Potential allelopathic azaphilones produced by the endophytic Chaetomium globosum TY1 inhabited in Ginkgo biloba using the one strain−many compounds method

Abstract On the basis of the one strain−many compounds strategy, seven azaphilones, including Chaetomugilin A (1), D (2), S (3), I (4), J (5), Q (6) and O (7), were isolated from the endophytic Chaetomium globosum TY1. Their structures were identified by NMR and HRESIMS spectrometry data. All azaphilones were evaluated for plant growth regulation using eight species of herbaceous plant seeds seedling growth bioassay, which showed the plant growth influence of the seedling. Among these compounds tested, Chaetomugilin O (7) with tetrahydrofuran exhibited higher response index and lower IC50 values than positive control glyphosate, a broad-spectrum systemic herbicide. 1–3 also showed better or similar inhibit activity to glyphosate. The structure−allelopathic activity relationship analysis of these isolated azaphilones indicates that both tetrahydrofuran and tetrahydrofuran combine with lactones ring groups give potent inhibition of seedling growth. Chaetomugilin O and Chaetomugilin A, D, S could be used to develop natural eco-friendly herbicides. Graphical Abstract


Introduction
Endophytes are bacteria or fungi that live in the intercellular spaces of the tissues of host plants without causing discernible manifestation of disease (Strobel 2002), they can be found in virtually all terrestrial plants and play an important role for the growth of hosts (Saikkonen et al. 2004). Recently, endophytes have been recognised as important sources of a variety of structurally novel active secondary metabolites with anticancer, antimicrobial and other biological activities (Yang et al. 2012;Li et al. 2016).
Fungi of the Chaetomium species, which belong to the family Chaetomiaceae, are the largest genus of saprophytic ascomycetes, with more than 350 Chaetomium species. Up to now, more than 200 metabolites with a wide range of bioactivities have been isolated from the genus Chaetomium, but compared with its richness of species, more bioactive secondary metabolites might be found in this fungus .
On the basis of the knowledge that the biosyntheses of secondary metabolites in endophytes are dependent on the culture parameters and available nutrition (one strain−many compounds) to improve the search for potent bioactive substances (Bode et al. 2002).
In our continuous screening for bioactive metabolites from Chaetomium sp. (Qin et al. 2009;Gao et al. 2013;Li et al. 2013), we investigated the metabolites of the extract of the fungus Chaetomium globosum TY1, leading to the isolation of five compounds (1-5) cultured in rice medium and two compounds (6-7) cultured in Potato Dextrose Broth medium ( Figure 1).

Identification of fungal metabolites
Chemical investigations of C. globosum TY1 crude extracts obtained from two different media led to the isolation of a series of azaphilones ( Figure 1) by multiple chromatographic procedures. 1 H and 13 C NMR data of compounds in CDCl 3 see Tables S1 and S2 (listed in supplementary material). Chaetomugilin A (1), D (2), S (3), I (4), J (5), Q (6) and O (7) were isolated and identified.

Evaluation of allelopathic activity
All isolated compounds were evaluated for allelopathic activity against C. sativus, B. campestris, E. sativa, L. sativa, D. carota, S. ningpoensis, S. oleracea and B. rapa seeds using previously reported assay ) by determining the germinate inhibition rate (GIR) ( Figure S2), IC 50 (Table S3) and seedling growth (shoot and root elongation) with respect to the control ( Figures S3 and S4), glyphosate, abroad-spectrum systemic herbicide. The RI was selected as an evaluation indicator, which ranges from −1 to 1, with positive values indicating stimulation by the treatments and negative values indicating inhibition by them.
As shown in Figure S2 and Table S3, Chaetomugilin O (7) with tetrahydrofuran can inhibit the germination of tested seeds, with GIR values ranging from 80 to 100% at 100 ppm, and being more active than the positive control glyphosate (GIR values ranging from 60 to 90% at 100 ppm), also lower (ranging from <10 to 73.85 ppm) than glyphosate (ranging from 19.03 to 81.52 ppm) against most of tested herbaceous plant seeds except S. ningpoensis (Table S3). Meanwhile, Chaetomugilin S (3) showed significant germination inhibit activity (GIR values ranging from 70 to 100% at 100 ppm), Chaetomugilin D (2) with GIR values ranging from 60 to 100% at 100 ppm, and Chaetomugilin A (1) with GIR values ranging from 50 to 100% at 100 ppm, better than or similar to positive control glyphosate. Compounds 7 and 2 showed the best activity with GIR value 100% at 200 ppm against all tested seeds, followed by 3 and 1 with GIR ranging from 90 to 100% at 200 ppm, even better than that of glyphosate (70 to 100% at 200 ppm). 1-3 with tetrahydrofuran combine with lactones ring, exhibited low IC 50 values ranging from <10 to 77.83 ppm, <10 to 82.01 ppm and 3.056 to 92.02 ppm, respectively, better or approximate to that of glyphosate (19.03 to 81.52 ppm). Both compound 4 (IC 50 : 64.61 to 139.8) and 5 (IC 50 : 51.00 to 331.6) showed weak inhibition, indicating that the unit -CH=CH-C=O-may decrease the activity. Above results showed that germination inhibit activity of 4-6 are lower than that of 1-3 and 7. Additionally, their inhibition effects are dependent on their concentrations. Among all tested herbaceous plant seeds, S. oleracea was found the most sensitive to isolated azaphilones, with IC 50 values from 13.07 to 71.10 ppm, and E. sativa was found the most insensitive seed in germination inhibit assay, with IC 50 values from 51.33 to 509.7 ppm.
As shown in Figures S3 and S4, Chaetomugilin O (7) was found to exhibit excellent inhibitory effects on the shoot and root elongation of all tested herbaceous plants. For example, Chaetomugilin O (7) showed prominent inhibition of shoot and root elongation (ranging from −0.7 to −1 and −0.6 to −1, respectively) at 100 ppm which is better than the positive control glyphosate (−0.3 to −0.8 and −0.3 to −0.8, respectively). In particular, 7 showed three-fold stronger inhibition of shoot elongation (RI = −0.91 at 200 ppm) than the positive control (RI = −0.34). Even at a lower concentration of 50 ppm, 7 exhibits two-to three-fold inhibition (RI ranging from −0.9 to −1) of shoot and root elongation of plants, C. sativus, D. carota and S. oleracea compared to that of glyphosate. Also, at 100 ppm, 1 and 2 showed high inhibition of shoot and root growth (RI values ranging from −0.5 to −0.1), and 3 showed high inhibition of shoot growth (RI values ranging from −0.5 to −0.1) to all plants and root growth (RI values ranging from −0.7 to −0.1) to most plants except only B. campestris (with RI = −0.3). What's more, at 200 ppm, 2 and 7 showed highest inhibition of shoot and root growth with RI ranging from −0.9 to −1 better than that of control glyphosate (−0.6 to −1), followed by 1 and 3 (RI ranging from −0.6 to −1).
In contrast, 5 and 6 containing a conjugated (-CH=CH-C=O-) unit showed weaker inhibition of shoot and root growth (RI values ranging from −0.2 to −0.8 at 100 ppm), and 4 showed the weakest inhibition (RI values ranging from −0.1 to −0.6 at 100 ppm). Interestingly at the lowest concentration 1-3, showed stimulation effect to tested herbaceous plants with RI values ranging from +0.5 to 0.
Above all, both germination and shoot and root elongation inhibit activity of 1-3 and 7 were found higher than that of 4-6. This growth-suppression activity may be ascribed to the presence of tetrahydrofuran. However, inhibit activity of 7 was higher than that of 1-3, which indicated that the lactone rings would like to decline the inhibitory effects.

Disclosure statement
No potential conflict of interest was reported by the authors.