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Structure-dependent activities of wheat allelochemicals on target and non-target organisms

Jona Ines Fritz1, Sylvia Bluemel2, Per Kudsk3, Francisco A. Macías4, Lars M. Hansen3 and Wieslaw Oleszek5

1Universität für Bodenkultur Wien, Department IFA-Tulln, Konrad Lorenz Str. 20, A-3430 Tulln, Austria. www.ifa-tulln.ac at E mail: ines.fritz@boku.ac.at
2
Austrian Agency for Health and Food Safety. Institute of Plant Health, Spargelfeldstr. 191, A-1226 Wien, Austria.
Email sbluemel@ages.at
3
Danish Institute of Agricultural Sciences, Research Centre Flakkebjerg, Dept. Of Crop Protection and Pests, DK-4200 Slagelse, Denmark. Email per.kudsk@agrsci.dk and larsm.hansen@agrsci.dk
4
Departamento de Química Orgánica, Facultad de Ciencias, Universidad de Cádiz, 11510 Puerto Real (Cádiz) Spain. Email famacias@uca.es
5
Department of Agricultural Microbiology and Department of Biochemistry, Institute of Soil Science and Plant Cultivation, ul. Czartoryskich 8, 24-100 Pulawy, Poland. Email sm@iung.pulawy.pl

Abstract

Wheat allelochemicals have been identified, isolated and tested for degradation and activity against several target organisms over the years. A number of researchers have investigated the fate and activity of benzoxazinones, especially BOA and DIMBOA. In this review the results relating to biological activity of a comprehensive research programme are summarised. The effect of the wheat allelochemicals and a selection of their most probable degradation metabolites on 26 different organisms in 28 biotests have been analysed by six research institutes during the course of a three year research project called FateAllChem, supported by the European Union (http://www.fateallchem.dk). The parent benzoxazinones BOA and MBOA showed almost no inhibition effects to the test organisms, their effect on plants, fungi and weeds could be assumed to be comparably weak. It was demonstrated that some of the metabolites with the phenoxazinone skeleton did affect the test organisms at much lower concentrations than the parent wheat benzoxazinones.

Media summary

Typical pests and other organisms widely used for ecotoxicity testing were exposed to wheat allelochemicals and to some of their degradation metabolites to study allelopathic interactions.

Key words

wheat allelochemicals, benzoxazinones, metabolites, weeds, ecotoxicity

Introduction

When searching for and identifying secondary metabolites of plants, one of the first questions we have to ask is for which purpose it may be produced (Niemeyer 1988). Potential target organisms are often identified very quickly. In many cases other plants, which are competitors for water, nutrients and room, may be suppressed by such allelochemicals (Korte 1992, Huang 2003). In addition, other pests such as insects or even micro-organisms may be repelled or inhibited by plant allelochemicals with or without synergistic effects of other secondary metabolites (Loayza-Muro 2000). To make the science of allelopathy even more complicated, the allelochemicals may not only have negative effects on other organisms, they can also attract for example beneficial insects which may, carry pollen from one flower to the next.

After some time of testing the function and the targets of a specific plant allelochemical are most likely well known. This is the case with wheat benzoxazinones, a class of chemicals produced in the roots of almost all wheat and maize varieties (Baumeler 2000, von Rad 2001) and known to suppress the growth of other plants, especially weeds. It is already known at which growth stage most benzoxazinones are synthesized, that they are stored and transported as glucoside and become activated as aglycon on demand, for example after wounding (Stochmal 2005, Villagrasa 2005). It was the goal of a large European research project (short name: FateAllChem) to investigate a number of aspects related to the utilisation of the wheat allelochemicals for biological weed control. The FateAllChem project focused on the target effects, non-target effects, fate, isolation and synthesis of the compounds, development of analytical methods, and influence of growth stages, varieties and growth conditions on benzoxazinone content in plants (Fomsgaard 2005a). The benzoxazinones should be exploited like pesticides after either isolation from natural sources or chemical synthesis. Alternatively growth of efficiently producing plants as intermediate vegetation is possible with the allelopathic effect initiated by shredding and digging in of the plants a short time before seeding of the next crop. In the course of this project it was necessary to test the efficiency of the wheat allelochemicals as well as their stability. Because it was already expected that the wheat benzoxazinones are easily biodegradable in soil, a set of known and newly identified metabolites was added to the list of substances for activity testing. Finally, any potential risk to the environment (non-target and beneficial organisms) should be analysed, with the same stringency as for conventional pesticides. The development of reliable analytical methods for quantification of benzoxazinones and their metabolites, the growth (production) of wheat plants under different climate conditions and the chemical synthesis of degradation metabolites for test purposes were part of the research project but are not part of this paper.

The purpose of this paper is to summarize the biotest data generated by several project partners in the course of the European project FateAllChem. The biotests that were included were tests on target organisms: weeds, insects and fungi, tests on non-target organisms: soil and water organisms together with bioassays on a number of plant species. The results are discussed in the light of other data from the literature.

Material and Methods

The substances used in the project FateAllChem are listed in table 1 with short and full chemical names. BOA, MBOA and DIMBOA are the parent substances; the other seven are their degradation metabolites. A degradation pathway was first suggested by Zikmundova et al. (2002) and extended by several authors (Etzerodt et al. 2005, Fomsgaard et al. 2004 and 2005b, Gents et al. 2005, Glenn et al. 2003, Understrup 2005). Schemes for BOA and MBOA degradation are given in figures 1 and 2.

Table 1. Wheat allelochemicals (short names in bold) and their degradation metabolites used for different sets of biotests. Not all substances were tested against all organisms.

Short

Chemical name

Type

No. biotests

DIMBOA

2,4-Dihydroxy-7-methoxy-2H-1,4-benzoxazin-3-one

parent substance

11

MBOA

6-Methoxy-benzoxazolin-2(3H)-one

parent substance

28

AMPO

2-Amino-7-methoxyphenoxazin-3-one

MBOA-metabolite

16

AAMPO

2-Acetamido-7-methoxyphenoxazin-3-one

MBOA-metabolite

19

BOA

Benzoxazolin-2(3H)-one

parent substance

27

AP

2-Aminophenol

BOA-metabolite

7

HPAA

2-Acetamidophenol

BOA-metabolite

10

HPMA

N-(2-Hydroxyphenyl)malonamic acid

BOA-metabolite

7

APO

2-Aminophenoxazin-3-one

BOA-metabolite

20

AAPO

2-Acetamidophenoxazin-3-one

BOA-metabolite

20

Selected results of the biotests are summarised in table 2. The data for the plant bioassays were published by Macías et al. (2005a; b), the data for the weed bioassays were published by Kudsk et al.(2005), the data for the aphid tests were published by Hansen (2005), the data for the bioassays on soil organisms were published by Coja et al. (2005a, b) and Idinger et al. (2005), the data for the fungi bioassays are published by Martyniuk et al. (2005) and the data for the aquatic bioassays are published by Fritz and Braun (2005). The footnote of table 2 lists the type of bioassays performed.

Figure 1. Degradation scheme for the wheat allelochemical MBOA according to Zikmundova et al. (2002) and Fomsgaard et al. (2004). The substances used for biotests are circled.

Figure 2. Degradation scheme for the wheat allelochemical BOA according to Zikmundova et al. (2002) and Fomsgaard et al. (2004). The substances used for biotests are circled.

Results

The original figures and units were just as diverse as the applied test systems. In many cases the calculation of an EC50 value was not possible as the necessary prerequisites had not been achieved for reasons of limited possibilities to repeat tests or for limitations in the test setup.

Because the solubility of most of the benzoxazinone metabolites was very low, it was not possible to apply concentrations high enough to cause inhibition effects in many biotests. Therefore what is given in table 2 is in about half of the cases more a rough extrapolation than a precise calculation. The available data did not allow following the strict rules given by standards (OECD 2003) or the recommendations provided by ecotoxicity textbook authors (Fendt 1998).

Uni- and multivariate statistical tests have been conducted to rank the effects of the substances and to find similarities and differences in the patterns of the biotest responses for the benoxazinones and their metabolites. The bar chart in figure 3 gives an overview of substance effects. High mean values, like those obtained from BOA and MBOA, represent low effects against the majority of the organisms. Low mean values, like those obtained from APO, AMPO and AAPO, stand for significant inhibition effects to the majority of the test organisms. Table 3 summarises the effect of the allelochemicals and their metabolites to groups of test organisms. The values should be interpreted the same way as described above for figure 3. Finally, a multivariate evaluation was done by applying a cluster analysis. Reliable results were achieved only if the data set was reduced to have at least not more than one missing value per row and per column in the data matrix. Some special evaluations were possible; the most general of these is given in figure 4, based on the results obtained from the aquatic biotests. The lower the connection distance between substances (as drawn in the graph), the more similar were their effects to the different organisms, and it could be assumed that their modes of action were also more similar.

Table 2. Summary of all EC50 and ED90 values (marked with *) obtained by the biotests. All values are given in µmol/l for aquatic tests and µmol/kg for terrestrial tests. In cases where no EC50 could be calculated the results are given as ‘> highest tested concentration’.

 

DIMBOA

MBOA

AMPO

AAMPO

BOA

AP

HPAA

HPMA

APO

AAPO

Daph

> 9.5

> 12

> 41

> 7

> 15

3.5

> 13

> 10

1.50

10

Chlo

> 47

> 61

8.8

> 7

> 74

< 92

> 13

> 51

1.34

27

Sel

> 47

> 61

0.22

> 7

> 74

3.0

> 13

> 51

0.73

1.7

Lumis

44

1.30

68

> 7

> 15

> 92

> 13

> 51

9.4

11.4

Fus

 

1640

 

> 100

3380

> 20

> 20

> 100

> 20

> 20

Ceph

 

812

 

> 100

1.40

13

> 20

> 100

2.7

18

Gaeu

 

467

 

> 100

852

7.3

> 20

> 100

3.7

9

Fols

9.0

12

2

18

16

 

13

 

2.4

10

Poec

> 9

> 600

619

528

741

 

660

 

47

39

Fol rep

9.5

6.1

0.4

18

15

 

13

 

2.4

> 8

Aphid

 

0.15

               

Echcg*

4730

4250

   

5380

         

Setvi*

2560

1690

   

2300

         

Poaan*

 

1250

   

2200

         

Apesv*

1160

1400

   

1650

         

Abuth*

 

2550

   

2590

         

Triin*

 

2050

   

3160

         

Amare*

1350

1880

   

1220

         

Coleop

 

41800

   

8740

     

91

83

Lep root

 

1249

> 1000

> 1000

1600

     

38

308

Lep sh

 

1080

> 1000

> 1000

5390

     

70

215

Lactuc

 

293000

> 1000

> 1000

399000

     

380

> 1000

Lyc root

 

444000

> 1000

> 1000

9180

     

153

> 1000

Lyc sh

 

147000

> 1000

> 1000

524

     

11

110

Allium

 

137000

> 1000

> 1000

406

     

73

306

Lolium

 

385

> 1000

> 1000

2130

     

24

501

Avena

 

2540

> 1000

> 1000

275

     

24

523

Echi

 

43

137

> 1000

> 1000

     

319

> 1000

* Results of the weed tests were provided as ED90 values.
Abbreviations used: Daph = acute immobilisation of Daphnia magna. Sel = acute growth inhibition (biomass) of Pseudokirchneriella subcapitata. Chlo = acute growth inhibition (biomass) of Chlorella sp. Lumis = acute inhibition of light emission of Vibrio fischeri. Fus = radial growth inhibition of Fusarium culmorum. Ceph = radial growth inhibition of Cephalosporium gramineum. Gaeu = radial growth inhibition of Gaeumannomyces graminis. Fols = mortality of Folsomia candida. Poec = mortality of Poecilus cupreus. Fol rep = reproduction of Folsomia candida. Aphid = mortality and reproduction of Sitobion avenae. Echcg = growth inhibition of Echinochloa crus-galli. Setvi = growth inhibition of Setaria viridis. Poaan = growth inhibition of Poa annua. Apesv = growth inhibition of Apera spica-venti. Abuth = growth inhibition of Abutilon theophrastis. Triin = growth inhibition of Tripleurospernum inodorum. Amare = growth inhibition of Amaranthus retroflexus. Coleop = Coleoptile elongation. Lep root = growth inhibition of Lepitium sativum roots. Lep sh = growth inhibition of Lepitium sativum shoots. Lactuc = growth inhibition of Lactuca sativa shoots. Lyc root = growth inhibition of Lycopersicon esculentum roots. Lyc sh = growth inhibition of Lycopersicon esculentum shoots. Allium = growth inhibition of Allium cepa roots. Lolium = growth inhibition of Lolium rigidum roots. Avena = growth inhibition of Avena fatua roots. Echi = growth inhibition of Echinochloa crus-galli.

Figure 3. Bar chart with mean values calculated from all EC50 and ED90 of the wheat allelochemicals. Standard deviations are shown, those for BOA and MBOA exceed the graph scale. The higher the bar, the lower was the toxicity or effect of the substance.

Figure 4. Dendrogram (cluster analysis) of a reduced set of biotest results (10 allelochemicals, 4 aquatic biotests) calculated with SPSS 10.0.7 based on distance matrix obtained by average linkage method (abbreviations used as in table 1).

Table 3. Biotest data summarised for groups of test types. The lower the values the higher was the effect of the 10 tested allelochemicals against the organisms.

Biotest type

No. organisms

No. substances

No. tests

Range of EC50 or ED90 [µmol/l] or [µmol/kg]

Aquatic biotests

4

10

40

0.2 - 12

Agar - fungi

3

8

24

1.4 - 3000

Terrestrial - insects

4

8 (1)

25

0.2 - 600

Terrestrial - weeds

7

2-3

18

1000 - 5000

Terrestrial - plants

10

6 (4)

58

11 - 440000

Discussion

The parent benzoxazinones BOA and MBOA showed almost no inhibition effects to the test organisms, their effect to plants, fungi and weed could be assumed to be comparably weak. The reason why almost no inhibitions could be observed may be either a low potential for interaction with living organisms because of the chemical structure (Lo Piparo et al. 2005) or by the comparably rapid biodegradation of the substances in soil and aqueous environments. If results from the 30-minute luminescent bacteria test are compared with those from the 72-hour algae test, it could be seen that there is an effect of MBOA in the first, short time test but almost no effect in the longer lasting test. No regular dose-response relation was obtained in the aquatic biotests for BOA (Fritz and Braun 2005). It is assumed that more stable and more effective metabolites may have been formed during the runtime of the tests, maybe APO and AAPO (see figure 2) but never in a reproducible way.

Significantly higher toxicity than for the benzoxazinones was observed if the degradation metabolites were used in the biotests. Especially the phenoxazinones (APO and AAPO) showed the highest inhibition effects, affecting all test organisms (figure 3). In addition, the multivariate statistical evaluation showed similar relations for those substances in the aquatic biotests (figure 4). One of the aquatic tests was designed in a way to be able to take samples for analytical purposes. In this case the formation of APO from 2-aminophenol within some minutes during the preparation of the test could be detected (Fritz and Braun 2005), maybe it was a spontaneous oxidation as described by Zikmundova et al. (2002). But the effect pattern for 2-aminophenol differed very much from those of APO and AAPO (figure 4). Therefore it is unclear whether the degradation pathway in the aquatic tests followed the scheme as given in figure 2, whether the path to HPAA or HPMA will be followed preferably or whether other, currently unidentified metabolites may have been formed even during the short preparation time or runtime of the biotest. Degradation studies in soil have shown that the degradation pathway in soil changes when the initital concentration of the parent compound is changed (Gents et al. 2005).

Acetyl-APO (AAPO) is a degradation product of APO, and acetyl-AMPO (AAMPO) is a degradation product of AMPO in soil. All four of those compounds were comparably stable, allowing direct assumptions about their toxicity, even in longer lasting biotests. In this study both of the acetylated compounds showed a lower inhibition effect than their respective non-acetylated predecessors. As this relation was observed for all types of organisms (bacteria, fungi, insects and plants) a more general rule about the effects of degradation products is suggested: the acetylation step could be done for biological detoxification.

For further testing the formation of metabolites and degradation products out of allelochemicals put into a biotest should be considered and followed more closely by appropriate analytical methods as it was done for this review.

When interpreting the summarised results for organism groups (table 3) one may conclude that the benzoxazinones and their metabolites had affected the aquatic organisms and the insects the most. The potential primary targets for benzoxazinones may be those groups or organisms closely related to them. It is open for discussion why benzoxazinones are produced and released by the plants but the degradation products have generally much higher effects. One of the next research goals should be to clarify the role of soil micro-organisms in activating wheat allelochemicals.

The data generated in the course of the project FateAllChem are the first known where effect studies on target organisms as well as a risk assessment to non-target organisms have been performed with parent compounds as well as with degradation products.

Acknowledgements

Special thanks go to all partners in the project FateAllChem and I would like to dedicate this paper to Inge Fomsgaard.

The research described in this paper was performed as part of the project FateAllChem, ‘Fate and Toxicity of Allelochemicals (natural plant toxins) in Relation to Environment and Consumer’. The project was carried out with financial support from the Commission of the European Community under the Work program Quality of Life, contract No. QLK5-CT-2001-01967.

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