Name: dry root rot disease assays in chickpea: a detailed methodology
Uploaded: 2021-01-17T13:00:00
Description: Watch this Scientific Journal Video about Dry Root Rot Disease Assays in Chickpea: a Detailed Methodology at JoVE.com

M.S.K 实验室的项目得到了国家植物基因组研究所核心资助的支持。VI 确认 DBT- JRF （DBT/2015/NIPGR/430）。我们感谢见习学生Rishika小姐、贾亚亨德拉扬先生和杜尔加德维小姐在录像拍摄期间提供技术帮助，并感谢桑迪普·迪克西特先生、安贾利小姐和阿瓦尼什·雷博士对原始数据和手稿文件进行批判性评估。我们感谢拉希姆·塔拉夫达尔先生和桑德·索兰基先生对实验室的帮助。我们感谢 DBT-e 图书馆联盟 （DeLCON） 和 NIPGR 图书馆提供访问电子资源和 NIPGR 工厂生长设施，用于植物生长支持/空间。

1. 分离 R. 巴塔蒂科拉 和储存
<ol><li>鹰嘴豆基因型和 DRR 症状的详细信息<ol>
<li>使用鹰嘴豆植物（基因型，JG 62），通常显示典型的DR症状，如干燥，脆原根没有侧根和微丝皮皮下树皮和皮1，3。</li>
	</ol>
</li>
	<li>收集和清洗<ol>
<li>背井离乡的植物，如干稻草色叶和脆原根与微分皮下表皮层下的症状。在连根拔起时，大部分根茎将留在土壤内，因?...

<ol>
	<li>Sharma, M., Ghosh, R., Pande, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Dry+root+rot+(Rhizoctonia+bataticola+(Taub.)+Butler):+an+emerging+disease+of+chickpea+-+where+do+we+stand.">Dry root rot (Rhizoctonia bataticola (Taub.) Butler): an emerging disease of chickpea - where do we stand.</a> Archives of Phytopathology and Plant Protection. 48 (13-16), 797-812 (2015).</li><li>Sinha, R., Irulappan, V., Mohan-Raju, B., Suganthi, A., Senthil-Kumar, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Impact+of+drought+stress+on+simultaneously+occurring+pathogen+infection+in+field-grown+chickpea.">Impact of drought stress on simultaneously occurring pathogen infection in field-grown chickpea.</a> Scientific Reports. 9 (1), (2019).</li><li>Nene, Y., Haware, M., Reddy, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Chickpea+diseases:+resistance+screening+techniques,+information+bulletins+No.+10.+Patancheru.">Chickpea diseases: resistance screening techniques, information bulletins No. 10. Patancheru.</a> Information Bulletin No. 10. Patancheru, A.P., India: International CroDS Research Institute for the Semi-Arid Tropics. , 1-10 (1981).</li><li>Pande, S., Krishna Kishore, G., Upadhyaya, H. D., Narayana Rao, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Identification+of+sources+of+multiple+disease+resistance+in+mini-core+collection+of+chickpea.">Identification of sources of multiple disease resistance in mini-core collection of chickpea.</a> Plant Disease. , (2006).</li><li>Pandey, P., Irulappan, V., Bagavathiannan, M. V., Senthil-Kumar, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Impact+of+combined+abiotic+and+biotic+stresses+on+plant+growth+and+avenues+for+crop+improvement+by+exploiting+physio-morphological+traits.">Impact of combined abiotic and biotic stresses on plant growth and avenues for crop improvement by exploiting physio-morphological traits.</a> Frontiers in Plant Science. 8, (2017).</li><li>Irulappan, V., Senthil-Kumar, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Morpho-physiological+traits+and+molecular+intricacies+associated+with+tolerance+to+combined+drought+and+pathogen+stress+in+plants.">Morpho-physiological traits and molecular intricacies associated with tolerance to combined drought and pathogen stress in plants.</a> Biotechnologies of Crop Improvement, Volume 3: Genomic Approaches. , (2018).</li><li>Jensen, A. B., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Standard+methods+for+fungal+brood+disease+research.">Standard methods for fungal brood disease research.</a> Journal of Apicultural Research. 52 (1), 1-39 (2013).</li><li>Agrios, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Plant Pathology: Fifth Edition. , 9780080473 (2004).</li><li>Coley-Smith, J. R., Cooke, R. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Survival+and+germination+of+fungal+sclerotia.">Survival and germination of fungal sclerotia.</a> Annual Review of Phytopathology. , (1971).</li><li>Nagamma, G., Saifulla, M., Sab, J., Pavitra, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Screening of chickpea genotypes against dry root rot caused by Macrophomina phaseolina (tassi) goid. 10 (4), 1795-1800 (2015).</li><li>Infantino, A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Screening+techniques+and+sources+of+resistance+to+root+diseases+in+cool+season+food+legumes.">Screening techniques and sources of resistance to root diseases in cool season food legumes.</a> Euphytica. 147 (1-2), 201-221 (2006).</li><li>Khaliq, A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Integrated+control+of+dry+root+rot+of+chickpea+caused+by+Rhizoctonia+bataticola+under+the+natural+field+condition.">Integrated control of dry root rot of chickpea caused by Rhizoctonia bataticola under the natural field condition.</a> Biotechnology Reports. 25, 00423 (2020).</li></ol>

印迹纸技术为在实验室条件下筛选鹰嘴豆基因型提供了一种简单的方法。滴接种使在时间基础上的相互作用调查，易于控制接种负荷（补充图1），并方便体外筛查。此外，即使是年轻的幼苗也可以使用。五天大的真菌培养（图1B）可以产生足够的幼崽来感染植物.液体中分含有 mycelia 和微分体（图 1D & E）。根腐症状<strong class="x...

干根腐烂（DRR）是鹰嘴豆1，2中具有经济意义的疾病之一。这是一种根特异性疾病，由Rhizoctonia巴塔蒂奥拉（电图，巨管，相相） 引起。受感染的植物缺乏横向根，并拥有脆脆的锥根和黄叶1，3。据报道，在干旱压力下，DRR对鹰嘴豆种植1、2、3构成一种新威胁。此外，据报道，在干旱压力下，在1、2、3的干旱压力下，DRR发生率会加剧。DRR在雨水灌溉地区比在灌溉田中更为普遍。利用耐药品种是战胜疾病和规避杀菌剂使用的方法。由于全球现有的鹰嘴豆种质具有遗传变异，因此对耐药/易感基因型的筛选和鉴定对于作物改良的分子育种至关重要。
强健、简单且具有成本效益的疾病检测对于调查鹰嘴豆的R.巴塔蒂奥拉感染模式至关重要。用于观察鹰嘴豆基因型对R.巴塔基奥拉感染反应的主要疾病分析是印迹纸技术1，4。这是一种简单的技术，可以使用液体真菌注射液，带根的幼苗和无菌印迹纸执行。但是，由于文献中没有提供分步协议，因此未充分利用此技术。
同时，病锅技术涉及准备潜在的病文化，并施加干旱压力。鉴于干旱压力加重了干旱病的发病率3，研究干旱压力下植物病原体相互作用6、7至关重要。病锅技术为这种同步研究提供了平台，促进种质筛选的更好可能性，并理解相互作用的机械基础。病理变化，如根长增加和横向根数减少（DRR疾病固有的）可以使用病锅技术1、3、7加以解决。
本文介绍了印迹纸和病锅技术的详细方案，可用于研究鹰嘴豆与 R.蝙 蝠豆和网眼鹰嘴豆种质之间的相互作用。研究中使用的材料的详细信息见《材料表》。

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>Fungus- Rhizoctonia bataticola</td>
			<td>Pathogen inoculum</td>
			<td>Indian Type Culture Collection No. 8365</td>
			<td>GenBank: MH509971.1, ITCC 8635 (https://www.iari.res.in/index.php?option=com_content&#38;view=article&#38; id=1251&#38;Itemid=1370)</td>
		</tr>
		<tr>
			<td>Soilrite mix</td>
			<td>Soil medium in the lab</td>
			<td>Keltech Energies Limited, Bangalore, India</td>
			<td>http://www.keltechenergies.com/</td>
		</tr>
		<tr>
			<td>Filter paper</td>
			<td>Blotting paper to support the plant growth</td>
			<td>Himedia</td>
			<td>http://himedialabs.com/catalogue/chemical2017/index.html#374</td>
		</tr>
		<tr>
			<td>Pot</td>
			<td>Growing plants</td>
			<td>10 and 30 cm size pots</td>
			<td>Routinely used nursery pots, for example, https://dir.indiamart.com/impcat/nursery-pots.html</td>
		</tr>
		<tr>
			<td>Potato dextrose agar/broth</td>
			<td>Culture and maintain the fungus</td>
			<td>Cat# 213400, DifcoTM, MD, USA</td>
			<td>https://www.fishersci.com/shop/products/bd-difco-dehydrated-culture-media-potato-dextrose-agar-3/p-4901946</td>
		</tr>
		<tr>
			<td>Incubator</td>
			<td>Culture the fungus</td>
			<td>LOM-150-2, S/N AI13082601-38, MRC, incubator, and shaker</td>
			<td>http://www.mrclab.com/productDetails.aspx?pid=91131</td>
		</tr>
		<tr>
			<td>Growth chamber</td>
			<td>Growing plants in controlled condition</td>
			<td>Model No. A1000, Conviron, Canada</td>
			<td>https://www.conviron.com/products/gen1000-reach-in-plant-growth-chamber</td>
		</tr>
		<tr>
			<td>Laminar airflow</td>
			<td>Carrying out aseptic exercises</td>
			<td>Telstar, Bio II advance, Class II cabinet, EN-12469-2000</td>
			<td>https://www.telstar.com/lab-hospitals-equipment/biological-safety-cabinets/bio-ii-advance-plus/, http://www.atlantisindia.co.in/laminar-air-flow.html</td>
		</tr>
		<tr>
			<td>Mesh</td>
			<td>Filtering the fungal mycelia</td>
			<td>Nylon mosquito net</td>
			<td>Mesh with 0.6-1 mm diameter pore size</td>
		</tr>
		<tr>
			<td>Autoclave</td>
			<td>Autoclaving media and chickpea seeds</td>
			<td>Autoclave</td>
			<td>http://www.scientificsystems.in/autoclave</td>
		</tr>
		<tr>
			<td>Microscopes</td>
			<td>Visualizing the infection ang fungal mycelia</td>
			<td>SMZ25 / SMZ18, Research Stereomicroscopes, Leica EZ4 educational stereomicroscope</td>
			<td>https://www.microscope.healthcare.nikon.com/products/stereomicroscopes-macroscopes/smz25-smz18 
			https://www.leica-microsystems.com/products/stereo-microscopes-macroscopes/p/leica-ez4/ 
			https://www.microscopyu.com/museum/eclipse-80i</td>
		</tr>
		<tr>
			<td>Weighing balance</td>
			<td>Weighing fungus and chemicals</td>
			<td>Sartorius Electronic Weighing Balance, BSA 4202S-CW</td>
			<td>https://www.sartorius.com/en/products/weighing/laboratory-balances</td>
		</tr>
		<tr>
			<td>WGA-FITC</td>
			<td>Fungus staining</td>
			<td>Sigma</td>
			<td>https://www.sigmaaldrich.com/catalog/product/sigma/l4895?lang=en&#38;region=IN</td>
		</tr>
		<tr>
			<td>Aniline blue</td>
			<td>Fungus staining</td>
			<td>Himedia</td>
			<td>http://www.himedialabs.com/intl/en/products/Chemicals/Dyes-Indicators-and-Stains/Aniline-blue-Water-soluble-Practical-grade-GRM901</td>
		</tr>
	</tbody>
</table>

dry root rot disease assays in chickpea: a detailed methodology

干根腐烂 （DRR） 疾病是世界各地鹰嘴豆种植的一种新兴生物压力威胁。它是由土壤传播的真菌病原体 Rhizoctonia巴塔蒂奥拉引起的。在文献中，关于疾病测定的全面和详细的分步协议是稀疏的。本文详细介绍了设置印迹纸技术以快速筛选基因型以抵抗 DRR 的步骤。印迹纸技术简单易用，成本更低。另一种基于病锅方法的方法是自然感染的模仿，可用于研究疾病三角中涉及的相互作用的成分——植物、病原体和环境。
此外，在自然界中，DRR主要发生在雨中鹰嘴豆种植区，那里的土壤水分随着作物生长的推进而消退。众所周知，干旱压力使鹰嘴豆植物容易患 DRR 疾病。对干旱压力下植物-病原体相互作用的病理和分子理解可以为从鹰嘴豆种质池中鉴定优质耐数 DRR 品种铺平道路。本文提供了一个逐步的方法，用于准备病锅和随后的疾病测定。总体而言，本文提供的信息将帮助研究人员准备 R.巴 塔基托真菌孵化，维持这种病原体，建立印迹纸技术，准备病菌培养和病锅，并评估鹰嘴豆植物的病原体感染。

本研究旨在展示印迹纸和病锅技术等技术，以促进病理和分子对干旱压力下植物-病原体相互作用的理解。为此，从鹰嘴豆田中采集出出现 DRR症状的植物 1、3、4，使用催眠技巧方法8分离真菌。R. 巴塔蒂托拉真菌培养在孵育后四天内在PDA板块上出现深灰色，在孵化后五...

Watch this Scientific Journal Video about Dry Root Rot Disease Assays in Chickpea: a Detailed Methodology at JoVE.com

Dry Root Rot Disease Assays in Chickpea: a Detailed Methodology

本研究提出了研究鹰嘴豆-瑞佐克托马基亚相互作用背后的病理形态和分子机制 的方法。印迹纸方法可用于快速研究鹰嘴豆基因型反应，而病锅基方法可用于同时 施加干旱和R.巴塔蒂奥拉感染 和筛选耐受基因型。

鹰嘴豆的干根罗特病测定：详细方法

Dry root rot (DRR) disease is an emerging biotic stress threat to chickpea cultivation around the world. It is caused by a soil-borne fungal pathogen, ...

本研究的主要目标是演示干根腐病检测的详细方法，如印迹纸技术和病锅技术。这些技术将有助于研究鹰嘴豆植物和Rhizoctonia蝙蝠菌丝之间的相互作用。了解鸡草植物与真菌病原体Rhizoctonia巴塔蒂奥拉的相互作用在农业上很重要。为此，需要多种用途和高度可重复的检测。在这里，演示了在我们的实验室中广泛使用的两种检测方法。其中一个是印迹纸为基础的，另一个是生病的锅为基础的测定。要执行印迹纸测定，准备Rhizoctonia巴塔蒂奥拉真菌接种，从倾斜培养到培养，然后到液体培养。准备鹰嘴豆苗圃，在一个大锅播种。把它们连根拔起，放在液体的花尿中，放在印迹纸上。观察疾病症状，即根部坏死、根部腐烂和接种8天后叶黄。这里显示的是一个简言之，在印迹纸技术的步骤。要执行生病的锅检测，接种鹰嘴豆粉与真菌。在30摄氏度下孵育15天后，获得病育。然后，粉他们和混合锅混合。在控制锅和病锅上播种。在以下几天，观察症状，即根部坏死，根腐烂，感染植物上的叶子发黄，播种后10天。病锅可以放大，并可用于大规模筛查。这里展示的是病锅技术中的步骤。里佐克托尼亚巴塔蒂奥拉是一种正在发育的病原体。这种真菌产生催眠和微细胞，在植物感染期间作为原生性。通过隔离法应用的纯文化用于测定。这些成功测定的关键点是最佳的中分负荷。印迹纸技术的演示从这里开始。为了准备液体Rhizoctonia巴塔提奥拉注射液，用三天前新鲜盘培养物的真菌加糖塞接种500毫升肉汤，并在28摄氏度的培养箱中孵化烧瓶5天。过滤出深灰色真菌菌体与微腹腔。悬浮在自 <3>分水中并取出介质。再次，过滤掉我的西莉亚，并去除水分通过印迹干燥我的西莉亚几分钟。将100克真菌注射在200毫升的自功能化水中，放在果酱瓶中，并正确混合。通过播种表面消毒种子来发展鹰嘴豆苗圃。播种8天后，将植物连根拔起，清洗并用自洗RO冲洗。拿一张印迹纸，折叠一次，放在纸盒上。用自动处理的水把它弄湿。采取植物和浸入孵化30秒间歇性向上和向下运动。将植物放在一个方式，根覆盖在薄板内，佛利亚留在外。折叠另一半，弯曲纸张。为了控制，将植物浸入自 <3> <3>化水中，将托盘放在生长室中八天。结果。观察叶黄、根腐烂、根部坏死和侧根数减少等症状。生病的锅技术的演示从这里开始。拿一公斤商业鹰嘴豆种子。用自来水清洗种子，再用RO水清洗。浸泡种子五个小时。再次，用RO水洗涤种子，在果酱瓶中加入300克。在 121 LVS 下连续两次进行 15 分钟的自动保存。冷却后，将瓶子通过层流转移。用三个真菌加子塞为瓶子接种大约四毫米的平方大小，并摇动它，以正确混合。将瓶子包裹在30摄氏度的孵化器中孵化15天。观察鹰嘴豆种子周围有深色木花生长的瓶子，并取出种子。在室温下干燥并粉粉。将Rhizoctonia巴塔蒂奥拉粉与索洛里特混合，并在室温下保持五天。表面对GT62种子进行消毒，并在两厘米深的地方播种。结果。观察病锅中90%死亡率。播种后10天，观察症状，如根坏死和减少侧根数和叶发黄在随后几天。病锅也可以在田间土壤中准备。干旱和里佐克托尼亚巴塔蒂奥拉感染。通过扣水在病锅中施加干旱压力。这里展示的植物受到控制，干旱，病原体，以及同时干旱和病原体压力。观察症状，即与仅使用病原体的治疗相比，在联合应激治疗中，多坏死、根腐和侧根数减少，根长。结论。膨胀纸技术可以在振动条件中进行。病锅技术可以在Vivvo条件下执行。在病港，非生物性压力，即干旱压力，可以随病原体感染一起施加。利用这些技术，可以研究病原体感染模式。大多数生理变化发生在鹰嘴豆-瑞佐克托尼亚巴塔蒂奥拉相互作用期间，还筛选鹰嘴豆基因型的耐药性。印迹纸测定对于生成用于显微镜和分子生物学研究的质量样品非常有用。病锅测定，模仿自然感染条件，适用于筛查鹰嘴豆种质。

Research

JoVE Journal

Biology

Protocol for RNAi Assays in Adult Mosquitoes (A. gambiae)

Protocol for RNAi Assays in Adult Mosquitoes A. gambiae

成蚊（冈比亚按蚊的RNAi实验议定书）

Reverse genetic approaches have proven extremely useful for determining  which genes underly resistance to vector pathogens in mosquitoes.  This video ...

科研

生物学

Preparation and Maintenance of Dorsal Root Ganglia Neurons in Compartmented Cultures

隔间文化背根神经节神经元的制备及维修

Neurons extend axonal processes that are far removed from the cell body to innervate target tissues, where target-derived growth factors are required for ...

Neurocircuit Assays for Seizures in Epilepsy Mutants of Drosophila

Neurocircuit Assays for Seizures in Epilepsy Mutants of Drosophila

Neurocircuit检测癫痫发作癫痫突变果蝇</em

Drosophila melanogaster is a useful tool for studying seizure like activity. A variety of mutants in which seizures can be induced through either physical ...

Isolation of Ribosome Bound Nascent Polypeptides in vitro to Identify Translational Pause Sites Along mRNA

Isolation of Ribosome Bound Nascent Polypeptides in vitro to Identify Translational Pause Sites Along mRNA

核糖体束缚新生多肽的分离<em&gt;在体外</em&gt;确定沿mRNA的翻译暂停网站

The rate of translational elongation is non-uniform. mRNA secondary structure, codon usage and mRNA associated proteins may alter ribosome movement on the ...

Using SecM Arrest Sequence as a Tool to Isolate Ribosome Bound Polypeptides

作为一种工具来隔离核糖体的束缚多肽SECM逮捕序列

Extensive research has provided ample evidences suggesting that protein folding in the cell is a co-translational process1-5. However, the exact pathway ...

Coupled Assays for Monitoring Protein Refolding in Saccharomyces cerevisiae

Coupled Assays for Monitoring Protein Refolding in Saccharomyces cerevisiae

耦合检测监控蛋白复性<em&gt;酿酒酵母（Saccharomyces cerevisiae）</em

Proteostasis, defined as the combined processes of protein folding/biogenesis, refolding/repair, and degradation, is a delicate cellular balance that must ...

Analysis of Skeletal Muscle Defects in Larval Zebrafish by Birefringence and Touch-evoke Escape Response Assays

Zebrafish (Danio rerio) have become a particularly effective tool for modeling human diseases affecting skeletal muscle, including muscular ...

A Fast Air-dry Dropping Chromosome Preparation Method Suitable for FISH in Plants

快速风干滴染色体制备方法适用于FISH在植物

Preparation of chromosome spreads is a prerequisite for the successful performance of fluorescence in situ hybridization (FISH). Preparation of high ...

Lateral Root Inducible System in Arabidopsis and Maize

Lateral Root Inducible System in Arabidopsis and Maize

侧根诱导系统<em&gt;拟南芥</em&gt;和玉米

Lateral root development contributes significantly to the root system, and hence is crucial for plant growth. The study of lateral root initiation is ...

Divergence of Root Microbiota in Different Habitats based on Weighted Correlation Networks

基于加权相关网络的不同栖息地根微生物群的差异

The root microbiome plays an important role in plant growth and environmental adaptation. Network analysis is an important tool for studying communities, ...