Chemical and Biological Characterization of Toxins Produced by

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Chemical and Biological Characterization of Toxins Produced by Weed Pathogenic Fungi as Potential Natural Herbicides Antonio Evidente Dipartimento di Scienze del Suolo, della Pianta e dell'Ambiente, Università di Napoli Federico II, Via Universitá 100, 80055 Portici, Italy

Several phytopathogenic fungi have been proposed for biocontrol of weeds, but few have been used. Recent studies were conducted to develop integrated biocontrol strategies using a combination of low-dose mycoherbicides and the phytotoxins they produce. This chapter describes the chemical and the biological characterization of phytotoxins produced by some Ascochyta, Pyrenophora and Phoma species proposed for the biocontrol of weeds (fat hen, annual grasses and sowthistle) infesting important agrarian crops, such as maize, sugar beet and cereals. Furthermore, the importance of phytotoxins in developing integrated weed control strategies is discussed.

Introduction Weeds are one of the most serious problems for agriculture and the environment. Infesting plants obstruct the normal flow of surface waters, alter the natural habitat, and cause heavy losses to crop production and the pasture industry. The control of weeds has been achieved with synthetic herbicides. They are usually used in large amounts in agriculture, causing problems to 62

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63 human and animal health, and environmental pollution. Therefore, many efforts have been made to biologically control weeds, using their natural antagonists such as insect s and/or microorganisms. Biological agents offer the advantage of being compatible with the environment, often with high specificity, and also represent a long term solution in the control of weeds with resistance to chemical herbicides. Recently, research was begun to isolate phytotoxins produced by some pathogenic fungi for weeds and on their use as natural herbicides. The goal of such studies is to use natural substances, their derivatives or synthetic analogues with increased efficacy and specificity to avoid microorganism release, and the possibility that they became pathogens of other organisms (7-5). Furthermore, the phytotoxins could be used indirectly as biomarkers for the improvement of fungi mycoherbicide properties. Phytotoxins are defined as microbial metabolites that are harmful to plants at low concentrations. These toxins often have low molecular weights and belong to several classes of natural products. The virulence of plant pathogens may depend on its capability to synthesize one or more toxins. Only few phytotoxins are known as host-specific toxins, and they are often phytotoxic to a broad range of plant species. In some cases, studies on their mode of action and their role as a "vivo-toxin" have also been carried out (4, 6-9).

Ascochyta caulina phytotoxins Ascochyta caulina (P. Karst.) v.d. A a and v. Kest. is a promising mycoherbicide for the control o f Chenopodium album (70), also known as common lambsquarters or fat hen, a world-wide diffused weed of arable crops such as sugar beet and maize throughout the world (77).

Isolation and Chemical Characterization of the Phytotoxins The phytotoxins produced in vitro by a standard strain of Ascochyta caulina (AC-1, kindly supplied by Dr. Piet Scheepens, Plant Research International, Wageningeen, The Netherlands) were purified by combination of cationicexchange, gel filtration and reversed phase T L C . Three toxins were isolated and characterized using spectroscopic and chemical methods. The main toxin, named ascaulitoxin (235 mg/Li, 1, Figure 1) appeared to be a JV^-p-Dglucopyranoside of the 2,4,7-triamino-5-hydroxyoctandioic acid (72). The other two toxins, which are non-proteinogenic amino acids like 1, were characterized as fraA«-4-amino-D-proline and ascaulitoxin aglycone (150 and 150 mg/L, 2 and 3, respectively, Figure 1) (13-14).

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COOH

HOOC—CH—CH — C H - C H -CH -~CH—COOH I I I | NH N H OH NH 7

2

2

2

2

2

3

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Figure 1. Ascochyta caulina phytotoxins. Any practical application of ascaulitoxin as a natural herbicide appears to be seriously limited by the very low amounts of this metabolite present in the fungal culture filtrates. Therefore, efforts must be first directed towards devising a convenient and simple method for total synthesis. The first attemps to realize this goal in now in progress. The preliminary determination of the relative configuration of the four chiral centers (C-2, C-4, C-5 and C-7) of ascaulitoxin (1) appears to be relevant to establish its absolute stereochemistry and to realize its stereoselective synthesis. The determination of the relative stereochemistry of ascaulitoxin molecule (1) was performed by N M R configurational analysis, based on the evaluation of ANC the homo (V _H) and hetero ( V C - H * J C - H ) nuclear coupling constants, in combination with R O E S Y (Rotating Overhauser Effect Spectroscopy) responses. This represents a success in the application of this method to the nitrogensubstituted chains. The conformation, and the relative stereochemistry of the four chiral centres of ascaulitoxin was determined as in 4 (Figure 2) ( 1 5 ) . 3

H

4

5

6

[/raws-L-Enantiomer (2S,4R)] [/raw-D-Enantiomer (2R,4S)

Figure 2. The relative stereochemistry of ascaulitoxin (4) and 4-aminoproline enantiomers (5 and 6).

The absolute stereochemistry of 2 was achieved using chemical and spectroscopic methods. In fact, the toxin was converted to its N^iV^-ditosyl methyl ester derivative, which showed different spectroscopic and chromatographic behaviour when compared to the analogue derivative of the cis-

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65 4-amino-L-proline. The latter was synthesized from fra>w-4-hydroxy-L-proline, the natural amino acid, according to reaction sequence already reported {16). Therefore, the toxin is the D-enantiomer (6, Figure 2) of the trans-4aminoproline, because an opposite optical rotation, with respect to that reported for the /nmy-4-amino-L-proline (5, Figure 2), was recorded (14).

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Biological Characterization of Phytotoxins When assayed at 30 (xg/droplet on punctured leaves of host and non-host weed and cultivated plants, ascaulitoxin showed a different degree of phytotoxicity on tested species (Table 1). Ascaulitoxin caused the appearance of necrotic spots surrounded by chlorosis on the leaves of fat hen. Clear necrosis also appeared both on weeds (common sowthistle, annual fleabane noogoora burr, tree of heaven) and on cultivated plants (pea and cucumber). Clear, but in reduced size, were necrosis on tomato and redroot pigweed (72). More intresting results were observed in testing the fra/w-4-amino-D-proline due to its selective phytotoxicity against dicot plants. Assayed at 1 mg/^1 on punctured leaves on the same plants (Table 1), the toxin (2) had a drastic effect on the host plant, causing the rapid appearance of a large necrosis area surrounding the puncture point. The toxin was active at a concentration 5 times lower, but caused less necrosis. On other dicot leaves, the phytotoxicity varied from large necrotic areas (poppy, annual mercury, cucumber, wild cucumber), through medium ones (tree of heaven, tomato, common sowthistle) to small necrotic spots (black nightshade). A n interesting aspect was the lack of toxicity of 2 when assayed on several monocots, both cultivated (wheat, oat, barley) as well as wild (canarygrass, slender foxtail, wild oat). When tested at up to 10" M on cut young fat hen seedlings, the toxin caused wide necrosis and dryness of cotyledons, while no effects could be seen on stems (13). 5

Toxins 1 and 2 lack antifungal and antibiotic activities when assayed (up to 50-100) |ng/disk on Geothchum candidum, Pseudomonas syringae subsp. syringae and E. coli, and they have no zootoxicity when tested at up to 40 fig/ml sea solution on brine shrimp larvae (Anemia salina L.) (12-13).

Greenhouse and Field Experiments A n alternative method based on ion exchange chromatography was developed to overcome difficulties and high cost to obtain each A. caulina toxin in a pure form. In fact, after mild acidification (formic acid) the culture filtrates were loaded on a cation-exchange chromatographic (Dowex 50, H form) column and water eluted to collect saccharose, which was used as carbon source in the culture medium, and other non basic compounds. Successive elution with +

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ammonium hydroxide allowed collection of only a mixture of the toxic metabolites (350 mg/1) (77). The efficacy of the toxin mixture was compared with that of culture filtrates alone or in combination with the fungus. In glasshouse experiments it showed the same toxicity as culture filtrates. Greenhouse experiments on young fat-hen plants also showed that the use of the toxin mixture solution (1 mg/ml) in conjunction with spores of A. caulina (at 10 /ml) improved the biocontrol efficacy of this fungus by more than 30%. Furthermore, the simultaneous application of toxins or fungal spores with low doses of herbicides, such as metribuzin and rimsulfuron, at 1/5 of labeled rate, gave better results than single-agent treatments (19). Formulations containing different combinations of A. caulina conidia, its phytotoxins and low dose herbicides have been tested. A significant improvement in the efficacy of the fungus was achieved in glasshouse trials with 6

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67 an aqueous formulation containing P V A , a polyvinyl alcohol, Psyllium, a plantderived polysaccharide, Sylgard 309, a surfactant, and nutrients and conidia (5 x 10 /ml). Field trials have investigated the performance of A. caulina conidia applied at different development stages of C. album, either as a single treatment or combined with sub-lethal doses of herbicides or with the fungal phytotoxins. With the available formulations, favourable weather conditions are needed to obtain infection in the field (18). 6

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Phytotoxin Analytical Methods and their Application as Biomarkers A specific method for the qualitative and quantitative analyses of A. caulina toxins (1, 2 and 3) by HP A C - P A D (High Performance Anionic Chromatography-Pulsed Amperometric Detector) on a Dionex chromatograph was developed (14). The toxins were well separated on an AminoPac PA1 analytical column. The mobile phase consisted of 23 m M sodium hydroxide and 7 m M sodium tetraborate solution. The developed method proved to be relatively simple, rapid, sensitive and reproducible. This method, applied to the culture filtrates of different strains of A. caulina for the qualitative and quantitative analyses of toxins produced in vitro required the preliminary separation of saccharose by cation-exchange chromatography, because its peak overlapped that of ascaulitoxin. The three toxins were well separated and a recovery rate close to 100% was observed for all toxins (14). This method was applied to quantitatively estimate the toxin content in the culture filtrates of 10 A. caulina strains (AC-1, AC-2, AC-14, Ac-16, AC-18, AC-21, AC-22, AC-31, AC-35, Ac-46) from different origins. The data obtained showed that the strains synthesized all three toxins: two of them (AC-2 and AC-14) were higher producers than the standard strain. Only a strain (AC46) was higher ascaulitoxin producer, with respect to the other toxins. It is also important to note to the wide range in toxin content between the tested culture filtrates of 74-298, 6-115, and 42-565 mg/1, for 1, 2 and 3, respectively, suggesting that there is no relation between the strain origin and their ability of to produce different toxins (14).

Pyrenophora

and Phoma Phytotoxins

Among the possible causes of loss in cereal yields, annual grass weeds, are one of the most important; this is due to their similarity in morphology, physiology and ecology to the crop species. A feature common to annual grasses is their prodigious seed production, which is responsible for their reproduction and diffusion, even if their viability is low.

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68 Methods to reduce the input of seed can improve long-term control of infesting grasses. One strategy is be the application of seed-borne pathogens as bioherbicides. Pathogens damaging the seed in the inflorescence or preventing flowering have also potential for biological control. Pyrenophora semeniperda (Brittlebank & Adam) Shoemaker, a seed-borne pathogen that causes several symptoms in infected plants, has been proposed as a bioherbicide (19). The fungus infects seeds and leaves of over 35 genera of grasses including all winter cereals and six dicotyledonous genera (20). In brome grass (Bromus spp.) and wheat (Triticum aestivum L.) it has been reported to cause the death of seed primordia and the subsequent abortion of seed (21). It is well known that other species of Pyrenophora produce toxins, some of which are potentially dangerous (22-23). Therefore, it seemed interesting to investigate the production of toxins by this species of Pyrenophora.

Isolation and Chemical Characterization of P. semeniperda Phytotoxins Preliminary in vitro experiments showed that the fungus in liquid culture produces low-molecular lipophilic phytototoxins. The isolation, chemical and biological characterisation of these phytotoxins are in progress. When grown on wheat kernels P. semeniperda does not produce such phytotoxins but rather cytochalasins, a large group of fungal metabolites having different biological activities (4, 24-25). This fungus synthesizes three new cytochalasins, named cytochalasins Z l , Z2 and Z3 (11, 11 and 1.6 mg/kg, 7, 8 and 10, respectively, Figure 3) together with four already known cytochalasins; cytochalasin B , produced in very large amounts (3.64 g/kg), cytochalasins F and T, and deoxaphomin (13,15, 9 and 16, respectively, Figure 3) (26). Cytochalasin Z l represents the first example of a 24-oxa[14]cytochalasan bearing a /?-hydroxybenzyl residue at C-3 of the perhydroisoindolyl-l-one moiety (24-25). Furthermore, cytochalasins Z l and Z3, structurally related to cytochalasins T and B , respectively, are the first two 24-oxa[14]cytochalasans with a lactonic macrocyclic ring deoxygenated at C-20 (26). Cytochalasin Z2, closely related to the well known cytochalasin T, is a 24-oxa-[14]cytochalasan showing for the first time, among all the [11], [13] and [14]cytochalasans, a hydroxymethyl group on the C-6 (26).

Biological characterization of cytochalasins produced by P. semeniperda In seedling assays (Figure 4) on wheat and on tomato, the most active compounds were cytochalasin B (CB), its 21,22-dihydroderivative (diHCB), cytochalasins F and Z3 (CF and CZ3), and deoxaphomin ( D E O X A ) . They were

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all able to reduce the root length by about 50%. In the puncture assay, only deoxaphomin, at the used concentration, showed the ability to produce small necrotic lesions. N o effects were produced in the immersion assay by any of the tested cytochalasins (26). This, together with the observed phytotoxicity of liquid culture filtrates, could mean that other metabolites are responsible for phytotoxic effects caused by the pathogen.

14 R,=H, R =OH 2

15 R,=OH, R =H 2

16

Figure 3. Pyrenophora and Phoma phytotoxins.

Further New Cytochalasins from Phoma exigua var. heteromorpha Phoma exigua var. heteromorpha (Schulzer et Saacc.) Noordless et Boerema, the causal agent of a severe foliar disease of Oleander (Nerium olender L.) observed in 1985 in a nursery near Bari (Italy), proved to be a good producer of cytochalasins. From liquid and solid cultures of this fungus, several known (cytochalasins A , B and F and deoxaphomin) and new (ascochalasin, 7O-acetylcytochalasin B , Cytochalasin T, U , V and W) cytochalasins were isolated, and their biological activities and S A R were investigated (4, 25). Because the potential applications and the availability of large amount of solid cultures of P. exigua var. heteromopha, new cytochalasins were isolated from

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this fungus. In fact, from its wheat kernel culture, three new cytochalasins were found and named Z4, Z5, and Z6 (1.43, 0.28 and 0.08 mg/kg, 11, 12 and 14, respectively, Figure 3) besides Z2 and Z3, and some of the above cited cytochalasins (27).

C F

C Z2

cytochalasins

Figure 4. Effect of cytochalasins on root elongation of tomato and wheat seedlings.

Cytochalasins Z4 and Z5 (11 and 12) are structurally related to cytochalasin B (13), the main toxin produced by both P. exigua var. heteromoprpha and P. semeniperda, as well as other important toxigenic fungi (24-25). Together with Z l (7), Z5 (12) represents a further example o f a 24oxa[14]cytochalasan bearing a /?-hydroxybenzyl residue at the C-3 o f the perhydroisoindolyl-l-one moiety whereas, in the same group, Z4 (11) is the first example o f a compound bearing an o-hydroxybenzyl residue attached to C-3 (24-25). Furthermore, Z6 (14) is the first 24-oxa[14]cytochaIasan showing at the same time the epoxy group located between C-6 and C-7 o f the perhydroisondolyl-l-one residue, the deoxygenation o f C-20, and the hydroxylation of C-19 as already observed for Z3 (27). Biological Characterization of Cytochalasins Produced by P. exigua var. heteromorpha 4

In the tomato seedling assay, at 10" M , only Z6 proved to be slightly active causing 30% inhibition of root elongation, whereas Z4 and Z5 were inactive. When assayed at the same concentration on brine shrimp, only Z5 caused a low mortality o f larvae (21%), whereas Z4 and Z6 were both inactive (27). The results of structure-activity relationship studies (25-26) suggest the important role o f the hydroxy group at C-7 in conferring the biological activity. This structural feature is present in 11 and 12 (Z4 and Z5) but not in 14 (Z6).

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71 Therefore, the lack of phytotoxicity in 11 and 12 is probably due to the orthoox para-hydroxy substitution of the benzyl residue attached at C-3 suggesting that the presence of an unsubstituted phenyl residue attached at C-10 is important for biological activity (27). Cytochalasins have been considered as potential mycotoxins (24-25). If high level of toxins were really produced in vivo, this could, in practice, make it hazardous to use P. semeniperda as a biological control agent against grass weeds. Hence, studies are in progress both to quantify the presence of such toxins in naturally infected seeds, as well as to estimate their stability and impact in the environment.

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Ascochyta sonchi phytotoxins Ascochyta sonchi (Sacc.) Grove is a natural pathogen isolated from necrotic leaves of sowthistle (Sonchus arvensis L.), a herbaceous weed occurring through the temperate regions of the world. It is being evaluated as a possible biocontrol agent (Berestetski, personal communication). Phytopathogenic fungi belonging to the genus Ascochyta are responsible for several diseases that cause necrotic lesions on leaves and stems (28). Some Ascochyta spp. have also been proposed as mycoherbicides for the biological control of noxious weeds (4, 14). Therefore, the production of toxic metabolites by A. sonchi, a promising pathogen, is of interest in view of their use as natural herbicides to be employed in addition to the use of the pathogen or an alternative to it. Isolation and chemical chracterization of A. sonchi phytotoxins A T L C analysis of the culture filtrates showed the presence of basic metabolites, probably bearing NH -groups, and amino acids and/or peptides. Purification of the crude culture filtrate by cationic exchange chromatography resulted in two fractions. The residue obtained by lyophilization of the basic eluate yielded a material containing the main metabolites in almost pure form. The residue, a yellowish highly water-soluble powder having phytotoxic activity, was further purified by a medium pressure silica gel column. The fraction containing the less polar metabolites, which proved to be phytotoxic on leaves, was further purified yielding a homogenous amorphous solid metabolite (1.2 mg/1, 17, Figure 5), which was named ascosonchine. Ascosonchine, whose structure was determined using essentially spectrocopic methods (IR, I D and 2D H and C - N M R and MS), was characterised as (Z)-2-hydroxy-3-(4-pyridyl)-2propenoic acid. The stereochemistry of the double bond present in ascosonchine was deduced by spectroscopic IR and N M R experiments. The IR absorption bands, 2

!

1 3

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72 in agreement with those reported for the Z-enol of phenylpyruvic acid derivatives, allowed assignment of a Z-configuration to the double bond. This Z configuration was further supported by the *H, C coupling constants recorded in the undecoupled C N M R spectrum of 17. In particular, the value of the vicinal H - C = C - C O O H [ c/ci-H3 3.7 Hz] is typical for a cis arrangement of the coupled nuclei in fragments C - C = C - C with similar sums of electronegative substituents. Presumably, the stability of the Z-enol form is due to the conjugation between the phenyl ring, olefinic and carboxyl groups. Therefore, ascosonchine can be formulated as (Z)-2-hydroxy-3-(4-pyridyl)-2-propenoic acid (17) (29). 13

1 3

1

13

3

=:

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!

l3

Figure 5. Structure of ascosonchine.

Biological characterization of Ascosonchine Tested with the leaf puncture assay on the host plant, 17 produced necrotic circular lesions after 2 days resembling those caused by the pathogen. The diameter of the necrotic area appeared very wide (up to 5 mm) when 15 or 3 jig/droplets (around 6 and 1.2 10" M , respectively) were applied to the leaf surface, and was still quite evident at a concentration five times lower. Assayed at 15 ^g/droplet on several weedy and cultivated plants (monocots and dicots) ascosonchine showed interesting selective properties. In fact, as shown in Table 2, it was completely ineffective on all the solanaceous species assayed (tomato, eggplant, red pepper, potato), was slightly (1-2 mm necrosis) active or almost inactive on leguminous (bean and chickpea) and cucurbitaceous (melon and zucchino) plants, but it caused severe (up to 10 mm) necrosis on many other species, such as Euphorbia, Salvia, Valerianella, or Triticum. Even i f further assessments are needed, this semi-selective toxin could have practical applications as a herbicidal compound. It is interesting to note that the toxin is still very active when used at a quite low concentration (29). In the antibiosis assay on G. candidum, ascosonchine assayed at concentrations up to 50 jig/disk proved to be completely inactive. The same negative result was observed when the toxin was tested on Pseudomonas syringae and Lactobacillus plantarum (a Gram- and a Gram+ bacteria, respectively). No effect was observed in the brine shrimp assay as tested at 3

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73 4

concentrations up to 10" M (29). Even i f father nontargeting tests of ascosonchine are required, the results from assays revealed only phytotoxic activity, whereas ascosonchine was completely ineffective on fungi, bacteria and arthropods. This could be very important from a practical point of view, and would confirm the hypothesis that more environmentally friendly and safe herbicides could be obtained by plant pathogenic fungi (4),

Table 2. Effect of ascosonchine in the leaf-puncture assay Common name

Scentific name

Family

Effect on leaves

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0

++ Amaranthaceae Alligatorweed Alternanthera philoxeroides Artichoke Compositae Cynara scolymus Bean Phaseolus vulgaris Leguminosae Convolvulaceae Convolvulus arvensis Bindweed + Cicer arietinum Leguminosae Chickpea Solanaceae Eggplant Solanum melongena + Four-o'clock Mirabilis jalap a Nyctaginaceae Poaceae ++ Foxtail millet Setaria italica +++ Valerianaceae Lamb's lettuce Valerianella locusta Melon Cucurbitaceae Cucumis melo Solanaceae Pepper Capsicum annum Solanaceae Potato Solanum tuberosum ++++ Labiatae Common sage Salvia officinalis +++ Asteraceae Sowthistle Sonchus arvensis + Spinach Chenopodiaceae Spinacia oleracea Euphorbiaceae Sun spurge ++++ Euphorbia helioscopia Solanaceae Tomato Lycopersicon esculentum Wheat +++ Triticum durum Poaceae Cucurbitaceae Zucchino Cucurbita pepo Toxicity determined with the following scale: - = no symptoms; + = necrosis with diameter around 1-2 mm; + + = necrosis 2-3 mm; + + + = necrosis 3-5 mm; ++++ = wider necrosis. Results of at least three replicates. a

Conclusions The results obtained recently on the development of weed biocontrol strategies are promising and encouranging, and further experiments are still in progress to overcome the important problems that arise during the practical application of phytotoxins in integrated crop management.

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4.

5. 6. 7. 8. 9. 10. 11.

12. 13. 14. 15. 16. 17. 18.

19.

20. 21.

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