DB43∕T 1957-2020 稻曲病菌分子检测技术规程(湖南省)

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ICS65.020CCSB16DB43湖南省地方标准DB43/T9157—2020稻曲病菌分子检测技术规程TechnicalRegulationsforMolecularDetectionofUstilaginoideavirens2020-12-29发布2021-03-29实施湖南省市场监督管理局发布

1DB43/T1957—2020目次前言························································································································Ⅲ1范围·····················································································································12规范性引用文件······································································································13术语和定义············································································································14原理·····················································································································15仪器设备与试剂······································································································15.1仪器设备·········································································································15.2主要试剂·········································································································16采样·····················································································································26.1样品采集·········································································································26.2样品贮运与存放································································································27操作方法···············································································································27.1样品DNA制备···································································································27.2检测···············································································································28结果判定···············································································································39样品保存与处理······································································································3附录A（资料性）稻曲病相关资料················································································4附录B（规范性）水稻稻曲病菌鉴定报告·······································································5I

2DB43/T1957—2020II

3DB43/T1957—2020前言本文件按照GB/T1.1—2020给出的规则起草。请注意本文件的某些内容可能涉及专利。本文件的发布机构不承担识别这些专利的责任。本文件由湖南省农业农村厅提出。本文件由湖南省农业标准化技术委员会归口。本文件起草单位：湖南省植物保护研究所、株洲市农业科学研究所。本文件主要起草人：陈岳、谭新球、李成刚、吴龙云、王淋玉、张卓、张鑫、史晓斌、李小娟、肖友伦、张德咏、刘勇。III

4DB43/T1957—2020IV

5DB43/T1957—2020稻曲病菌分子检测技术规程1范围本文件规定了稻曲病菌PCR检测的样品制备、检测技术和操作程序。本文件适用于土壤、水稻种子和稻谷中稻曲病菌的分子检测。2规范性引用文件下列文件中的内容通过文中的规范性引用而构成本文件必不可少的条款。其中，注日期的引用文件，仅该日期对应的版本适用于本文件；不注日期的引用文件，其最新版本（包括所有的修改单）适用于本文件。GB/T6682分析实验室用水规格和试验方法3术语和定义下列术语和定义适用于本文件。稻曲病菌无性态为Ustilaginoideavirens，属半知菌亚门、腔孢纲、瘤座孢目、绿核菌属，病原有性阶段有性态为Clavicepsoryzaesativae，属子囊菌门、子囊菌纲、麦角菌属、稻麦角菌。稻曲病主要危害水稻穗部并形成稻曲球，严重影响水稻的产量和稻米品质。见附录A。PCR（PolymeraseChainReaction），聚合酶链式反应，是指在DNA聚合酶催化下，以母链DNA为模板，以特定引物为延伸起点，通过变性、退火、延伸等步骤，体外复制出与母链模板DNA互补的子链DNA的过程。是一项DNA体外合成放大技术，能快速特异地在体外扩增任何目的DNA。4原理核糖体基因转录间隔区（InternaltranscribedspacerITS）进化速率较编码区快，在真菌种间存在着丰富的变异，而在种内不同菌株间却高度保守，可以为病原菌的分子检测提供理想的靶序列。根据稻曲病菌ITS的特异序列，合成1对特异性引物，获得稻曲病菌特异性扩增片段，用于土壤、水稻种子和稻谷中稻曲病菌的快速分子检测。5仪器设备与试剂5.1仪器设备PCR检测仪、凝胶成像仪、土壤取样器、台式离心机、纯水仪、电泳仪、旋涡振荡器等。5.2主要试剂1

6DB43/T1957—2020苯酚/氯仿/异戊醇、乙醇、二甲基亚砜、氢氧化钠、脱脂奶粉、DNAmarker、吐温20、PCR试剂盒等。除特别说明以外，本文件所用试剂均为分析纯，实验用水应符合GB/T6682的规定。6采样6.1样品采集6.1.1土壤水稻田W型5点取样，取1-10cm深度的50g土壤样品。6.1.2水稻种子和稻谷将稻种或稻谷混匀后称取50g。6.2样品贮运及存放样品采集后，放入密闭的容器内，送实验室。将取样结果进行记录，记录表格见附录B。处理的样品应保存在-20℃以下，避免反复冻融。7操作方法7.1样品DNA制备7.1.1土壤中DNA的提取土壤风干后，制成100目的土壤样品，取10g土壤加入5mL无菌水，冷冻抽干24-48h，加入少量石英砂，倒入液氮充分研磨，将研磨后的土壤分装至1.5mL离心管中，每管加入500μl0.4％脱脂奶粉溶液，涡旋混匀。12000rpm/min离心10min。取上清加入等体积浓度为20μg/mL的蛋白酶K缓冲液，55℃水浴2h。水浴结束后，加入1/2体积的7.5MNH4Ac溶液，混匀。12000rpm/min离心10min。吸上清液加入2倍体积无水乙醇，-20℃沉淀0.5h，12000rpm/min离心10min，去除上清液，70％乙醇洗涤沉淀物，室温晾干。提取的DNA用20μl含RNA酶的TE缓冲液溶解，-20℃保存备用。土壤DNA也可用土壤DNA提取试剂盒提取。7.1.2种子和稻谷中DNA的提取将混匀后的稻种液氮研磨成粉末，称取0.1g于2mL管子中，加入800μlCTAB提取液，65℃水浴15min，期间反复摇匀；加入等体积苯酚/氯仿/异戊醇，混匀后摇床轻摇20min；12000rpm/min离心10min，移上清500μl，加入2倍体积的无水乙醇，-20℃沉淀0.5h，12000rpm/min离心10min，去除上清液，70％乙醇洗涤沉淀物，室温晾干。提取的DNA用20μl含RNA酶的TE缓冲液溶解，-20℃保存备用。7.2检测7.2.1PCR检测2

7DB43/T1957—20207.2.1.1反应体系针对水稻稻曲病菌与其他属种病菌ITS序列差异设计了一对特异性引物。UV-F：5’-CTTGTGTTTTCCAATGCATGT-3’；UV-R：5’-CAAGTGCGAGGATAACTGAAT-3’。引物由生物公司合成。稻曲病菌PCR检测反应条件为：反应体积为25μL，其中包括10×PCRbuffer2.5μl，10μmol/L的引物对各0.2μl，MgCl2(5mM)2.5μl，dNTPs（10mM）2.0μl，TaqPolymerase1U，DMSO0.5μl，DNA模板溶液各1μl，10×梯度稀释的稻曲病菌基因组DNA作为阳性模板，阴性对照采用无菌水，另设一个不加任何模板的空白对照。7.2.1.2反应条件PCR扩增程序：94℃预变性3min；94℃变性30sec，60℃退火30sec，72℃延伸30sec，32个循环；72℃延伸7min。7.2.1.3琼脂糖凝胶电泳将5-10μlPCR产物用1％琼脂糖电泳分离（电压为120V，时间为20min），经溴化乙锭染色后于紫外灯下根据扩增产物的大小判定结果。8结果判定在阳性对照、阴性对照和空白对照结果均正常的情况下：如待测样品扩增出346bp左右的条带，则判定为该样品水稻稻曲病菌PCR鉴定结果为阳性；如待测样品未扩增出346bp左右的条带，则判定为该样品水稻稻曲病菌PCR鉴定结果为阴性。9样品保存与处理样品经登记和经手人签字后妥善保存。对稻曲病菌PCR检测结果为阳性的样品应保存于-20℃冰箱中，以备复核。该样品保存期满后须经高压灭菌（121℃15min）后处理。3

8DB43/T1957—2020附录A（资料性）稻曲病相关资料A.1分布地区稻曲病的发生分布较为广泛，在中国、日本、美国、缅甸、印度、菲律宾等近40个国家均有发生。国内江南、华南、西南及东北稻区已有该病的发生报道。湖南稻区中、晚稻稻曲病发生普遍，尤以中稻发生严重。A.2病状稻曲病主要危害水稻穗部并形成稻曲球（见图A.1）。病菌侵染初期，小穗基部逐渐隆起且呈现白色；随后病菌突破颖壳，膨大，外层被膜包裹；病粒逐渐膨大呈圆球状，淡黄色，外部开裂；稻曲球继续生长膨大，被膜完全破裂，露出病原菌的后垣孢子，形成黄色稻曲球。稻曲球最初为橘黄色，后逐渐加深，变为黄绿色、暗绿色、墨绿色至黑色，最后病粒外层覆盖一层戎状厚垣孢子粉。根据稻曲球总病菌发育程度的不同，从内到外可以分为3层：最里层浅黄色，由密集的菌丝体和大量的厚垣孢子组成；中间层是橘黄色，由菌丝和厚垣孢子构成；最外层墨绿色，由成熟的黑色厚垣孢子和老熟的菌丝组成。图A.1水稻稻曲病菌危害状A.3侵染与病害流行稻曲病的发生流行与多种因素有关，主要包括品种、田间菌源量、气候条件及栽培管理措施等，其中气候条件尤其是水稻孕穗至破口期的湿度条件是影响稻曲病菌侵染的重要因素之一，在水稻孕穗期至破口期，雨量多、日照少利于稻曲病的发生。不同品种对稻曲病菌的抗性也存在差异，一般晚熟品种比早熟品种发病重，糯稻比粳稻感病，杂交稻比常规稻感病。田间稻曲病菌的菌源量与稻曲病的发生有显著相关性，老病区、重病区比新稻区发病重。4

9DB43/T1957—2020附录B（规范性）水稻稻曲病菌鉴定报告样品种类采样时间采样地点样品数量采样部位样品来源送检日期送检人送检单位联系电话检测鉴定方法：检测鉴定结果：备注：鉴定人（签名）：审核人（签名）：鉴定单位盖章：年月日注：本单一式两份，检测单位和受检单位各一份。本检测结果只对送检样品负责。5