Natural Compounds for Novel Strategies of Parasitic Plant Management

Chapter 6

Natural Compounds for Novel Strategies of Parasitic Plant Management

Downloaded by TUFTS UNIV on October 18, 2016 | http://pubs.acs.org Publication Date: May 25, 2006 | doi: 10.1021/bk-2006-0927.ch006

Maurizio Vurro and Angela Boari Institute of Sciences of Food Production, CNR, Via Amendola 122/O, 70125 Bari, Italy

Parasitic plants are among the worst weed problems, being responsible for major losses to many crops. Considering that seed germination is one of the key phases for parasite development, natural compounds could play interesting roles in the management of parasitic plants. For example, fungal toxins produced by phytopathogenic fungi could be used, depending on their biological and chemical characteristics, as natural herbicides or as biomarkers to select more efficacious mycoherbicides. Alternative strategies, such as the use of natural amino acids to cause physiological disorders of germinating seeds, the selection of more virulent fungi producing toxins, and the use of metabolites isolated from host root exudates for soil application, to stimulate "suicidal germination" of seeds and reduce the seed bank, are also possible.

The Problem Species of the genus Orobanche, commonly called broomrapes, are among the worst parasitic weed species of the world, being responsible for severe losses to vegetable, legume and sunflower crops in Europe, including the Balkans and Russia, the Middle East and North Africa (7), by interfering with water and mineral intake and by affecting photosynthate partitioning. 76

© 2006 American Chemical Society Rimando and Duke; Natural Products for Pest Management ACS Symposium Series; American Chemical Society: Washington, DC, 2006.


77 Difficulties in broomrape control are due to the large amount of seeds produced, that can remain viable for many years. They germinate only i f stimulated by host root exudates, and produce a germ tube that, i f it attaches to the host root, develops a haustorium penetrating the root and forming a tubercle. This is followed by the most damaging phase, a long underground one, during which the parasite withdraws water, nutrients and photosynthates from the host. Parasite emergence occurs only when most of the damage has already been produced. Traditional control methods have been tried on different crops, but none has proved to be effective. Orobanche spp. cannot usually be managed by persistent selective herbicides, since herbicides are not able to differentiate between the crop and the parasite, except on herbicide-tolerant transgenic crops (2). Multiple applications of low rates of crop-degraded herbicides can provide a modicum o f control and may be more useful when integrated with other methods (J). Seed eradication by solarization or soil fumigation is effective but expensive, and soil sterilization by fumigation with methyl bromide and ethylene dibromide is mostly banned due to environmental risks (4). Furthermore, mechanical control is impossible, except by removing flower stalks to reduce seed production and dispersal, because these weeds attach to crop roots. Although many promising agents have been isolated, no pathogenic organisms have been developed as mycoherbicides for Orobanche biocontrol (J, 6). Considering that the germination of seeds is a key phase of the parasitic plant life cycle, searching for natural compounds able to inhibit seed germination or to stimulate it in absence of the host, consequently reducing the seed bank, seems to be an attractive and environmentally friendly approach.

Use of natural compounds as herbicides Fungi represent an enormous source of metabolites, mostly still unexplored. Toxic metabolites produced by fungal pathogens can: belong to different chemical families (i.e.: glycosides, peptides, phenolics, terpenoids); have different ecological and environmental roles, such as to be important factors for pathogenicity or virulence; have different behaviors with respect to the host, varying from strictly host-specific to completely non-specific compounds; and act with different mechanisms, affecting several sites in the host. Fungal species belonging to the same genus are able to produce a wide variety of metabolites, e.g.: Alternaria, Claviceps or Fusarium species, for which more than one hundred of secondary metabolites have been isolated. A

Rimando and Duke; Natural Products for Pest Management ACS Symposium Series; American Chemical Society: Washington, DC, 2006.


78 further source of variability is that toxins belonging to the same structural group can be produced by different microorganisms belonging to many different genera. This is the case, for example, of cytochalasins, produced by more than 30 different fungal species (7); or trichothecenes, a group including more than 100 different compounds, produced by different genera, such as Fusarium (more than 25 different trichothecenes), Myrothecium (producing roridins and verrucarins), and Trichoderma (trichodermins) (8)\ or destruxins, metabolites known for their herbicidal and insecticidal properties, produced in tens of different forms (9). Phytotoxins from fungal pathogens of crops have received considerable attention mainly in the understanding of disease development and in setting up strategies for disease control, but much less attention has been given to the secondary metabolites produced by weed pathogens. Usually bioactive compounds produced by plant pathogenic fungi, including those attacking weeds, are considered risky compounds, because they have been intensively studied mainly in relation to the negative effects to human and animal health when these toxins accumulate in agricultural commodities and are absorbed through nourishment. Besides a few families of metabolites, such as ochratoxins, aflatoxins, trichothecenes, zearalenols, fumonisins and alkaloids of Claviceps, which have proved to be responsible for severe human and animal poisonings, there are many other metabolites that are not so dangerous, and some of them have been proposed to be used as natural herbicides (10, 11). With regard to infesting broomrapes, Zonno and Vurro (12) have recently shown that some toxins produced by fungi of the genus Fusarium were able to inhibit germination of O. ramosa seeds, and proposed their practical use for parasitic plant management. A n assay on stimulated germinating seeds was used. Briefly, O. ramosa seeds are sterilized in sodium hypochlorite. Seeds are then rinsed with sterile tap water and conditioned on wet glass microfibre filters in Petri dishes, at 26° C in the dark for 3 weeks. Filters are then cut in small pieces, each containing around 100 seeds. The pieces are placed on another filter moistened with 2 ml of the assay solution, containing the toxin and GR24 (a synthetic stimulant) and kept at 25° C in the dark. After 4 days, the percentage of seed germination is determined and compared with that of the control, prepared with the same procedure as the treatments but without the toxin. Eighteen toxins were assayed, and seven of them (diacetoxyscirpenol, HT-2 toxin, neosolaniol, T-2 toxin, fusarenon X , deoxynivalenol, nivalenol) caused 100% inhibition of germination when assayed at 10" M . A l l the other toxins were active at different levels when assayed at 10" M , ranging between 63 % of germination (moniliformin) to almost inactive (around 92% of germination) in case of enniatin. The strongest toxins at 10" M , T-2, 5

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HT-2, neosolaniol and diacetoxyscirpenol, caused an almost complete inhibition of seed germination (12). Some of those toxins are powerful mammalian mycotoxins, and the risk of introducing mammalian toxic compounds into the environment should be carefully ascertained. A l l four most active compounds belong to the trichothecene A group (Figure 1), a well known group of mammalian mycotoxins (13, 14)\ the other three most active compounds belong to the trichothecene B group (Figure 2).

Figure 1. Chemical structure of T-2 toxin, belonging to the trichothecene A group.

Figure 2. Chemical structure of deoxynivalenol, belonging to the thricothecene B group.

Thus, the common chemical structure of those compounds seems to play an important role in blocking the germination of stimulated seeds. The trichothecenes are a family of tetracyclic sesquiterpenoid substances produced by several species of Fusarium. More than 100 compounds are known. They cause a wide variety of biological effects owing to the diversity of chemical structures within the group. They all are potent inhibitors of protein synthesis in eucaryotic cells (15). The results are in agreement with a previous study, using 14 fungal toxins on germination of seeds of Striga hermonthica, a hemiparasitic weed infesting cereals (16). In fact, T-2 and deoxynivalenol were also the most active, at very low concentrations against Striga. Enniatin and beuvericin were slightly more active against Striga, whereas nivalenol was much stronger against Orobanche. The activities of fosaric and dehydrofiisaric acid, as well as of their respective methyl esters, were much stronger against Striga than against Orobanche. More recently, many fungi were isolated from diseased O. ramosa plants during extensive field surveys carried out in Southern Italy, and some of them proved to be promising potential mycoherbicides for biological control of



80 broomrapes (6). Fifty-three isolates tested for virulence were also grown in vitro both on liquid and solid media with the main aim to find new metabolites having the ability to inhibit the induced germination of O. ramosa seeds (77). Among them, one strain of Myrothecium verrucaria and one of Fusarium compactum were able to produce some metabolites very active in inhibiting the germination of O. ramosa seeds, when grown in liquid culture. Eight metabolites were isolated from M. verrucaria culture extracts. Seven o f them, identified by spectroscopic methods, proved to be macrocyclic trichothecenes, namely verrucarins A , B , M and L acetate, roridin A , isotrichoverrin B and trichoverrol B (Figure 3). The main metabolite was identified as verrucarin E (Figure 4), a disubstituted pyrrole not belonging to the trichothecene group. The main metabolite produced by F. campactum was identified as neosoloaniol monoacetate (Figure 4), a further well known trichothecene (18). Verrucarins A , B , M and L acetate belong to a subgroup of macrocyclic trichothecenes having a differently functionalized lactone ring located between C-4 and C-15. This macrocycle was substantially different and open, respectively, in roridin A , isotrichoverrin B and trichoverrol B , which belong to two other subgroups of the macrocyclic trichothecene family (19, 20, 21). Assayed on O. ramosa seeds at 10" M , all the metabolites caused the total inhibition o f the stimulated germination, except verrucarin E , not belonging to the trichothecene group, which proved to be inactive. A t 10* M all the trichothecenes were still highly active, causing total inhibition of seed germination, except isotrichoverrin B , which was slightly less toxic. A t a concentration 10 times lower, most of the metabolites were still active, except verrucarin M , which proved to be almost inactive, besides isotrichoverrin B and verrucarin E . Particularly noteworthy is the activity, at this concentration, of roridin A and neosolaniol monoacetate, both able to cause 100% inhibition of seed germination (18). Although the evaluation of the in vitro activity of hazardous metabolites appears to be important, and could also represent a preliminary assessment of the risk due to the introduction of mycoherbicides into the environment, the "real" fate of those metabolites when applied in the environment should be carefully ascertained, both to prevent contamination that would have a significant impact on the environment and consequently a negative impact on public perceptions, but also to prevent discarding useful compounds or the producer microorganisms because of concerns about their use. For example, neither intact macrocyclic trichothecenes nor toxic metabolites in plant tissues after treatment o f kudzu (Pueraria montana) with the proposed mycoherbicide M. verrucaria could be detected (22). Considering the efficacy of some toxins at very low concentrations, the possibility of using fungal toxins as natural herbicides to inhibit germination of 4

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parasitic plant seeds seems to be not so remote. Many toxins are not selective, being able to cause the same toxic effects both on host as well as on non-host plants. For this reason, the toxicity to crop plants has to be ascertained, even i f the application is at very low concentration and their quick degradation after inhibition of seed germination could avoid toxic effects. Fungal culture extracts could be an interesting source of new compounds acting as natural and original germination inhibitors.

Isotrichoverrin B, 6

Trichoverrol B. 7

Roridin A, 5

Figure 3. Chemical structure of macrocyclic trichothecenes.

Neosolaniol monoacetate

Figure 4. Chemical structure of verrucarin E and neosolaniol monoacetate.

From a practical point of view, considering that most of the crop plants parasitized by broomrape are irrigated, toxins could be introduced by drip irrigation systems, in very low amounts near the host roots. This could prevent the germination of the seeds only where needed, preventing attachment of


82 haustorium to the host roots. This should minimize environmental risks, reducing the amount of toxins to be applied and avoiding toxin dispersal. Furthermore, being natural products, those metabolites should be easily bioinactivated soon after their application, reducing risks of drifting and dispersal. In any case, it is of utmost importance to evaluate the fate of the metabolites, including the assessment of the stability and their movement into the soil, in order to consider the real risk of their introduction in the environment.


Use as biomarkers A different potential use of toxic metabolites produced by weed pathogens is as "biomarkers". If toxins proved to be virulence factors, meaning there is a positive correlation between toxin production and aggressiveness of the candidate mycoherbicide, more virulent strains of the pathogens could be selected simply by testing the production in vitro of toxic metabolites and choosing the highest toxin producers. In this regard S3 strains, isolated from diseased O. ramosa plants during extensive field surveys carried out in Southern Italy, were grown in both in liquid and solid media (17). The ability of the extracts to inhibit seed germination was evaluated, and the production in vitro of fusaric and dehydrofusaric acids (FA and D F A , respectively) by Fusarium strains was estimated. The possible correlation of toxin content and toxicity of extracts with virulence of strains was ascertained. Almost all the strains that proved to be highly virulent were also able to produce liquid cultures with a very high efficacy in the O. ramosa seed germination assay. Only two of the tested strains (one of F. camptoceras and one of F. oxysporum) were highly virulent without any production of bioactive metabolites from either liquid or solid cultures. Conversely, only one fungus, a strain of M. verrucaria appeared to be a very interesting source of toxic compounds, although it proved to be only weakly virulent. Some other strains were neither virulent nor producers of bioactive metabolites, and it could be they were saprophytes occasionally isolated from Orobanche tissues. In that study, no correlation was found between the content of F A and D F A and the toxicity of the extracts of liquid cultures, with the exception of a strain of Fusarium sp. whose liquid extract had both high contents of D F A (204 mg/1) and F A (76 mg/1), as well as high toxicity. In fact, the extract of a strain of F. oxysporum, which had one of the highest contents of F A (more than 166 mg/1), was inactive on seed germination, whereas that of another strain of F. oxysporum, with one of the lowest F A contents (29 mg/1), caused about 66% inhibition of seed germination. The same was true for D F A . In fact, the extract of one strain of F. oxysporum, having one of the highest contents (more than 195


83 mg/1), caused 31% inhibition, around the same caused by a strain of F. proliferatum, one of the weakest D F A producers (around 17 mg/1) (77). Moreover, among F. oxysporum strains, comparison of the production of metabolites with the virulence of the strains did not show a positive correlation. Two strains that were among the best F A and D F A producers caused no disease. Conversely, another strain, that produced quite low amounts of toxins, proved to be one of the strongest pathogens of O. ramosa (77).


Use of amino acids This approach is modeled on the mechanism of action of the so called 'Trenching disease", a disease of tobacco caused by saprophytic bacteria growing on the roots, overproducing isoleucine (23). The end products of the branched chain amino acid pathway (valine, leucine, and isoleucine) allosterically regulate the activity of acetolactate synthase (ALS). The isoleucine overproduction inhibited the activity of A L S in the tobacco, shutting down synthesis of valine and leucine, which disrupted essential protein metabolism. Several modern chemical herbicides also inhibit single biosynthetic enzymes in plants (including A L S ) , rendering treated plants incapable of producing a metabolite essential for plant growth. The idea, in this case, is to supply high or unbalanced amounts of essential amino acids to cause disorders in the physiological processes that occur during the germination of seeds, inhibiting the process of germination or that of germ tube elongation. Some preliminary experiments carried out using the biological assay described above showed the ability of some amino acids to inhibit germination of stimulated parasitic seeds. In particular, in the case of O. ramosa, the most interesting results were obtained supplying proline, arginine, histidine and methionine, whereas for Striga hermonthica, a parasitic weed attacking mainly cereals, encouraging preliminary results were obtained using leucine, threonine and tyrosine (24). In particular, methionine was able to inhibit germination of O. ramosa seeds i f applied within three days after the addition of the stimulant to the seeds, whereas its inhibitory activity was overcome by seed washing. Furthermore, both the length and the shape of the germinated seeds treated with different concentrations of methionine seemed to be influenced by the concentration of the amino acid supplied (24). Amino acids are not normally toxic to humans or to the environment, and are rapidly metabolized by soil microorganisms, so that they could be "safe" method of parasitic weed management. Studies, carried out by the authors in collaboration with Prof. David C. Sands, Montana State University, Bozeman, U S A , are in progress to evaluate the possible use of this interesting approach to


84 inhibit the germination of parasitic weed seeds and to understand physiological mechanisms of action.

the


Obtaining more virulent mycoherbicides due to toxin overexpression The development of pathogens with enhanced biocontrol activity by selection or by the introduction of genes responsible for toxin or other metabolite biosynthesis seems a reasonable possibility. Several genes in the biosynthetic pathways of fungal toxins have already been identified and cloned, such as many of those encoding the biosynthetic pathway of trichothecenes (25 and ref. therein cited) and toxins produced by several species of different fungal genera such as Fusarium and Trichoderma. The protein NEP1 is a potent phytotoxin isolated for the first time from culture filtrates of a strain of Fusarium oxysporum pathogenic for Erythroxylum coca, and later found as a product of many other strains of the same species (26). The toxin, inducing necrosis in leaves, is responsible for the natural virulence of the species producing it, and the gene nepl, encoding that proteinaceous toxin, has been identified. Recently, Amsellem et al. (27) have transferred the nepl gene to a weak pathogenic strain of Colletotrichum coccodes, a potential mycoherbicide for the biological control of Abutilon theophrasti. Compared to the wild type, the obtained transgenic strain was more virulent, faster in causing the disease, and with a reduced requirement for the dew period. With regard to the possibility of obtaining mycoherbicides overproducing amino acids, variants of F. oxysporum f. sp. cannabis resistant to valine analogs were obtained (23). These resistant variants excreted 10-55 times more valine than their wild type parent, and proved to be more virulent to Cannabis sativa than the wild type parent. The wild type strain resulted in 25% control of the target plant, while the valine mutants increased control to 70-90%. In addition, the development of wilt disease was more rapid in the plants infested with the valine overproduces. Thus, overproduction of an essential amino acid could provide a highly effective means of enhancing the virulence of a biocontrol agent (23).

Use of seed germination stimulants Considering that the germination of seeds of parasitic plants depends on chemicals exuded from the roots of the host plant, an alternative approach for the management of parasitic weeds could be the reduction of the amount of parasite



85 seeds in the soil by "suicidal" germination. This can be obtained by the application of a germination stimulant to the soil, in absence of the host. The parasite seeds will germinate but, in absence of the host, they will die. The chemical nature of such germination stimulants is well known in the case of Striga spp. (28), whereas less information is available for other parasitic plants. The Striga spp. germination stimulants reported, isolated from both host and non-host plants, belong to different chemical groups, most of them being sesquiterpenes. Strigol is the most representative compound of the largest group, named strigolactones (28) and was the first germination stimulant characterized (29). Two stimulants of O. minor seeds, alectrol and orobanchol, have been isolated from the root exudates of Trifolium pratense, the host plant (30). More recently, six sunflower sesquiterpene lactones were tested as O. cumana germination stimulants, and two of them significantly increased seed germination. The effect of these two compounds is species-specific, showing no germination stimulant activity on other Orobanche spp. tested (37). Strigol has been the starting point for the development of the synthetic G R family, among which GR24 is the commonest compound used for lab studies. More recently, another synthetic stimulant, named Nijmegen 1, has been developed. This compound can be more easily synthesized compared to other compounds and, because of its biological activity in germinating parasitic weed seeds and its easier preparation, it has been proposed for a practical use in the field (32). Considering that large amounts of stimulants should be available for their practical application at a field level, limiting factors for their marketing and use are: long, difficult and highly costly synthesis processes, in the case of synthetic compounds; and difficulties to obtain large amounts and high cost of purification, in the case of natural compounds. Alternative strategies to obtain low-cost natural stimulants could be, for example, the use of metabolites stimulating the suicidal germination produced by fungi (33), suitable for mass production, or the purification from residual nutrient solutions obtained by N F T (Nutritive Film Technique) tomato crops (24).

Conclusions The use of natural compounds could offer more environmentally friendly and more efficacious approaches for the management of parasitic weeds. Natural metabolites could be used directly, as natural herbicides, and indirectly, as biomarkers. Alternative strategies, such us the use of metabolites isolated from host root exudates and from fungal cultures for soil application to stimulate the "suicidal germination" of seeds and reduce the seed bank, or of natural


86 amino acids to cause physiological disorders, is also possible. scientific efforts are needed to develop these strategies.

Concerted

References 1.

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Natural Compounds for Novel Strategies of Parasitic Plant Management

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