Abstract

Media summary

Key Words

Introduction

Some cereal plants produce a series of benzoxazinoids compounds (cyclic hydroxamic acids). The number of this group of natural products is small, but they possess diverse biological activities. These compounds are involved in the defense of plants against fungi and insects as well as in allelopathic interactions (Frey 1997). The most important benzoxazinoids reported are 2,4-dihydroxy-(2H)-1,4-benzoxazin-3(4H)-one (DIBOA) and 2,4-dihydroxy-7-methoxy-(2H)-1,4-benzoxazin-3(4H)-one (DIMBOA), which are present in wheat, maize, and rye and have been found in members of different families. Moreover, these aglycones are unstable in solution and soil, being transformed to 2-benzoxazolinone (BOA), 7-methoxy-2-benzoxazolinone (MBOA), and other degradation products (Fomsgaard 2004). These transformations depend on the chemical and biological conditions. Some of these transformation products are more biologically active than the original ones (Friebe 1998).

Transport of allelochemicals to the soil can occur mainly by leaching of the foliar parts, exudation from root, decomposition of plant residues by microbial action (Chou 1976), or by direct transformation by microbes associated to the roots (Zikmudova 2002). Previous publications have dealt with the isolation, characterization, and biological activity of the degradation products (Chase 1991; Gagliardo 1992). However, the dynamic aspects of the degradation processes of these compounds during bioassays, or the influence of other microbial substrate on degradation have not been researched. Here, we report the stability and degradation studies of DIBOA and its derivatives in different conditions and target plants during bioassays. Our objectives were to study the influence of soil microbes in the degradation dynamics of these compounds. Understanding this process will allow us to elucidate the influence of these microbes on chemical defense strategies in plants that produce hydroxamic acids and to propose which chemical structures are responsible for the allelopathic behavior observed.

Methods

Donor and target plants.

Donor plants (Triticum aestivum L. and Secale cereale L.) and target plants (Avena fatua L., Lolium rigidum L. and Sinapis alba L.) seed surfaces were sterilized with 12 % calcium hypochlorite and pre-germinated in Petri dishes at 24 °C, by using agar-agar as substrate, and under photoperiod conditions (16 h light, 8 h darkness).

Bioassay-hydroponic culture

The obtained plants were aseptically transferred to sterilized glass containers (2 L). Glass perlite, rock wool or commercial soil were used as substrates (Figure 1). Bioassays were carried out in both sterile and non-sterile conditions. Different donor plant densities were co-cultured with target seedlings using Hoagland culture media (Sigma-Aldrich Co., 1.6 g/L, pH=5.6).

Allelochemicals analysis

Donor plants (roots and shoots), culture solutions or substrates, and target plants, were extracted with methanol (1%AcOH x 4) and analyzed by HPLC-DAD in previously validated conditions (Eljarrat 2004). Under these instrumental parameters, representative allelochemicals, responsible for the allelopathic potential 2,4-dihydroxy-2H-1,4-benzoxazin-3-(4H)-one (DIBOA) (Reberg-Horton 2005), in addition to their degradation products 2-aminophenol (APH), benzoxazolin-2(3H)-one (BOA) and 2-amino-3H-phenoxazin-3-one (APO) (Macías 2004), are separated, identified and quantified simultaneously. The identification was made by comparison of retention times and UV-VIS spectra at two wavelengths (95 % confidence) with pure standards, and standards addition. Quantification was made by the external standard method.

Results

Exudation of allelochemicals

Allelochemicals can be present in several parts of plants including roots, rhizomes, leaves, stems, pollen, seeds and flowers and can be released into the environment by root exudation, leaching from aboveground parts, and volatilization and by decomposition of plant material. Most of benzoxazinones are actively liberated to the field by exudation (Pérez 1991) and their degradation products are involved in allelopathic potential of donor plants. The release of hydroxamic acids from roots of rye seedlings can probably be affected by biotic stresses. It has been shown that the release of hydroxamic acids through root exudates is affected by defoliation of rye seedlings. The result of repeated defoliation of rye seedlings, was an increase in the allocation of hydroxamic acids to roots and root exudates. Other biotic stress can be the presence of specific micro-flora associated with seeds before germination.

In order to establish a correlation between biotic activity of soil and root exudation of hydroxamic acids here we present the results of two assays made in sterile and non-sterile conditions where concentrations of DIBOA and DIMBOA are analyzed (figure 1).

(a)	(b)

Fig. 1.- Time-study of exudation of DIBOA in Sinapis alba L. (a) and DIMBOA in Triticum aestivum L. var. Astron (b) under sterile and non-sterile conditions.

Measured levels of production of DIBOA are ten times bigger than levels of DIMBOA but in both cases less production can be obtained when seeds germinated under non-sterile conditions, which suggest an active microbial degradation of both hydroxamic acids. This fact could reduce drastically allelopathic potential of crops if biological conditions of soil are not the most suitable for stability of allelochemicals.

Degradation during bioassays

In order to know the evolution of the inoculated allelochemicals during the time of the bioassay, these are studied for high-pressure liquid chromatography (HPLC-DAD). The levels of the assayed compounds and those coming from the decomposition spontaneous are quite different seven days after the start of growth of target plants. In this case (figure 2) initial compounds DIBOA and BOA are degraded into APH and APO.

Fig. 2.- HPLC-DAD (253.1 nm) chromatogram obtained for allelochemicals and degradation products in the initial growth solution and after 7 days bioassays in sterile and non-sterile conditions.

Degradation of inoculated allelochemicals is also recorded in sterile conditions, which supposes an evidence of possible physical and chemical transformation, but is accelerated in non-sterile conditions. This fact is shown by the quantification of this allelochemicals in the growth solution after 7 days (figure 3). DIBOA is linearly degraded in almost four days, which is follows by increasing in concentration of BOA and APO with low quantities of APH. This last compound is an intermediate degradation product which quickly produces the final allelochemical APO.

(a)	(b)

Fig. 3.- Time-study in Sinapis alba L. as target plant of evolution under sterile (a) ad non-sterile (b) conditions of allelochemicals and degradation products in the growth solution.

First consequence of the evolution of these compounds during bioassays is that, in those assays which involve different times, it will imply necessarily a different concentration of each of them, which will have different bioactivity levels. In this way, two assays made in 48 h or 144 h will not reproduce the same conditions. Responses of bioassays will change with time, so it is necessary to assume new concept in bioassays: time-evolution.

Quantification analysis shows that there are other unidentified compounds involved in these degradation processes, which is demonstrated by the fact that quantities of BOA and APO are clearly lower than DIBOA ones. More isolation and structural elucidation studies are required to identify all intermediates and final compounds related to the evolution of benzoxazinones.

Assimilation and Bioactivity

In order to determine if the analyzed compounds are involved in allelopathic interactions, the responses of three target species cultivated with the same initial concentration of DIBOA are measured. After quantifying the accumulation levels into roots and stems of seedlings, it is possible to obtain different situations (figure 4). All compounds can be measured into seedlings but levels are higher when target species are those which are not considered producer of hydroxamic acids. In this way, Lolium perenne L. tends to accumulate initial compound DIBOA which suggest a slow degradation of this compound or a rapid assimilation for this seedlings. For Avena fatua L. an intermediate situation is obtained and secondary metabolite BOA reaches highest levels under non-sterile conditions, but not when microbial activity is present. Finally, the assimilation of analyzed compounds is very weak in the producer of them, Sinapis alba L. This fact suggests a prevention of re-assimilation of exudates, even the degradation compounds.

In other way, incorporated levels of APO under non-sterile conditions are slightly higher than levels measured in sterile ones. This situation can be explained by the fact that under microbiological processes, the degradation from DIBOA to BOA and this to APO is accelerated, so higher levels of the final compound is available for target plants.

Fig. 4.- Assimilation of allelochemicals and degradation compounds in three different target species in sterile and non-sterile conditions.

Bioactivity of hydroxamic acids is well known, especially with standard target species, but activity profiles must be completed with data of action over weeds or even over the same producer plants. Activity on the growth of seedlings of three target species has been measured using root and shoot lengths of plants exposed to a initial concentration of 1 mM of DIBOA. Total effects on the growth is higher under non-sterile conditions, which suggest the presence of the degradation compounds, although high concentrations of DIBOA can have also a high inhibitory activity on the roots even under sterile conditions. It is also important the high inhibition measured for the growth of seedlings of Sinapis. These data can be explained by the lack of a proper detoxification process for high levels of DIBOA or even for the presence of the degradation compounds such APO or other still non-identified compounds.

Fig. 5.- Bioactivity of allelochemicals on the root and shoot growth of three target plants.

Conclusions

Measured levels of allelochemicals liberated from donor plants by exudation processes can be ten times higher when assays are carried under non-sterile conditions. Studies of potential allelopathic of new compounds and new allelopathic plants must take in consideration the microbial action. This one may play an important role in the increasing or decreasing of the levels of the allelochemicals available for target species. There is a clear correlation between degradation of DIBOA to BOA, and this to APO mediated by APH. This process occurs in both cases of sterile or non-sterile assays, but it can be drastically accelerated in the last situation. Quantification analysis shows that there are other unidentified compounds involved in these degradation processes. The evolution of assayed compounds during bioassays forces to assume new concept in bioassays: time-evolution. Similar bioassays which involve different times can be really made with quantitative and qualitative different concentration of allelochemicals. This fact can be correlated with different bioactivity levels and the lack of reproducibility of assays; even could be the cause of non-clear results. There is a clear inhibitory effect of high concentration of DIBOA over the growth of Sinapis alba L., suspected allelopathic crop by the active liberation of hydroxamic acids, so it is possible to conclude than this plant lacks of effective detoxification method for high quantities of this allelochemical, or even than other bioactive unidentified degradation compounds are involved in this inhibition. More isolation and structural elucidation studies are required to identify all intermediates and final compounds related to the evolution of benzoxazinones.

Acknowledgements

This work was financially supported by the European Union. FATEALLCHEM. Contract No. QLRT-2000-01967. Fellowships from Universidad de Los Andes-Venezuela (A. O. B.), European Commission, and the Regional Government of Andalusia-Spain (D. M.) are also gratefully acknowledged.

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