Financial support: CSIC Nº203 and CONICYT Nº122.

Keywords: antifungic activity, interespecies variability, mycotoxins, sugar esters.

Morphological variation in type IV trichomes in Ss and Ng was studied through SEM. The differences can be related to chemical differences in the excreted sugar esters. Ng trichomes exude two fractions, one of glucose tri-esters and the other one of sucrose tetra-esters, in a 3:7 ratio. The main acid found forming these esthers, is 3-methylvalerianic acid, in consonance to those secreted by other Solanaceae. Esters from Ss are novel structures, which can also be separated into three fractions, two of arabinoxylans, and the other one of arabinose, all glycosilated with β-hydroxipalmitic acid and sterified with the C₁₂-C₁₆ acids. All five fractions have antifungic activity at μg/cm² concentrations, both against common and mycotoxigenic fungi, such as A. niger, A. flavus, P. chrysogenum y P. expansum. A. flavus does not grow in the presence of the SE of Ss and is not insensible to those from Ng, but these last are more effective in the inhibition of P. expansum, the other mycotoxigenic fungi studied. The differential antifungal activity observed gives the plant protection against a wide spectrum of fungi, resulting in a better adaptation to the environment. Both plants are common weeds, with the potential of contributing to germplams lines in the improvement programmes of crops such as L. esculentum, and their extracts can be used as natural fungicides to protect crops and plantations.

70% of all Solanaceae are pubescent, and the detailed study of the leaf ultrastructure shows the presence of hairs and glandular trichomes, structures that have a well established defensive role in the interaction of plants and their ecosystems (Goffreda et al. 1989; Juvik et al. 1994; Harborne, 2001). The chemical secretions of Type IV peduncular trichomes are composed by complex mixtures of esters of short chain fatty acids and simple sugars such as glucose and sucrose (King et al. 1990; Steffens, 2000; van der Hoeven and Steffens, 2000). The conventional improvement programmes for tomato and potato have been aimed at the selection of vegetal materials where these trichomes were present, as they give the plants natural resistance to pests (Goffreda et al. 1989; Shapiro et al. 1994). In a systematic screening to identify new native Solanaceae germplasm sources with sugar esters (SE), we have described the insecticidal properties of Ss and Ng extracts (Cesio et al. 2000). The present work is the continuation of the studies on the chemical and biological properties of their exhudates, that represent results of possible agricultural interest.

Isolation of trichomes secretions

In large quantities. The aerial parts, which contain the trichomes, are submerged 30" in acetonitrile. The solvent is removed by rotaevaporation and the residue is fractioned by CC.

Exclusively from the trichomes. 1) The aerial parts, frozen in liquid nitrogen, are mixed with ground dry ice and shaken in a vortex mixer at high speeds. The vegetal material is removed and the dry ice is allowed to sublime. The isolated trichomes are extracted with acetonitrile. 2) The contents of the heads of the trichomes is extracted with a microsyringe using a magnifying glass.

Structural elucidation. The extracts are fractionated by CC using a CHCl₃:MeOH 100:0-90:10 gradient. The SE fractions are studied by conventional procedures to establish their sugar and fatty acid contents. The pure compounds are studied by spectroscopical methods to establish their structures [NMR (400MHz); MALDI-TOF-MS; GC-EI-MS].

Antifungal activity. A quantitative bioautographic method was used (García et al. 1997).

SEMs were as gold replicas.

The peduncular trichomes of the two species are different (Figure 1). The variability is observed in their morphology, in their location in the plants and in the chemical compositions of their secretions, which present different antifungal activities.

Trichome location and morphology

Ng trichomes are only present in the inflorescences. Morphologically they have a long pluricellular (n > 5) peduncle and can break from their base. They have also globular heads that grow turgid as they fill with exudate. Ss trichomes are on both faces of the leaves, being denser in the abaxial side. They have fewer cells (n < 4) in their peduncles, do not break off at the base, and have oval multilayered heads.

Exudates chemical composition

The exudates were selectively extracted to verify their origin in the trichomes under study. It this way it could be established that the SE isolated from the trichomes and present in the epicuticular waxes were identical for each species. There is an interspecific variation in there compositions, as Ng excretes esters of sucrose and glucose in a 7:3 ratio with 3-methylvalerianic acid as main component (78%). Figure 2 shows the complexity of these fractions, which could be resolved by careful analysis of the NMR and MS spectra, and comparison with bibliographic data (Severson et al. 1994; Ohya et al. 1996). The main component, which is representative of the whole fraction, is sucrose -2,3,4 tri(3-methylvalerianate) -1'monoacetate. The other constituents of the fraction have other acids linked to the sugar. In the case of the glucose esters the main component is the trivalerianate.

The esters of Ss are novel structures, composed by arabinose and arabinoxilanes glicosilated with β-hydroxipalmitic acid, esterified with fatty acids such as lauric, miristic and palmitic. Three groups of components can be separated by CC, M1, M2 and M3 (5%, 28% y 67%). By NMR COSY, HMQC and HSQC-TOCSY bidimensional experiments, M2 could be established to be β-xylopyranosil (1-5)-α-furoarabinose esterified with palmitic acid and glycosilated with one unit of β-hydroxipalmitic acid. In the case of M3 is the non-esterified glycoside (Figure 3). The structure of M1 corresponds to a arabinose polyesterified with lauric, miristic and palmitic acids, and glycosilated with β-hydroxipalmitic acid. The molecular weight was established by MALDI, and the sugars were analysed by the conventional alditol technique.

Antifungal tests

In most cases phytopathogenic fungi have to penetrate the epicuticular wax layers before invading a plant (Kolattukudy, 1995). Due to this it is common to find antifungal compounds in the external surface of plants (Kennedy et al. 1992). The traditional agar diffusion assay to evaluate antifungal compounds is not applicable for lypophilic compounds (García et al. 1997). Such substances do not diffuse in agar, a circumstance that can result in false negatives. Prior work from our group with a modification of Rahalison's technique (Rahalison et al. 1991; Larramendi et al. 1998) has shown that Bioautography on TLC is particularly useful to assay bioactive compounds present in complex mixtures, as shown in Figure 4, with a quantitative perspective.

The results can be presented in mass/area units, with valuable information on the bioactivity of the substance(s) under study. The antifungal assays are shown in Table 1.

The fractions show differential activity to the assayed fungi, linked to the different chemical structures involved. This is particularly interesting in the case of fungi that contaminate foods and are mycotoxigenic, as A. flavus and P. Expansum (Pitt and Hocking, 1999). A. niger is inhibited by all fractions, although those from Ng are most active. P. chrysogenum is inhibited by Ng esters and by the M3 and M1 fractions of Ss. The Ng fractions are the most active. The two arabinoxilanes from Ss, M2 y M3, inhibit aflatoxin producing A. flavus. This is the first report of inhibition of A. flavus by SE. A. niger and P. expansum producer of the carcinogenic mycotoxine patulin, are inhibited by all fractions assayed. (Paster et al. 1995). To evaluate their possible commercial use, both common food preservatives and the Ng fractions were tested in conditions used for preservatives. The activity of these SEs was similar to those of propylparaben and orto phenylphenol (Table 2).

The morphological diversity of the trichomes of the two plants is also expressed in the chemical composition of their SEs and the antifungal activity of these secretions. The fungal inhibition is of the same level as that of common commercial food preservatives. In general, the compounds from Ng are more active than those from Ss. In spite of this, the only natural SEs described in the literature capable of inhibiting the growth of A. flavus are M2 and M3 from Ss. The other mycotoxigenic fungi, patulin producing P. expansum, is inhibited by all SEs tested. These results have two possible uses: the use of SEs extracts as natural antifungals in postharvest treatments, mainly in "organic farming", and as source of new germplasm with antifungic properties, that could be used to introduce these resistance characters in Solanaceae (Pragnell, 2003) crops, such as potatoes, tomatoes or aubergines.

Bachs. Horacio Pezzaroglio and Daniel Lorenzo are great fully acknowledged for taking NMR and GC-MS spectra and Bach. Gabriel Gonzalez for the technical assistance in the graphic design.

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