹出一个单独的“结果窗口”,显示整个面积和%面积,分别代表步骤5中定义的正方形单位的轮廓叶子的表面积和叶子的受感染面积百分比。
关闭“结果窗口”时,您可以选择保存结果。

致谢


该协议描述了由Karki等人先前发表的实验中使用的晚疫病感染检测的详细信息。(2020年)。该工作得到了美国农业部NIFA / NSF植物生物相互作用计划的资助,编号为:2018-67014-28488。


参考


行政长官Caten和JL的Jinks(1968)。疫霉菌单个菌株的自发变异性。一,文化差异。Can J Botany 46(4):329-48。
Fisher,MC,Henk,DA,Briggs,CJ,Brownstein,JS,Madoff,LC,McCraw,SL和Gurr,SJ(2012)。对动物,植物和生态系统健康的新真菌威胁。自然484(7393):186-194。
Haverkort,AJ,Boonekamp,PM,Hutten,R.,Jacobsen,E.,Lotz,LAP,Kessel,GJT,Visser,RGF和Van Der Vossen,EAG(2008)。马铃薯晚疫病的社会成本和通过顺生基因改造获得持久抗药性的前景。马铃薯研究杂志51(1):47-57。
Haverkort,AJ,Boonekamp,PM,Hutten,R.,Jacobsen,E.,Lotz,LAP,Kessel,GJT,Vossen,JH和Visser,RGF(2016)。通过同基因成因获得的动态品种使马铃薯具有持久的晚疫病抗性:DuRPh项目的科学和社会进展。马铃薯研究59(1):35-66。
Karki,HS,Jansky,SH和Halterman,DA(2020)。野马铃薯的筛选确定了晚疫病抗性的新来源。工厂Dis :PDIS06201367RE。
康涅狄格州鲁登(Rueden),辛德林(J. ImageJ2:用于下一代科学图像数据的ImageJ。BMC生物信息学18(1):529。
斯图尔特,H 。E. (1990 )。种植年龄和接种浓度对分离马铃薯小叶对疫霉致病性的主要基因表达的影响。Mycol Res 94(6):823-826。
Vleeshouwers,VGAA,van Dooijeweert,W.,Paul Keizer,LC,Sijpkes,L.,Govers,F。和Colon,LT (1999)。多种茄科植物对疫霉菌的抗药性的实验室分析反映了现场情况。Eur J植物病理学105(3):241-50。
沃森,JH,范阿克尔,G。,伯杰沃特,M。,乔,KR,雅各布森,E。和维瑟,RG(2016)。在龙demissum R8晚疫病抗性基因已在抗晚疫病品种在全球部署的SW-5同系物。 理论理论杂志129(9):1785-1796。
张航,H 。,徐,楼。,吴,Y 。,胡,H 。和Dai,X.(2017)。中国马铃薯主食研究与产业发展进展。ĴINTEGR银RIC 16(12):2924至2932年。
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引用:Karki, H. and Halterman, D. A. (2021). Phytophthora infestans (Late blight) Infection Assay in a Detached Leaf of Potato. Bio-protocol 11(4): e3926. DOI: 10.21769/BioProtoc.3926.
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