First report on the occurrence of benzoxazinoids in an invasive weed plant, woolly cupgrass (Eriochloa villosa (Thunb./Kunth)

Woolly cupgrass (Eriochloa villosa /Thunb./ Kunth) is a common weed in East Asia and North America. This weed plant spreading quickly in Europa. There are several reasons for the successfulness, especially its germination properties. As other Panicoideae species contain benzoxazinoids it was supposed that woolly cupgrass also contains these chemicals. For this reason the benzoxazinoid content of plants at the stage of flowering was investigated. As it was supposed, woolly cupgrass contain benzoxazinoids, which is a novel occurrence of these chemicals in Poaceae family.


Introduction
Woolly cupgrass (Eriochloa villosa /Thunb./ Kunth) is a common weed plant in East Asia. In the middle of the twentieth century it was appeared in North America, in the United States, especially as a corn weed. Soon it was spread and also appeared in the south part of Canada at the start of the twenty-first century. At the same time it was came out in Europe too (Romania, Ukraine, Hungary) [1]. In the last few years this problematic weed plant was found from other Western and Eastern European countries [2]. In Hungary there are numerous regions where this invasive plant was found [3]. The problems of controlling this annual weed originating from its seed germination properties, namely that the seeds emerge from deeper soil layers and long-continued time of the growing season [1]. In addition the plants are less susceptible for some herbicides used against other annual grass weeds in corn [4]. Echinochloa, Tripsacum and Zea [6]. Ten days old Echinochloa plants for example contained DIBOA in glycosidic form in amount of 194-267 μg g -1 fresh weight [7]. Five week old Echinochloa crus-galli plants in turn contained DIMBOA and DIBOA in form of their glucosides, in amount of 8.7 -18.85 and 4.05 -7.5 μg g -1 fresh weight respectively [8].
HMBOA was not demonstrated from this plant species. Corn and Coix spp. however contain HMBOA in considerable amount [9]. Up to now there is no data about the benzoxazinoid content of Eriochloa villosa which was investigated in our experiment.

Materials and Methods
Seeds of woolly cupgrass were collected from field grown plants in Hungary, in the region of Hajdúság, near to town Debrecen, in 2020. Ten seeds were shown at the 20 th of May, 2021, in 14 cm diameter pots, filled with universal soil for garden use, obtained from commercial trade. After germination 3 plants were left in each pot. We had independent four pots. The plants were grown in special growing house, on the open air. Water supply was applied to the 65 % of the field water capacity, every two days. The sample collection was carried out at the start of flowering (Picture 1.). The sample preparation and HPLC determination was carried out according to Lyons et al. [10]. DIMBOA and DIBOA standards were isolated from etiolated corn and rye seedlings by using the method of Hartenstein et al. [11]. HMBOA standard was synthetized and provided by Pierre Mateo (Universität Bern, Institute of Plant Sciences). The DIMBOA and DIBOA standards were checked and identified by their melting points and by 1 H NMR spectra recorded for CD3OD solutions. The applied equipment was Bruker 360 (360 MHz) spectrometer. Chemical shifts were referenced to CH3OH (δ 3.31 ppm). The

Results and discussion
The occurrence of benzoxazinoids was demonstrated in a new plant species -woolly cupgrass (Eriochloa villosa /Thunb./ Kunth) -by our laboratory. Table 1 demonstrates the measured maximal benzoxazinoid contents in different parts of woolly cupgrass plants. DIMBOA. These compounds are present in intact tissues in glycosidic form [6], but as a consequence of our sample preparation method, glycosidase enzymes released aglucones, which were measured and presented.
Compared to earlier results [7,8] the measured concentrations are at the same range (data are not presented) than the concentrations in shoots of ten days old Echinocloa plants.
The young parts of older plants can contain benzoxazinoids in as high concentration as young plant [12], and the examined shoot and root parts were the youngest parts of the plants. Pethő [8] measured benzoxazinoids in lower concentrations in five week old seedlings of barnyard grass, but the plant material was the whole shoot of the plants, not the only the youngest parts.
As it is well known, the distribution of various types of benzoxazinoids is vary amongst different species [13], so there is the possibility of variation even in a plant subfamily (Panicoideae). As woolly cupgrass is an invasive plant, with high ability for spreading and high competition capacity, one of the reason of this plant's success can be the benzoxazinoids production, which allow the higher ability to tolerate both biotic and abiotic stress factors. Moreover the benzoxazinoids producing plants exudate these chemicals through their roots to the rhizosphere [12], and these exudates serve as allelochemicals, regulate the germination and growth of competing plant species.