Chaga mushroom (Inonotus obliquus) is a pathogenic fungus that grows mostly on birch species (Betula pendula Roth and B. pubescens Ehrh.) and has traditionally been used as an anticancer medicine. This study aimed to compare the chemical composition and cytotoxic activity of chagas growing on both Betula spp. on various cancer cell lines. The freeze-dried extracts contained triterpenes inotodiol, lanosterol betulin, and betulinic acid typical to conks growing on Betula species. The cytotoxic activity of chaga growing on Betula pendula and B. pubescens 80% ethanolic extracts against 31 human cancer cell lines was evaluated by a sulforhodamine B assay. Chaga extract showed moderate activity against all cancer cell lines examined; it did not result in high cytotoxicity (IC₅₀ ≤ 20 µg/mL). The strongest inhibitions were observed with chaga (growing on B. pendula) extract on the HepG2 and CAL-62 cell line and with chaga (from B. pubescens) extract on the HepG2 cell line, with IC₅₀ values of 37.71, 43.30, and 49.99 μg/mL, respectively. The chaga extracts from B. pendula exert somewhat stronger effects on most cancer cell lines studied than B. pubescens extracts, which can be attributed to a higher content of inotodiol in B. pendula extracts. This study highlights the potential of chaga as a source of bioactive compounds with selective anticancer properties. To the best of our knowledge, this study is the first investigation of the chemical composition of I. obliquus parasitizing on B. pubescens.

Chaga (Inonotus obliquus) is a pathogenic fungus that grows mostly on birch species (Betula pendula and B. pubescens) and has traditionally been used as an anti-cancer medicine. The study aimed to compare the chemical composition and cytotoxic activity of chagas growing on both Betula spp. on various cancer cell lines. The freeze dried extracts contained triterpenes inotodiol, lanosterol betulin and betulinic acid typical to conks growing on Betula species. The cytotoxic activity of chaga growing on Betula pendula and B. pubescens 80% ethanolic extracts against thirty-one human cancer cell lines has been evaluated by a sulforhodamine B assay. The strongest inhibitions were observed with chaga (growing on B. pendula) extract on the HepG2 and CAL-62 cell line, and with chaga (from B. pubescens) extract on HepG2 cell line, with IC50 values of 37.71, 43.30, and 49.99 μg/mL, respectively. The chaga extracts from B. pendula exert somewhat stronger effects on most cancer cell lines studied than B. pubescens extracts. This study highlight the potential of chaga as a source of bioactive compounds with selective anti-cancer properties, encouraging further exploration of its therapeutic mechanisms and potential applications in cancer treatment.

Key message: This is the first study to investigate the success of inoculation of Alnus incana and A. glutinosa with Inonotus obliquus (Ach. ex Pers.) Pilát, providing new insights about the potential cultivation of this valuable fungus under field conditions. Results show that conks developed slowly under natural conditions. The development of the fungus observed in A. glutinosa suggests that this species may be as suitable a host for I. obliquus than birches. Furthermore, visible stem symptoms proved unreliable for detecting infection, highlighting the importance of molecular diagnostics for accurate assessment. Context: Inonotus obliquus (Ach. ex Pers.) Pilát (commonly known as Chaga) is a medicinal fungus traditionally harvested from birch trees. Artificial cultivation is increasingly considered due to a rising commercial demand. However, our knowledge remains limited regarding infection dynamics and conk development on different host species. Aims: We assessed the success of I. obliquus inoculation in Betula pendula , B. pubescens , Alnus incana , and A. glutinosa stands 2 years after inoculation in order to: (i) identify the most susceptible host tree species and the most effective I. obliquus strains for cultivation; (ii) assess the frequency of conk formation under hemiboreal conditions; investigate whether (iii) visible symptoms on stems resulted from I. obliquus infection and (iv) mechanical wounding influenced fungal establishment. Methods: The experiment was conducted at two locations in Estonia, encompassing a total of six forest sites, with inoculation success assessed using species-specific qPCR. Sterile conk development was additionally monitored visually 4 years post-inoculation. Results: Inoculation success differed between the two locations, with an infection rate of 18% observed at Järvselja and 3% at Võrumaa . Three fungal strains consistently achieved higher infection rates among the six tested ones. Conks developed only on A. glutinosa trees and with specific strains. Only the identity of the fungal strains resulted in a significant effect on infection ess. Visible stem symptoms, such as bleeding and bulging, were unreliable indicators of the infection rate. Conclusion: I. obliquus conks developed more rapidly on A. glutinosa compared to B. pendula , B. pubescens , and A. incana . Moreover, fungal strains play a crucial role in determining cultivation success.