Gandolfina Farina

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.

Effects of a triazolic fungicide on maize plant metabolism: modifications of transcript abundance in resistance-related pathways

Using a molecular approach, the effects of tetraconazole (TC), a triazolic inhibitor of P-450-based enzymes, has been investigated in maize, focusing on the resistance-related biosynthetic pathways involving one or more microsomial P-450 mono-oxigenases, putative secondary targets of the fungicide. Several observations were made. First, TC affected the phenylpropanoid-flavonoid biosynthesis, increasing the anthocyanin content and the mRNA abundance of the genes involved in the later steps of the pathway (A1 and C2), while the expression of another gene, PAL, decreased in the roots. Second, the activity of a hydroxyproline-rich glycoprotein gene involved in root cell wall structures was enhanced. Finally, maize seedlings acquired increased resistance to drought, which corresponded with modified levels of abscissic acid (ABA) and the overexpression of an ABA regulated gene. Taken together, these results suggest that TC act as a potential activator of plant defence-responses to both abiotic and biotic challenges and represents a powerful tool for elucidating the regulatory interconnections between different biosynthetic pathways.

Analysis of genotypic diversity inCercospora beticola Sacc. field isolates

Genetic variability and population structure ofCercospora beticola, the causal agent of Cercospora leaf spot in sugarbeet, from four sugarbeet-growing regions of Greece were investigated using growth rate, pathogenicity, and mini- and microsatellite DNA fingerprinting. Mycelial growth and pathogenicity were very diverse within and between groups, and no correlation was found between these features and the geographic origin of the isolates. High diversity was found by micro- and minisatellite fingerprinting, with an average gene diversity of 0.21, and no significant differences among populations. Among the 46 isolates, 45 different genotypes were identified, showing a high degree of genotype diversity. Analysis of the genetic profiles provided no evidence for regional patterns of variation (ΦFST=0.01, P=0.261) and the analysis of molecular variation (AMOVA) revealed that genetic variability was due mainly to variations within (99%) rather than between (1%) populations. Such a low level of genetic differentiation is reflected by a migration rate value Nm of 4.7. The high migration rate cannot be referred to splash dispersed conidia. To justify the absence of a regional structure in these C.beticola populations, we must suppose the existence of a long-distance means of dispersal, such as seed transmission and/or man mediated transmission.

Comparative antifungal effect and mode of action of tetraconazole on Ustilago maydis

Abstract Tetraconazole, a new, recently introduced antifungal triazole, has been assayed in parallel with a number of standard analogues on various sensitive strains of Ustilago maydis. The values of EC50 and EC90 tetraconazole concentrations, determined on strain ATCC 14826 in agar, were 0.5 × 10−6 and 3.5 × 10−5 m , respectively, in reasonable agreement with those needed to inhibit by 50% and 90%, respectively, the ergosterol biosynthesis in broth cultures. Squalene and 12 sterols have been extracted from the latter, characterized and quantified. Accumulation of 14α-methylsterols and reduction of ergosterol and other late precursors are consistent with the inhibition of 14α-demethylase caused by the title compound.

Synthesis and antifungal activity of 2-hydroxy-4,5-methylenedioxyaryl ketones as analogues of kakuol.

In a study aiming to determine the structural elements essential to the antifungal activity of kakuol, we synthesized a series of 2‐hydroxy‐4,5‐methylenedioxyaryl ketones, and we assayed their in vitro antifungal activity. The most sensitive target organisms to the action of these class of compounds were Phytophthora infestans, Phytium ultimum, Cercospora beticola, Cladosporium cucumerinum, and Rhizoctonia solani. Most of the analogs showed a remarkable in vitro activity, and some of them appeared significantly more effective than the natural product. The biological activity was mainly affected by introducing structural modification on the carbonyl moiety of the natural‐product molecule. In particular, compound 5a, bearing a CC bond conjugated to the CO group, was found active with a MIC value of 10 μg ml−1 against Cladosporium cucumerinum. The results suggest that 2‐hydroxy‐4,5‐methylenedioxyaryl ketones can be considered promising candidates in the development of new antifungal compounds.

Characterization of field-isolates and derived DMI-resistant strains of Cercospora beticola

Cercospora beticola strains with laboratory induced resistance to tetraconazole were compared with their parental WT sensitive strains to evaluate the effects of resistance on fitness and assess whether any change in the sterol biosynthetic pathway was associated to the reduced fungicide sensitivity. In vitro growth rate on agar media and pathogenicity were found to be negatively affected by resistance. The main functional sterols in C. beticola WT strains under investigation were ergosterol, brassicasterol and ergosta-7,22-dienol. Resistant strains showed the same qualitative sterol composition, ruling it out as, per se, a cause for resistance. On the basis of the sterols detected both in sensitive and resistant strains, a possible biosynthetic pathway to the three functional sterols is proposed. Tetraconazole treatment caused, in all sensitive strains, the immediate accumulation of 14alpha-methylated sterols, which, for inhibitor concentrations up to EC50 values, were, in order of abundance, 14alpha-methylergosta-8,24(28)-dien-3beta,6alpha-diol, eburicol and obtusifoliol. However the data do not support a critical role of the 14-methyl-3,6-diol derivative in the growth arrest of C. beticola. As main difference between sensitive and resistant strains, the formers were found to accumulate higher amounts of 14alpha-methylated sterols. Although the data do not allow to establish a specific mechanism for resistance, some molecular mechanisms such as target site alterations and sterol biosynthetic pathway can be ruled out as a possible cause for reduced sensitivity.

Natural Products as Sources of New Fungicides: Synthesis and Antifungal Activity of Zopfiellin Analogues

The synthesis of a series of cyclooctadiene anhydrides, analogues of the natural compound zopfiellin, was performed to assay their in vitro and in vivo antifungal activity on a set of plant pathogenic fungi. Most of the synthesized compounds possessed a broad spectrum of activity. In particular, the anhydrides 2 and 5a were very effective against the Oomycete diseases such as Phytophthora infestans and Pythium ultimum, reaching a level of activity well comparable with that of commercial fungicides in use. Preliminary in vivo evaluation of their protectant activity is also reported.

Heat sensitivity of mycelium and haustoria in bean leaves infected with bean rust

Bean leaves experimentally inoculated with bean rust were subjected to heat treatment at 50° for 20 s, at different times after inoculation, and the modifications induced by heating in the pathogen and host cells were studied by electron microscopy, at different times after the treatment. This induced no alterations either in healthy or infected leaf cells. On the contrary, it caused severe ultrastructural alterations in the pathogen. These alterations were already visible in the haustoria and hyphae at the end of the treatment, became progressively more severe with time, and led to the death of the pathogen two days after the treatment. From this time onwards the haustoria appeared shrunken, extremely electron-dense, and encased in a mass of granular material, seemingly secreted by the host cell. No encasement was ever found around haustoria apparently dead by senescence in untreated leaves.