Black root rot is a soilborne fungal disease of various plant species reported worldwide. After infection, dark necrotic lesions are observed on the roots of infected plants. Growth of infected plants are suppressed. Although a causal agent of this disease was designated as Thielaviopsis basicola previously, it was later reclassified and transferred to a new genus: Berkeleyomyces (Nel et al. 2018). Additionally, the fungi were divided into two species based on their phylogenetic characteristics: B. basicola and B. rouxiae (Nel et al. 2018). In Japan, the occurrence of black root rot disease on various plant species, e.g. pansy (Viola × wittrockiana) (Kasuyama and Tanina 2008; Horita and Ohgami 2015), okra (Abelmoschus esculentus) (Kobayashi and Kotani 1987), tobacco (Nicotiana tabacum) (Otani 1962), sweet pea (Lathyrus odoratus) (Kasuyama and Tanina 2008), Japanese honewort (Cryptotaenia japonica) (Kasuyama and Tanina 2008), and carrot (Daucus carota) (Horita and Ohgami 2015) have been reported. Most recently, in 2016, black root rot of lettuce was reported for the first time in Japan (Nakane et al. 2019). It is noteworthy that all identified black root rot pathogens in Japan were B. rouxiae (Nakane et al. 2019; Nakane and Usami 2020). No isolate of B. basicola has been reported to date.
In fact, B. rouxiae has spread worldwide and has been reported as a black root rot pathogen of various plant species (Nel et al. 2018, 2019). Nevertheless, no findings have been reported about the relation among geographical distribution, isolation source, pathogenicity, and phylogenetic characteristics of the respective isolates. That knowledge is important for epidemiological research of the disease, such as elucidating the lettuce black root rot which has occurred suddenly in Japan. For elucidation of the infection route and source of the disease, Japanese lettuce isolates should be compared with Australian (O'Brian et al. 1994) and American (Koike 2008) isolates reported earlier. Comparison between Japanese lettuce isolates and isolates from other plant species is also important. To conduct such epidemiological investigations and to undertake other studies of fungal population, molecular genetic tools must be developed for individual identification and phylogenetic analyses of fungal isolates.
Gelodenhuis et al. (2004) used internal-short sequence repeat (ISSR)-PCR to discover 14 microsatellite-like sequences in the T. basicola genome. Although 7 of these sequences were not polymorphic, they used another 7 DNA markers which were designed from the microsatellite-like sequences to analyzepopulation diversity (Gelodenhuis et al. 2006). Nevertheless, most of these markers were not useful for the analysis of B. rouxiae. Some primers of these DNA markers amplified no PCR product; others were not polymorphic. Therefore, for this study, we developed polymorphic DNA markers that are useful for phylogenetic analysis, epidemiological population analysis, identification of individual isolates, and for other genetic investigations of B. rouxiae.