Bipolaris sorokiniana, Shoemaker

Ube, Naoki, Harada, Daiyu, Katsuyama, Yuhka, Osaki-Oka, Kumiko, Tonooka, Takuji, Ueno, Kotomi, Taketa, Shin & Ishihara, Atsushi, 2019, Identification of phenylamide phytoalexins and characterization of inducible phenylamide metabolism in wheat, Phytochemistry 167, pp. 1-9 : 2

2.1. Detection of inducible metabolites in wheat leaves infected by B. sorokiniana

B. sorokiniana conidia were inoculated to the third leaves of 3-week-old wheat seedlings. We extracted metabolites from the leaves with 80% methanol 48 h after inoculation, and subjected the extracts to HPLC analysis. As shown in Fig. 1 , the accumulation of compounds 1–6 was induced in leaves inoculated with B. sorokiniana . Compounds 3–6 were collected by preparative HPLC and subjected to LC-MS analysis. 3–6 showed [M+H] + ions at m/z 251, 293, 291, and 277, respectively. Comparison of the detected ions and retention time by HPLC analysis with those of authentic compounds revealed 6 was CouAgm. LC-MS/MS analysis of 3–6 showed different fragment ions at m/z 88 in 3, m/z 130 in 4, m/z 128 in 5, and m/z 114 in 6 in addition to a commonly detected fragment ion at m/z 147 corresponding to coumaroyl moiety ( Fig. S1 ). The fragment ion at m/z 114 in 6 corresponded to an agmatine moiety. Differences in fragment ions among 3–5 were due to the conversion of a guanidine group to amine (−42), the addition of a hydroxy group (+16), and dehydrogenation (−2) in an agmatine moiety, respectively. Accordingly, we predicted 3–5 as p -coumaric acid amides of hydroxyputrescine, hydroxyagmatine, and hydroxydehydroagmatine, respectively. The induction of these compounds was previously shown in wheat and barley. p -Coumaroyl-3-hydroxyputrescine accumulated in wheat leaves inoculated with rust fungus ( Stoessl et al., 1969). p -Coumaroyl-3-hydroxyagmatine and CouAgm accumulation was induced under snow cover in wheat ( Jin and Yoshida, 2000). p -Coumaroyl-3- hydroxydehydroagmatine accumulation was induced in response to powdery mildew in barley leaves ( von Röpenack et al., 1998), as well as CouAgm and p -coumaroyl-3-hydroxyagmatine. Compounds 1 and 2 in the upper left inset showed [M+H] + ions at m/z 321 and m/z 305, respectively. These ions did not correspond to those of the inducible compounds previously reported in wheat.

Figures

Fig. 1. High-performance liquid chromatography (HPLC) analysis of extracts from B. sorokiniana-inoculated, control, and intact leaves. A suspension of B. sorokiniana conidia (inoculation) or distilled water containing 0.25% Tween20 (control) was placed on the third leaves of 3-week-old wheat seedlings. The leaves were extracted 48 h after inoculation and subjected to HPLC analysis. Untreated leaves (intact) were also extracted and analyzed by HPLC. Insets were zoom up views of the chromatograms.