Data sets for symbiotic interactions between Rosellinia necatrix and weed roots assist the spread of white root rot in soil

We hypothesized that symbiotic colonization of Rosellinia necatrix in weed roots could facilitate fungal spread in the soil of Japanese pear orchards. Our data show that some weeds can form symbiotic relationships with R. necatrix and probably assist the spread of the white root rot fungus in soil. To investigate whether any weed is colonized with R. necatrix at its roots, two Japanese pear orchards, known for infestation of white root rot for more than two decades, were selected for sampling weed plants. Twenty weed species, at least one sample per species, were collected from the orchard floor from May through September of 2014. Roots of each weed sample were thoroughly washed with tap water and dried under room condition for 4 d. Total DNA of each root sample was extracted using MagExtractor (Toyobo Co., Osaka, Japan) in accordance with the manufacture’s instruction. Nested polymerase chain reaction (PCR) was performed for every DNA sample, in accordance with the method of Shishido et al. (2012). To confirm our hypothesis above, we used concrete frames in the field of Chiba University Experiment Farm located at Matsudo-shi, Japan. Four concrete frames had been fallowed for three years, and 8 weed species grew naturally in these frames. A 10-cm-long dried Japanese pear twig, 1 cm in diameter, was autoclaved and aseptically inoculated with R. necatrix K64 in an Erlenmeyer flask, followed by incubation at 25°C for two months. The pear twig, completely colonized with the fungus, was inserted into the center of the concrete frame as the inoculum source. One week after twig-insertion, up to 20-cm-deep soil samples were collected using a 20-cm-long plastic tube at points 3, 10, 20, and 30 cm from the twig, with four replicates at right angles to each other. Four replicated soil samples of the same distance from the inoculum twig were pooled and thoroughly mixed. Total DNA was extracted from 500 mg of each soil mixture, using ISOIL for beads beating (Nippon Gene Co., Tokyo, Japan). A similar nested PCR for detecting R. necatrix was carried out as described above. Soil sample collection and nested PCR assays were conducted every two weeks for seven weeks after twig-insertion. To confirm results using concrete frames, i.e., promotion of the spread of R. necatrix by weed roots, we conducted another inoculation experiment with artificially growing weed plants in planter boxes under greenhouse condition. Rescue grass was used in this experiment because the weed most commonly accommodated R. necatrix in the Japanese pear orchards. Seeds were sown at 3-cm intervals in two lines, 8 cm apart. The non-weed control received no seeds. Three months after sowing, soil samples were collected from the points at 3, 25, and 50 cm from the inoculum twig. Soil DNA was extracted using ISOIL for beads beating, and nested PCR for detecting R. necatrix DNA was performed as described before.

我们提出假说：杂草根系内的褐座坚壳菌（Rosellinia necatrix）共生定殖，可促进该真菌在日本梨园土壤中的扩散。本研究数据显示，部分杂草可与褐座坚壳菌形成共生关系，并可能协助该白根腐病菌在土壤中传播。为调查是否存在杂草的根系被褐座坚壳菌定殖，我们选取了两处已有二十余年白根腐病侵染史的日本梨园，用于采集杂草样本。2014年5月至9月期间，我们从梨园地面采集了20种杂草，每种至少采集1份样本。每份杂草样本的根系经自来水彻底冲洗后，于室温条件下晾干4天。采用磁珠法DNA提取试剂盒（MagExtractor，日本大阪东洋纺公司），按照制造商说明书提取每份根系样本的总DNA。参照Shishido等（2012）的方法，对每份DNA样本开展嵌套式聚合酶链式反应（Nested polymerase chain reaction）检测。为验证上述假说，我们在日本千叶大学松户市实验农场的田间混凝土框中开展了实地试验。此前有4个混凝土框已休耕3年，其内自然生长了8种杂草。将直径1cm、长10cm的干燥日本梨枝条经高压灭菌后，在三角烧瓶中以无菌方式接种褐座坚壳菌K64菌株，随后于25℃下培养2个月，获得完全被该真菌定殖的梨枝条作为接种源。将该接种枝条插入混凝土框中心，随后在距接种枝条3cm、10cm、20cm、30cm的位置，使用20cm长的塑料套管采集深度达20cm的土壤样本，每个位置设置4个相互垂直的重复采样点。将同一距离处的4份重复土壤样本混合并充分混匀。称取500mg每份混合土壤样本，采用珠磨法土壤DNA提取试剂盒（ISOIL for beads beating，日本东京日生基因公司）提取总DNA。参照前述方法开展嵌套式聚合酶链式反应检测，以鉴定褐座坚壳菌的存在。自接种枝条插入后，每两周采集一次土壤样本并开展相关检测，持续7周。为验证混凝土框试验所得结论——即杂草根系可促进褐座坚壳菌的扩散，我们开展了另一项接种试验：在温室条件下的种植箱中人工种植杂草并开展接种实验。鉴于在日本梨园中褐座坚壳菌最常定殖于该类杂草，本次试验选用了扁穗雀麦（Rescue grass）。将种子以3cm的间距播种于两行中，两行间距8cm；无杂草对照组不播种。播种3个月后，在距接种枝条3cm、25cm、50cm的位置采集土壤样本。采用前述珠磨法土壤DNA提取试剂盒提取土壤总DNA，并开展嵌套式聚合酶链式反应检测以鉴定褐座坚壳菌DNA。