Dihydrowyerone derivatives as components of the furanoacetylenic phytoalexin response of tissues of Vicia faba

Changes in concentrations of 7 wyerone derivatives in bean tissues undergoing resistant reactions to Botrytis cinerea or B. fabae and in cotyledons in response to mercuric chloride have been examined using high performance liquid chromatography. The proportion of derivatives occurring in their saturated (dihydro) forms varied between cotyledon, leaf and pod tissues and with time after inoculation. Unsaturated derivatives were always present in greater concentrations than their dihydro analogues.     

Biosynthesis of the phytoalexin wyerone in Vicia faba

In contrast to earlier results [1-¹⁴C] acetate, [2-¹⁴C] malonate and [n 9,10-³H] oleate show significant incorporations into wyerone and related Vicia faba phytoalexins, following infection by Botrytis cinerea.     

The effect of pH and composition of test solutions on the inhibitory activity of wyerone acid towards germination of fungal spores

Wyerone acid at 100μg/ml prevented germination of conidia of Botrytis cinerea Fr. in aqueous extracts of leaves of Vicia faba L., but it was inactive in water and some synthetic nutrient solutions though antifungal activity appeared when the pH of these media was adjusted to 4.0 or 4.5. In pollen diffusates (V.faba or Nicotiana glutinosd) wyerone acid had little activity even at pH 4.0. In malt extract at pH 4.5 wyerone acid was active against several other plant pathogenic fungi but not against B. cinerea. It was concluded that both pH and unknown components of natural media affected the fungitoxicity of wyerone acid.     

Changes in wyerone acid concentrations in leaves of Vicia faba after infection by Botrytis cinerea or B.fabae

Wyerone acid was produced by leaves of Vicia faba in response to infection by both Botrytis cinerea and B.fabae. Host cell death caused by either fungus appeared to be the trigger for rapid wyerone acid synthesis, although the phytoalexin was not confined to brown cells. At B. cinerea inoculation sites wyerone acid concentration increased rapidly, at the time of fungal invasion of the epidermis, to levels greater than that completely inhibitory to mycelial growth. Wyerone acid is therefore probably the primary cause of the inhibition of B. cinerea within infected tissue. The partial blackening of B.fabae inoculation sites and surrounding peripheral tissues was accompanied by an increase in wyerone acid. There followed a striking decrease as tissues became completely blackened and invaded by B.fabae. B.fabae appeared to metabolize wyerone acid and prevent its accumulation in invaded tissues. Mycelial growth of B. fabae was less sensitive to wyerone acid than was B. cinerea. The differing abilities of B.fabae and B. cinerea to spread from lesions after both have induced wyerone acid production probably depend on both their differing sensitivities to the phytoalexin and their abilities to metabolize it to less toxic products.     

Wyerone Acid Phytoalexin Synthesis and Peroxidase Activity as Markers for Resistance of Broad Beans to Chocolate Spot Disease

Infection of broad bean (Vicia faba L.) leaves with Botrytis fabae (Sard.) resulted in wyerone acid phytoalexin accumulation and increase in peroxidase activities in resistant and susceptible broad bean cultivars. Rapid wyerone acid accumulation was observed in leaves of resistant cultivars that reached levels greater than twofold of the susceptible cultivars. Following infection of broad bean cultivars, either resistant or susceptible to B. fabae, wyerone acid synthesis in resistant cultivars peaked at 5 days then declined, whereas in susceptible cultivars synthesis gradually decreased. While the ethanol‐extract of wyerone acid significantly reduced B. fabae spore germination with increased concentrations, the phytoalexin had no effect on in vitro B. fabae germ‐tube growth. Peroxidase activity in infected leaves of resistant was 10 times higher than the susceptible cultivars and eight times higher in uninfected leaves of the resistant than the susceptible cultivars. Peroxidase activity in infected leaves was greater than twofold in resistant cultivars and less than twofold in susceptible cultivars, when compared with uninfected leaves. The ratio of peroxidase activity in infected to uninfected leaves increased over time in susceptible cultivars but remained unchanged in resistant cultivars. Peroxidase activity in uninfected and infected leaves and wyerone acid biosynthesis in infected broad bean plants were successfully used to ascertain the resistance and susceptibility of four broad bean cultivars to B. fabae. Using wyerone acid synthesis and peroxidase activity as preliminary markers for resistance of broad bean to chocolate spot disease, caused by B. fabae, is suggested in this study.     

Furanoacetylene and isoflavonoid phytoalexins in Lens culinaris

The ability of two Lens species to synthesise the furnaoacetylenes wyerone and wyerone epoxide, as well as the pterocarpan variabilin, has been demonstrated. This links Lens more close to Vicia than to the remaining genera of the tribe Vicieae.     

Fungal modification of pterocarpan phytoalexins from Melilotus alba and Trifolium pratense

Medicarpin (3-hydroxy-9-methoxypterocarpan), an isoflavonoid phytoalexin characteristic of sweetclover (Melilotus alba), is metabolised by three fungi, Botrytis cinerea, Colletotrichum lindemuthianum (race β) and C. coffeanum (isolates CCA and CCP) to afford products with decreased antifungal activity. Peterocarpan phytoalexins from red clover (Trifolium pratense) are similarly modified. A fourth organism, Helminthosporium carbonum apparently lacks this ability. Fungal-mediated tranformation principally involves hydroxylation, methylation and demethylation although ring fission has also been noted.     

Chemical markers in Veronica sect. Hebe. III

In a continued chemosystematic investigation of the water-soluble compounds in Veronica sect. Hebe, we have investigated two more of the species formerly classified as Parahebe. Both species contained mannitol in considerable amounts and in addition some glucosides of iridoid acids. Veronica cheesemanii was characterised by aucubin and its esters: 2′-O-benzoylaucubin and an aucubin diester named cheesemanioside. The main iridoid compounds in Veronica hookeriana were catalpol and its ester verminoside, but this species also contained the sugar ester methyl 1-O-benzoyl-3-α-glucuronosylglycerol and a caffeoyl phenylethanoid glycoside (CPG) named parahebeoside, a 2′-O-β-xylopyranosyl derivative of the known plantamajoside. The results show that the studied species of the former genus Parahebe are very different with regard to their chemical content. This is in agreement with the DNA sequence data and implies the genus was polyphyletic as previously circumscribed.     

Phenolic acids in the genus Lycopodium

The dihydric phenolics from the leaves of 170 Ecuadorian Lycopodium specimens representing 49 species and varieties have been examined as semiquinones by EPR spectroscopy. An ester of dihydrocaffeic acid was observed in 137 extractors of specimens all confined to the subgenus Urostachya. The ester was not present in the subgenera Lycopodium and Lycopodiella, where the dominant phenolic acid was chlorogenic acid. The concentrations of the “Urostachyaester” were found to increase with increasing altitude. Hydroquinone and protochatechuic acid have been observed in a few specimens.     

Biosynthetic relationship between tetrahydroanthracene and anthraquinone in Aloe saponaria

The incorporation of Me¹⁴COONa into aloesaponol I, laccaic acid D methyl ester and aloesaponarin I was demonstrated. The biosynthetic relation between aloesaponol I and aloesaponarin I was established, but incorporation of aloesaponol I into laccaic acid D methyl ester, or vice versa was not demonstrated and this result was confirmed by an investigation using labelled laccaic acid D methyl (¹⁴CH₃) ester. It was possible to show that aloesaponol I and laccaic acid D methyl ester were biosynthesized in parallel in Aloe saponaria.     

Properties of gallic acid-induced extracellular laccase of Botrytis cinerea

The properties of the extracellular laccase produced by Botrytis cinerea induced by garlic acid are compared with the laccase produced by Botrytis in the presence of grape juice. The extra- and intracellular laccases are compared and found to be very similar.     

Two novel stilbene-2-carboxylic acid phytoalexins from Cajanus cajan

Three phytoalexins were isolated from leaves of pigeon pea which had been challenged with Botrytis cinerea. One was identified as pinostrobin chalc     

Purification and properties of laccase from Botrytis cinerea

The partial purification of an extracellular laccase from Botrytis cinerea is described. Specificity of the enzyme, its K_m for a number of substrates and sensitivity to some inhibitors are described. The enzyme is a typical laccase but has an exceptionally low pI and great stability to acid pH. On gel electrophoresis two isoenzymes could be detected.     

Iridoid and other constituents of Canthium subcordatum

The main constituent of the stem bark of Canthium subcordatum was found to be shanzhiside methyl ester, an iridoid recently described for two Mussaenda species; it is accompanied by the new iridoid shanzhisin methyl ester gentiobioside. No alkaloids were detected.     

Very-long-chain iso and anteiso branched fatty acids in N-acylphosphatidylethanolamines from a natural cyanobacterial mat of Calothrix sp.

A combination of TLC, ESI-MS/MS and GC-MS was used to identify unusual molecular species of N-acylphosphatidylethanolamines containing very-long-chain anteiso branched fatty acids (VLCFAs) from Calothrix sp. collected in Antarctica and determine their component VLCFA up to 33-methyltetratriacontanoic acid as picolinyl ester derivatives using GC-MS.     

Induction of laccase formation in Botrytis

Optimal conditions for laccase excretion by Botrytis cinerea were determined. Addition of gallic acid to the culture medium induced maximal enzyme production and excretion. Other inducers previously reported for other organisms were ineffective. The relationships between internal and external laccase formation were determined.     

Steroidal alkaloids of Marsdenia rostrata

Some of the main constituents of Marsdenia rostrata, originating from two localities, were isolated and identified. The sample from the Toonumbar State Forest in northern New South Wales contains the known alkaloid anabasine and two new steroidal ester alkaloids, rostratine and dihydrorostratine. They were identified as O-acetyl-O-nicotinoylsarcostin and O-acetyl-O-nicotinoyldihydrosarcostin. The sample from the South Coast of N.S.W. yielded the two new ester alkaloids and a number of neutral polyhydroxy pregnane aglycones, one of which has been identified as metaplexigenin, but there was no anabasine.     

Natural products as sources of new fungicides (V): Design and synthesis of acetophenone derivatives against phytopathogenic fungi in vitro and in vivo

A series of acetophenone derivatives (10a–10i, 11, 12a–12g, 13a–13g, 14a–14d and 15a–15l) were designed, synthesized and evaluated for antifungal activities in vitro and in vivo. The antifungal activities of 53 compounds were tested against several plant pathogens, and their structure–activity relationship was summarized. Compounds 10a–10f displayed better antifungal effects than two reference fungicides. Interestingly, the most potent compound 10d exhibited antifungal properties against Cytospora sp., Botrytis cinerea, Magnaporthe grisea, with IC₅₀ values of 6.0–22.6?µg/mL, especially Cytospora sp. (IC₅₀?=?6.0?µg/mL). In the in vivo antifungal assays, 10d displayed the significant protective efficacy of 55.3% to Botrytis cinerea and 73.1% to Cytospora sp. The findings indicated that 10d may act as a potential pesticide lead compound that merits further investigation.     

Degradation of wyerone,the phytoalexin of faba beans byRhizobium leguminosarum

Wyerone is the predominant phytoalexin produced byVicia faba. At a concentration of 100 µM, wyerone prolonged the lag phase before the onset of exponential growth of different strains ofRhizobium leguminosarum andBradyrhizobium japonicum. The response to wyerone was dose dependent, with a shorter lag phase occurring at lower concentrations. Wyerone was only moderately inhibitory towards the phytopathogenic bacteriumErwinia carotovora cv. atroseptica. HPLC analysis of the medium during bacterial growth indicated thatRhizobium leguminosarum was able to metabolize wyerone. The identification of the product as hydroxyester wyerol was confirmed by GC/MS analysis.