Biotransformation of 2-Benzoxazolinone to 2-Amino-(3<Emphasis Type="Italic">H</Emphasis>)-Phenoxazin-3-one and 2-Acetylamino-(3<Emphasis Type="Italic">H</Emphasis>)-Phenoxazin-3-one in Soil

An alternative to the use of synthetic pesticides is to exploit the natural defense chemicals produced by cereals. An important class of allelochemicals is cyclic hydroxamic acids and related benzoxazolinones. A prolonged degradation experiment of the allelochemical compound from rye 2-benzoxazolinone (BOA) was carried out for up to 90 d at 15°C at three different concentration levels, 3, 3000, and 30,000 nmol BOA· g soil^–1, respectively, in a sandy loam soil. Two main degradation products, 2-amino-(3H)-phenoxazin-3-one (APO) and 2-acetylamino-(3H)-phenoxazin-3-one (AAPO), were identified and quantified by LC-ESI-MS-MS. The half-life of BOA increased with higher levels of BOA added to the soil. Half-lives of BOA, APO, and AAPO were determined by fitting a single first-order model to the degradation data. Half-life of BOA was determined to be 0.6 d in the 3 nmol BOA g soil^–1 treatment. Half-lives of BOA, APO, and AAPO were 3.1, 2.7, and 2.1 d, respectively, in the 3000 nmol BOA· g soil^–1 treatment. In the 30,000 nmol BOA· g soil^–1 treatment, the half-lives were 31 d for BOA and 45 d for APO. The microbial community structure was not affected by addition of BOA to the soil as investigated by analysis of signature fatty acids. The results suggest that the exploitability of BOA for crop protection is dependent on the existing concentration of BOA in the soil and the timing of incorporation of hydroxamic acid synthesizing crops into the soil.     

2,2′-OXO-1, 1 ′-azobenzene A microbially transformed allelochemical from 2,3-Benzoxazolinone: I

2,2-Oxo-1,1 -azobenzene (AZOB), a compound with strong herbicidal activity, was isolated and characterized from a soil supplemented with 2,3-benzoxazolinone (BOA). A parallel experiment with 6-methoxy-2,3-benzoxazolinone (MBOA) yielded AZOB as well as its mono-(MAZOB) and dimethoxy-(DIMAZOB) derivatives. These compounds were produced only in the presence of soil microorganisms, via possible intermediates, I and II, which may dimerize or react with the parent molecule to form the final products. In the case of MBOA, it was shown that demethoxylation precedes the oxidation step. Although BOA and 2,4-dihydroxy-1,4(2H)-benzoxazin-3-one (DIBOA) were leached out of rye residues, there were no detectable amounts of the biotransformation products in the soil. When BOA was mixed with soil and rye residue, either under field conditions or in vitro, AZOB was detected. Levels of free BOA in the soil were greatly reduced by incubation with rye residue. AZOB was more toxic to curly cress (Lepidium sativum L.) and barnyardgrass (Echinochloa crusgatti L.) than either DIBOA or BOA.Journal Article No. 12943 of the Michigan Agricultural Experiment Station.     

Hydroxamic Acid Content and Toxicity of Rye at Selected Growth Stages

Rye (Secale cereale L.) is an important cover crop that provides many benefits to cropping systems including weed and pest suppression resulting from allelopathic substances. Hydroxamic acids have been identified as allelopathic compounds in rye. This research was conducted to improve the methodology for quantifying hydroxamic acids and to determine the relationship between hydroxamic acid content and phytotoxicity of extracts of rye root and shoot tissue harvested at selected growth stages. Detection limits for an LC/MS-MS method for analysis of hydroxamic acids from crude aqueous extracts were better than have been reported previously. (2R)-2-β-d-Glucopyranosyloxy-4-hydroxy-(2H)-1,4-benzoxazin-3(4H)-one (DIBOA-G), 2,4-dihydroxy-(2H)-1,4-benzoxazin-3(4H)-one (DIBOA), benzoxazolin-2(3H)-one (BOA), and the methoxy-substituted form of these compounds, (2R)-2-β-d-glucopyranosyloxy-4-hydroxy-7-methoxy-(2H)-1,4-benzoxazin-3(4H)-one (DIMBOA glucose), 2,4-hydroxy-7-methoxy-(2H)-1,4-benzoxazin-3(4H)-one (DIMBOA), and 6-methoxy-benzoxazolin-2(3H)-one (MBOA), were all detected in rye tissue. DIBOA and BOA were prevalent in shoot tissue, whereas the methoxy-substituted compounds, DIMBOA glucose and MBOA, were prevalent in root tissue. Total hydroxamic acid concentration in rye tissue generally declined with age. Aqueous crude extracts of rye shoot tissue were more toxic than extracts of root tissue to lettuce (Lactuca sativa L.) and tomato (Lycopersicon esculentum Mill.) root length. Extracts of rye seedlings (Feekes growth stage 2) were most phytotoxic, but there was no pattern to the phytotoxicity of extracts of rye sampled at growth stages 4 to 10.5.4, and no correlation of hydroxamic acid content and phytotoxicity (I₅₀ values). Analysis of dose–response model slope coefficients indicated a lack of parallelism among models for rye extracts from different growth stages, suggesting that phytotoxicity may be attributed to compounds with different modes of action at different stages. Hydroxamic acids may account for the phytoxicity of extracts derived from rye at early growth stages, but other compounds are probably responsible in later growth stages.     

Cyclic hydroxamic acid accumulation in corn seedlings exposed to reduced water potentials before,during, and after germination

Cyclic hydroxamic acids are innate compounds associated with pest resistance in several grass species. The major cyclic hydroxamic acids of com, 2,4-dihydroxy-7-methoxy-2H-1,4-benzoxazin-3-one (DIMBOA) and 2,4-dihydroxy-3H-1,4-benzoxazin-3-one (DIBOA), were measured in seedlings after exposure to various water stress treatments. Both DIMBOA and DIBOA were found in greater quantities in plants experiencing a water deficit stress than in nonstressed plants. The increased cyclic hydroxamic acid coincided with a reduction in seedling growth, suggesting that cyclic hydroxamic acids are stress metabolites. Plants grown under conditions that restrict growth, such as water deficit stress, contain higher cyclic hydroxamic acids, which should make them more resistant to herbivorous pests and pathogenic microorganisms.     

Aneugenic 2,4-dihydroxy-7-methoxy-1,4-benzoxazin-3-one (DIMBOA) and 2,4-dihydroxy-1,4-benzoxazin-3-one (DIBOA) in sprouts of Triticumaestivum cultivars – A ‘safety health food’?

The concentrations of the benzoxazinoids (BAs), 2,4-dihydroxy-7-methoxy-1,4-benzoxazin-3-one (DIMBOA) and 2,4-dihydroxy-1,4-benzoxazin-3-one (DIBOA) were determined in four selected Triticum aestivum cultivars. The impact of the cultivation period on the overall concentrations in the cultivars was determined by HPLC. Furthermore, the distribution of the two BAs was analysed in aerial and subterranean parts of the seedlings after different time periods. The highest levels were detected after 3 days. Subsequently, the levels of DIMBOA declined in all cultivars (reduction between 84% and 93%), while DIBOA was not detectable after 7 days. The distribution between aerial parts and roots varied substantially in the different cultivars. In addition, DIMBOA amounts, expressed in per cent, as well as in mg per seedling, were determined in one selected cultivar during the entire time course for better understanding of eventual dilution effects during plant growth. Our findings indicate that the consumption (up to several 100 g) of fresh sprouts (as suggested by suppliers) may already lead to exposure levels of up to 470 mg DIMBO and 190 mg of DIBOA per 100 g each. The potential adverse health effects of these doses are discussed.     

Bread from common cereal cultivars contains an important array of neglected bioactive benzoxazinoids

Bread is consumed in large quantities all over the world, and rye bread is especially popular throughout the Nordic countries. Wholemeal bread is highly recommended as a basic ingredient in daily food, because wholemeal food products generally promote good health due to their vitamin, mineral and fibre content. The literature suggests that wholemeal products have other health-promoting effects even if the ingredients responsible have not been identified. Benzoxazinoids are a group of natural products that have not previously been reported in mature grains. Here, we report for the first time the identity and quantity of 10 compounds of the benzoxazinoid family in mature grains, hydrothermally processed grains of durum wheat (Triticum durum, cv. Kamut), a commercial variety of rye (Secale cereale cv. Picasso) and an old Nordic rye landrace (S. cereale, Svedjerug), as well as in bread baked with flour milled from those grains. Concentrations of the 10 benzoxanoids were determined using LC–MS/MS and ranged from 0 to 348 nmol g⁻¹ for conventional flour, to 772–1177 nmol g⁻¹ in bread baked with flour from hydrothermally processed grains and to 3116–5570 nmol g⁻¹ in flour from hydrothermally processed grains. Benzoxazinoids possess documented physiological effects, and research into the importance of these compounds in the daily diet is therefore needed. Ongoing studies in our lab on the uptake and transformation of benzoxazinoids in mammals will be reported in the near future.     

Growth Inhibition and Root Ultrastructure of Cucumber Seedlings Exposed to Allelochemicals from Rye (Secale cereale)

Inhibition of "Calypso" cucumber seedling growth by rye allelochemicals, 2(3H)-benzoxazolinone BOA and 2,4-dihydroxy-1,4(2H)-benzoxazin-3-one DIBOA, was studied by analyzing the growth of seedling tissues and organs. Light and electron microscopy of seedling root cells were also carried out to investigate the mechanism(s) of root growth inhibition and mode of action of these compounds. BOA inhibited root elongation and reduced the number of cucumber lateral roots by 77 and 100% at 0.1 and 0.43 mg BOA/ml deionized (DI) water, respectively. DIBOA also inhibited root growth, but did not affect the number of lateral roots. BOA increased size of cucumber cortical root cells fivefold, but DIBOA had no effect. Both compounds reduced the regeneration of root cap cells and increased the width of cortical cells resulting in increased root diameter. BOA and DIBOA caused increased cytoplasmic vacuolation, reduced ribosome density and dictyosomes, reduced number of mitochondria, and reduced lipid catabolism. Starch granules in amyloplasts of seedling roots treated with BOA and DIBOA were also greatly reduced compared to the control. Changes in cellular ultrastructure indicated that BOA and DIBOA reduced root growth by disrupting lipid metabolism, reducing protein synthesis, and reducing transport or secretory capabilities.     

CHANGES OVER TIME IN THE ALLELOCHEMICAL CONTENT OF TEN CULTIVARS OF RYE (<Emphasis Type="Italic">Secale cereale</Emphasis> L.)

Published studies focused on characterizing the allelopathy-based weed suppression by rye cover crop mulch have provided varying and inconsistent estimates of weed suppression. Studies were initiated to examine several factors that could influence the weed suppressiveness of rye: kill date, cultivar, and soil fertility. Ten cultivars of rye were planted with four rates of nitrogen fertilization, and tissue from each of these treatment combinations was harvested three times during the growing season. Concentrations of a known rye allelochemical DIBOA (2,4-dihydroxy-1,4-(2H)benzoxazine-3-one) were quantified from the harvested rye tissue using high performance liquid chromatography (HPLC). Phytotoxicity observed from aqueous extracts of the harvested rye tissue correlated with the levels of DIBOA recovered in harvested tissue. The amount of DIBOA in rye tissue varied depending on harvest date and rye cultivar, but was generally lower with all cultivars when rye was harvested later in the season. However, the late maturing variety Wheeler retained greater concentrations of DIBOA in comparison to other rye cultivars when harvested later in the season. The decline in DIBOA concentrations as rye matures, and the fact that many rye cultivars mature at different rates may help explain why estimates of weed suppression from allelopathic agents in rye have varied so widely in the literature.     

Role of benzoxazinones in allelopathy by rye (Secale cereale L.)

Two phytotoxic compounds [2,4-dihydroxy-1,4(2H)-benzoxazin-3-one (DIBOA) and 2(3H)-benzoxazolinone (BOA)] were previously isolated and identified in 35-day-old greenhouse-grown rye shoot tissue. Both compounds were also detected by TLC in greenhouse-grown root and fieldgrown shoot tissue. The concentration of DIBOA varied in the tissues, with the greatest quantity detected in greenhouse-grown shoots. DIBOA and BOA were compared with -phenyllactic acid (PLA) and -hydroxybutyric acid (HBA) for activity on seed germination and seedling growth and were consistently more toxic than either compound. Dicot species tested, including lettuce (Lactuca sativa L.), tomato (Lycopersicon esculentum Mill.), and redroot pigweed (Amaranthus retroflexus L.), were 30% more sensitive than the monocots tested. Of the two benzoxazinone compounds, DIBOA was most toxic to seedling growth. DIBOA and BOA reduced chlorophyll production inChlamydomonas rheinhardtii Dangeard, by 50% at 7.5 × 10^–5 M and 1.0 × 10^–3 M, respectively. Both DIBOA and BOA inhibited emergence of barnyardgrass (Echinochloa crusgalli L. Beauv.), cress (Lepidium sativum L.), and lettuce when applied to soil, indicating their potential for allelopathic activity.Journal Article No. 11945 of the Michigan Agricultural Experiment Station.