Data sets for symbiotic interactions between <i>Rosellinia necatrix</i> and weed roots assist the spread of white root rot in soil
We hypothesized that symbiotic colonization of <i>Rosellinia necatrix</i> 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 <i>R. necatrix</i> and probably assist the spread of the white root rot fungus in soil. To investigate whether any weed is colonized with <i>R. necatrix</i> 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 <i>R. necatrix</i> 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 <i>R. necatrix</i> 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 <i>R. necatrix</i> 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 <i>R. necatrix</i> 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 <i>R. necatrix</i> DNA was performed as described before.