Gemma Assante

Screening of the genus Cercospora for secondary metabolites

Abstract Screening of 61 species of Cercospora grown on a potato-agar medium showed the presence of the phytotoxin cercosporin in 24 of them, and of dothistromin in 8. Some strains of C. beticola produce a yellow phytotoxin (CBT). The new metabolites cercosporin esters, ligustrone A, B, C, taiwapyrone, 3-methoxy-2,5,7-trihydroxy-1,4-naphthaquinone, cis -4,6-dihydroxymellein and ( − )-11-acetyldehydrocurvularin were isolated besides the known cynodontin, ( − )-dehydrocurvularin, (+ )-mellein and cis -3 S ,4 S -4-hydroxymellein.

Histological studies on the mycoparasitism of Cladosporium tenuissimum on urediniospores of Uromyces appendiculatus.

Interactions between the mycoparasite Cladosporium tenuissimum and the bean rust Uromyces appendiculatus were studied through light and electron microscopy in vitro at the host-parasite interface. Urediniospore germination decreased on contact with ungerminated C. tenuissimum conidia, possibly due to antibiosis mechanisms. C. tenuissimum grew towards the bean rust spores and coiled around their germ tubes. Penetration of the urediniospores occurred either enzymatically and/or mechanically, through appressorium or infection cushion structures, from which a thin penetrating hypha was generated. Enzyme production by the mycoparasite was suggested by the loosening of the matricial components of the spore wall, which sometimes left chitin fibrils visible. Mycoparasite hyphae grew within the host spore, emptied its content, and emerged profusely forming conidiophores and conidia. C. tenuissimum was able to grow on media containing laminarin, suggesting the ability of producing glucanases, but not when chitin was used as the sole carbon source. Conidia that had been grown on a sugar-rich medium, filtered, and extracted with organic solvents, were found to contain cladosporol and related compounds. Complete control of the bean rust disease was achieved by application of C. tenuissimum culture filtrates but not by conidial suspensions. This is the first report of parasitism by C. tenuissimum on U. appendiculatus. These investigations provide additional observations on a genus besides Melampsora and Cronartium from which this fungus has been isolated and tested to date. The possible role of environmental factors for the exploitation of this organism as a biocontrol agent is also mentioned.

Perylenequinones from cucumber seedlings infected with Cladosporium cucumerinum

Abstract The structure of cladochrome A, a perylenequinone pigment isolated from etiolated cucumber seedlings infected with fungal spores of Cladosporium cucumerinum, has been revised and established as that of a diester of 3-hydroxybutyric acid with ent-isophleichrome. Another pigment from the same source, cladochrome B, is the corresponding ester of ent-isophleichrome with 3-hydroxybutyric and benzoic acids.

Antagonism of the Two-Needle Pine Stem Rust Fungi Cronartium flaccidum and Peridermium pini by Cladosporium tenuissimum In Vitro and In Planta.

ABSTRACT Selected isolates of Cladosporium tenuissimum were tested for their ability to inhibit in vitro aeciospore germination of the two-needle pine stem rusts Cronartium flaccidum and Peridermium pini and to suppress disease development in planta. The antagonistic fungus displayed a number of disease-suppressive mechanisms. Aeciospore germination on water agar slides was reduced at 12, 18, and 24 h when a conidial suspension (1.5 x 10(7) conidia per ml) of the Cladosporium tenuissimum isolates was added. When the aeciospores were incubated in same-strength conidial suspensions for 1, 11, 21, and 31 days, viability was reduced at 20 and 4 degrees C. Light and scanning electron microscopy showed that rust spores were directly parasitized by Cladosporium tenuissimum and that the antagonist had evolved several strategies to breach the spore wall and gain access to the underlying tissues. Penetration occurred with or without appressoria. The hyperparasite exerted a mechanical force to destroy the spore structures (spinules, cell wall) by direct contact, penetrated the aeciospores and subsequently proliferated within them. However, an enzymatic action could also be involved. This was shown by the dissolution of the host cell wall that comes in contact with the mycelium of the mycoparasite, by the lack of indentation in the host wall at the contact site, and by the minimal swelling at the infecting hyphal tip. Culture filtrates of the hyperparasite inhibited germination of rust propagules. A compound purified from the filtrates was characterized by chemical and spectroscopic analysis as cladosporol, a known beta-1,3-glucan biosynthesis inhibitor. Conidia of Cladosporium tenuissimum reduced rust development on new infected pine seedlings over 2 years under greenhouse conditions. Because the fungus is an aggressive mycoparasite, produces fungicidal metabolites, and can survive and multiply in forest ecosystems without rusts, it seems a promising agent for the biological control of pine stem rusts in Europe.

The proteome of exudates from germinating Lupinus albus seeds is secreted through a selective dual-step process and contains proteins involved in plant defence

The general knowledge of defence activity during the first steps of seed germination is still largely incomplete. The present study focused on the proteins released in the exudates of germinating white lupin seeds. During the first 24 h, a release of proteins was observed. Initially (i.e. during the first 12 h), the proteins found in exudates reflected the composition of the seed, indicating a passive extrusion of pre‐formed proteins. Subsequently, when the rate of protein release was at its highest, the composition of the released proteome changed drastically. This transition occurred in a short time, indicating that more selective and regulated events, such as secretory processes, took place soon after the onset of germination. The present study considered: (a) the characterization of the proteome accumulated in the germinating medium collected after the appearance of the post‐extrusion events; (b) the biosynthetic origin and the modalities that are the basis of protein release outside the seeds; and (c) an assessment of antifungal activity of these exudates. The most represented protein in the exudate was chitinase, which was synthesized de novo. The other proteins are involved in the cellular mechanisms responding to stress events, including biotic ones. This exudate was effectively able to inhibit fungal growth. The results of the present study indicate that seed exudation is a dual‐step process that leads to the secretion of selected proteins and thus is not a result of passive leakage. The released proteome is involved in protecting the spermosphere environment and thus may act as first defence against pathogens.

Cladosporol A stimulates G1-phase arrest of the cell cycle by up-regulation of p21waf1/cip1 expression in human colon carcinoma HT-29 cells

Cladosporols, purified and characterized as secondary metabolites from Cladosporium tenuissimum, display an antifungal activity. In this study, we tested the antiproliferative properties of cladosporol A, the main isoform of this metabolite family, against human cancer cell lines. By assessing cell viability, we found that cladosporol A inhibits the growth of various human colon cancers derived cell lines (HT‐29, SW480, and CaCo‐2) in a time‐ and concentration‐dependent manner, specifically of HT‐29 cells. The reduced cell proliferation was due to a G1‐phase arrest, as assessed by fluorescence activated cell sorting analysis on synchronized HT‐29 cells, and was associated with an early and robust over‐expression of p21waf1/cip1, the well‐known cyclin‐dependent kinases inhibitor. This suggests that the drug may play a role in the control of cancer cell proliferation. Consistently, cyclin D1, cyclin E, CDK2, and CDK4 proteins were reduced and histone H1‐associated CDK2 kinase activity inhibited. In addition to p21waf1/cip1, exposure to 20 µM cladosporol A caused a simultaneous increase of pERK and pJNK, suggesting that this drug activates a circuit that integrates cell cycle regulation and the signaling pathways both involved in the inhibition of cell proliferation. Finally, we showed that the increase of p21waf1/cip1 expression was generated by a Sp1‐dependent p53‐independent stimulation of its gene transcription as mutagenesis of the Sp1 binding sites located in the p21 proximal promoter abolished induction. To our knowledge, this is the first report showing that cladosporol A inhibits colon cancer cell proliferation by modulating p21waf1/cip1 expression.

Secondary mould metabolites. Part 16. Stemphyltoxins, new reduced perylenequinone metabolites from Stemphylium botryosum var. Lactucum

The structure and stereochemistry of Stemphyltoxins I–IV (1)–(4), four epoxy derivatives of reduced perylenequinones isolated from the fungus Stemphylium botryosum var. Lactucum, have been elucidated, mainly on the basis of n.m.r. evidence. A further metabolite, stemphyperylenol (5), is a hexahydro-1,4,7,10-tetrahydroxyperylene-3,9-quinone, a structure which suggests an unusual head-to-tail biosynthetic coupling of two pentaketide units.

Mycochromone and mycoxanthone: Two new metabolites from Mycosphaerella rosigena☆

Abstract Two new metabolites, dimethyl 2-(2-ethenyl-5-hydroxy-4H-1-benzopyran-4-onyl-3)-malonate (mycochromone 1a ) and methyl 2-methoxy-8-hydroxy-9-oxo-9H-xanthene-1-carboxylate (mycoxanthone 6a ), have been isolated and identified from the mycelium of Mycosphaerella rosigena grown on potato-agar.

Asteromine, a bioactive secondary metabolite from a strain of Mycosphaerella asteroma

Abstract The structure of asteromine, a new 6,6′-binaphtho-α-pyrone metabolite, has been elucidated by means of NMR spectroscopy. In bioassays, asteromine exhibits phytotoxic effects on Cucumis sativus , a low antibacterial and antifungal activity, and is lethal to Artemia salina shrimps at 10 −4 M.

Three sesquiterpenes produced by the fungus Laurilia sulcata

Abstract The structures of sulcatine B, 5- epi -armillol and armillol, three protoilludene sesquiterpenes isolated from still liquid cultures of Laurilia sulcata , have been assigned on the basis of one- and two-dimensional (HETCOR and COLOC) 1 H and 13 C NMR studies. The relative configuration of the metabolites was deduced from NOE experiments and the magnitude of the 1 H- 1 H coupling constants.