ICS 65.020 CCS B 16 湖 DB43 南 省 地 方 标 准 DB43/T 1 9 5 9—2020 辣椒炭疽病菌检测技术规程 Technical Regulations for Detection of Colletotrichum complex of Capscium annuum L. 2020-12-29发布 湖南省市场监督管理局 2021-03-29实施 发 布 DB43/T 1959—2020 目 次 前言························································································································ Ⅲ 1 范围 ····················································································································· 1 2 规范性引用文件 ······································································································ 1 3 术语和定义 ············································································································ 1 4 原理 ····················································································································· 1 5 仪器设备与试剂 ······································································································ 2 5.1 仪器设备 ········································································································· 2 5.2 主要试剂 ········································································································· 2 6 采样 ····················································································································· 2 6.1 样品采集 ········································································································· 2 6.2 样品贮运与存放 ································································································ 2 7 操作方法 ··············································································································· 2 7.1 样品 DNA 制备 ··································································································· 3 7.2 检测 ··············································································································· 3 8 结果判定 ··············································································································· 4 9 样品保存与处理 ······································································································ 4 附录 A（资料性） 辣椒炭疽病菌相关资料 ······································································· 5 附录 B（规范性） 辣椒炭疽病菌样本鉴定报告 ································································· 7 I DB43/T 1959—2020 II DB43/T 1959—2020 前 言 本文件按照 GB/T 1.1—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。 本文件主要起草人：谭新球、张鑫、满益龙、陈岳、郑立敏、张卓、欧阳超、刘思珍。 III DB43/T 1959—2020 IV DB43/T 1959—2020 辣椒炭疽病菌检测技术规程 1 范围 本文件规定了辣椒炭疽病菌高效、快速检测和种类鉴定的样品制备、检测技术和操作程序。 本文件适用于土壤、辣椒种子、辣椒植株和果实中炭疽病菌的检测和种类鉴定。 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/T 6682 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。 辣椒炭疽病菌属半知菌亚门、类腔菌纲、黑盘菌目、刺盘孢属真菌（Colletotrichum Complex）,目 前我省已经报的辣椒炭疽菌有辣椒胶孢炭疽菌（Colletotrichum gloeosporioides）、辣椒尖孢炭疽菌 （ Colletotrichum acutatum ）、 辣 椒 平 头 孢 炭 疽 菌 （ Colletotrichum truncatum ）、 辣 椒 红 色 炭 疽 菌 （Colletotrichum capsici）和辣椒短孢炭疽菌（Colletotrichum brevisporum）等 5 个种，存在不同的地理 分布特征。辣椒炭疽病主要危害辣椒的茎秆、叶片、果实，形成典型的病斑，严重影响辣椒的产量和品 质。见附录 A。 4 原理 利用真菌高度保守的核糖体基因转录间隔区（Internal transcribed spacer, ITS），结合病原菌的单核 苷酸重复序列（Simple sequence repeat, SSR）和真菌基因组中诱导坏死基因（Necrosis-inducing secreted gene，NIS）进行相应特异性扩增片段比较，确定辣椒炭疽病菌的种类和种内菌株的差异。 4.1 ITS 进化速率较编码区快，在真菌的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靶序列。根据辣椒炭疽病菌 ITS 的特异序列，合成特异性引物，获得辣椒 炭疽病菌特异性扩增片段，用于辣椒种子、茎秆、叶片和果实中炭疽病菌的检测。 4.2 SSR 在真菌基因中比例较高，而且稳定性好，基于辣椒炭疽病菌种类比较多且普遍存在复合侵染， ITS-PCR 鉴 定 不 能 区 分 不 同 种 和 相 同 种 菌 株 间 差 异 。 根 据 辣 椒 胶 孢 炭 疽 病 菌 （ Colletotrichum gloeosporioides）基因组，以 6 个碱基为重复单元，以 100-300 个核苷酸大小为靶标序列，设计合成 7 对特异性引物，获得辣椒炭疽病菌的特异性扩增片段，用于辣椒种子、茎秆、叶片和果实等样品中炭疽 病菌种类鉴定和种内菌株差异性的检测。 4.3 NIS 基因在真菌遗传中比较保守，但是不同菌株之间存在明显差异，为病原菌的分子检测提供靶 1 DB43/T 1959—2020 序列。根据辣椒炭疽病菌 NIS 的特异序列，合成特异性引物，获得辣椒炭疽病菌特异性扩增片段，用于 辣椒种子、茎秆、叶片和果实中炭疽病菌的检测和田间复合种群的检测。 5 仪器设备与试剂 5.1 仪器设备 PCR 检测仪、凝胶成像仪、台式离心机、土壤取样器、纯水仪、电泳仪、旋涡振荡器等。 5.2 主要试剂 苯酚/氯仿/异戊醇、乙醇、二甲基亚砜、氢氧化钠、脱脂奶粉、DNA marker、吐温 20、PCR 试剂盒 等。 除特别说明以外，本文件所有试剂均为分析纯，实验用水应符合 GB/T 6682 的规定。 6 采样 6.1 样品采集 6.1.1 土壤 田块 W 型 5 点取样，取 1-10cm 深度的 50 g 土壤样品。 6.1.2 辣椒种子 将辣椒种子混匀后称取 50 g。 6.1.3 辣椒茎秆和叶片 每块田 W 型选 5 个点，取田间发病植株上部相对幼嫩的茎秆和叶片，每点取 5 株植物的茎秆和叶片， 混合后取样 20g。 6.1.4 辣椒果实 每块田 W 型选 5 个点，取田间发病辣椒果实，称取发病组织样本 20g。 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 经 100 目过筛后的土壤加入少量无菌水（10g 土壤加入 5 mL 无菌水）后冷冻抽干 24-48h