Lorenzo Sparapano

A phytotoxic pimarane diterpene of Sphaeropsis sapinea f. sp. Cupressi, the pathogen of a canker disease of cypress

Abstract A phytotoxic metabolite, named sphaeropsidin A, is produced in vitro by Sphaeropsis sapinea f. sp. cupressi , a fungus that causes a canker disease of Italian cypress ( Cupressus sempervirens ). The spectroscopic data indicates that sphaeropsidin A is identical with a pimarane diterpenoid antibiotic previously isolated from some Aspergillus spp. When absorbed by severed twigs of three species of Cupressus and cuttings of two herbaceous plants, sphaeropsidin A at 0.1 mg ml −1 produced leaf yellowing, browning and dieback and yellowing of leaf, necrosis and epinasty, respectively. Subperidermal injection of a 0.1 mg ml −1 solution of sphaeropsidin A into young cypress trees caused longitudinal fissures and dark brown discolouration of cortical tissues. The compound showed antimicrobial activity towards 12 fungal species when assayed at a concentration range from 10 to 100 μg ml −1 . This is the first report showing that sphaeropsidin A is a non-selective fungal phytotoxin.

Two Naphthalenone Pentakides from Liquid Cultures of «Phaeoacremonium aleophilum», a Fungus Associated with Esca of Grapevine

Several phytotoxic metabolites were extracted from culture filtrates of Phaeoacremonium aleophilum, a fungus associated with the esca of grapevine and related diseases. Two of these metabolites were identified by chemical and spectroscopic methods as scytalone (23.9 mg l-1) and isosclerone (2.4 mg l-1), two naphthalenone pentaketides already known as fungal metabolites. Assayed on detached leaves of grapevine cv. Italia, scytalone at 0.05 mg ml-1 caused light green to chlorotic, rounded to irregular, interveinal or marginal spots, and isosclerone at 0.1 mg ml-1 caused large, coalescent chlorotic and necrotic spots followed by distortion of the lamina and withering. This is the first report on the production in vitro of scytalone and isosclerone by P. aleophilum, and on the phytotoxic activity of these compounds.

Infection of grapevines by some fungi associated with esca. II. Interaction among Phaeoacremonium chlamydosporum, P. aleophilum and Fomitiporia punctata

Cross-inoculation experiments were designed to study the interaction among the three fungi, Phaeoacremonium chlamydosporum (Pch), P. aleophilum (Pal) and Fomitiporia punctata (Fop) most commonly associated with esca of grapevine. The experiments were carried out in southern Italy on grapevines cv. Italia and Matilde, and the inoculations were performed in January 1999 by infecting trunks (cv. Italia) and branches (cv. Matilde) through wounds. Fop, Pch and Pal were inoculated singly or in groups of two or three in all possible combinations. Pch, Pal or both were inoculated above or below the inoculation site of Fop. From the results obtained in the first eight months of experiments planned for three years, the following conclusions can be drawn. All fungi inoculated singly spread along the woody tissue and caused brown streaking downward and upward along the trunk and branches of inoculated vines. On the basis of internal symptoms, vines cv. Italia were more susceptible than those cv. Matilde; in particular, the brown wood-streaking induced by P. aleophilum was much more severe on cv. Italia. Co-infection with the two tracheiphilous species of Phaeoacremonium caused wood gummosis and discoloration, whereas the lignicolous basidiomycete F. punctata alone caused wood deterioration and decay (white rot). Severity of internal symptoms, assessed as extent and colour of the discoloured wood, varied with the growth and interaction of the inoculated fungi. The wood discoloration caused by F. punctata was not hampered by P. chlamydosporum, but it was always limited by P. aleophilum. A similar interaction was observed in vitro with cultures of the three fungi together, which showed a marked antagonistic effect of P. aleophilum against F. punctata. Although not consistent, foliar symptoms (interveinal and marginal chlorotic areas) developed within six months on ‘Matilde’ vines co-inoculated with either species of Phaeoacremonium, or within three months after a syringe containing a liquid culture of P. chlamydosporum was inserted into current-season ‘Italia’ shoots.

Sphaeropsidins B and C, phytotoxic pimarane diterpenes from Sphaeropsis sapinea f.sp. cupressi and Diplodia mutila.

Abstract Two phytotoxic pimarane diterpenes, named sphaeropsidins B and C, were isolated from two phytopathogenic fungi causing canker diseases of Italian cypress ( Cupressus sempervirens L.): Sphaeropsis sapinea f. sp. cupressi and Diplodia mutila . S. sapinea f. sp. cupressi produced both phytotoxins, whereas D. mutila produced sphaeropsidins C and A, the latter also being reported as the main phytotoxin of S. sapinea f. s. cupressi . Characterized by chemical and spectroscopic methods, sphaeropsidin B proved to be a known fungal metabolite for the first time isolated from S. sapinea f. sp. cupressi , while sphaeropsidin C is a new tricyclic acid pimarane diterpene produced by both fungi. Assayed on severed twigs of cypress and oak, sphaeropsidins B and C caused dieback on C. macrocarpa , browning and necrosis on C. sempervirens and yellowing on C. arizonica , necrosis on Quercus cerri and Q. ilex , and browning and necrosis on Q. robur . When injected into cortical tissues of cypress and oak seedlings mentioned above, they caused on the former dark brown discolouration, browning of internal tissues and browning, and brown-blackish spots or necrotic lesions on the latter. On non-host plants like tomato and oat, sphaeropsidins B an C caused necrosis on cuttings and brown discolouration or stewing on the stem. In the antimicrobial assay, both compounds showed an inhibitory effect on mycelial growth of seven test fungi.

Three-Year Observation of Grapevines Cross-Inoculated with Esca-Associated Fungi

Cross-inoculation experiments were carried out in southern Italy (Apulia) on grapevines cv. Italia and Matilde in order to study interactions among the white rot fungus Fomitiporia punctata (Fop) and two tracheiphilous hyphomycetes, Phaeoacremonium aleophilum (Pal) and Phaeomoniella chlamydospora (Pch), commonly associated with esca and brown wood-streaking of grapevine. In January 1999 the three fungi were inoculated through wounds on the trunks and spurs of cv. Italia, and branches and spurs of cv. Matilde. Pch, Pal and Fop were inoculated singly or in groups of two or three in all possible combinations. From the results so far obtained, the following conclusions can be drawn: 1. all three fungi are pathogenic; 2. inoculation of the above fungi on standing grapevines produces symptoms on trunks, shoots, leaves and berries that are reminiscent of those shown by esca-affected vines in the vineyard. In particular, symptoms in the wood (brown streaking, gummosis or “black goo”, and wood decay or white rot), and on the leaves (various forms of chlorosis, reddening and necrosis, distortion of the lamina) and berries (black measles) were reproduced; 3. severity of internal symptoms, assessed as length of brown streaks and intensity of colour change of the infected wood, varied with the growth and interactions between the inoculated fungi; 4. the wood streaking which developed after inoculation with Pal or Pch on the spurs extended to the supporting shoot or branch, and even to the stem; 5. a non-synergistic, competitive association of the two hyphomycetes, and an inhibition of Fop growth by Pal was observed in planta. A similar marked antagonistic effect of Pal against Fop was previously shown to occur when examining interactions between the three fungi in vitro; 6. the wood discolouration caused by Fop was not hampered by Pch, but it is always limited by Pal; 7. on the basis of internal symptoms, grapevines cv. Italia were more susceptible to the esca fungi than grapevines cv. Matilde.

Infection of Grapevines by Some Fungi Associated with Esca. I. "Fomitiporia punctata" as a Wood-Rot Inducer

Inoculation experiments with three strains of Fomitiporia punctata on grapevine cv. Sangiovese and on grafted ‘Italia’ rootstocks were carried out in southern Italy in 1992-1993. Inoculations were performed on fresh wounds made on the spurs, branches and trunks of vines showing no symptoms of esca. The fungus developed in the discoloured wood around the inoculation site, and caused white rot within two years. No symptoms were induced on foliage or fruit of the infected vines, nor was there any significant difference in virulence of the strains of F. punctata. After 2 years, re-isolation of F. punctata from the diseased woody tissues was successful, whereas no other species of fungi suspected to act as a “precursor” of wood decay were isolated. In 1999, further experiments were carried out with one strain of F. punctata on standing vines cv. Italia and Matilde free of any sign of wood deterioration. The development of internal symptoms was recorded monthly. The results indicated that the cv. Matilde was less susceptible than the cv. Italia. The first signs of spongy wood decay appeared 6 months after inoculation on both cultivars. F. punctata was re-isolated from the infected vines, whereas no species of Phaeoacremonium or other wood-decaying fungi were isolated from either inoculated or non-inoculated vines. These findings suggest that F. punctata behaves as a primary pathogen, being able to cause wood deterioration and spongy decay both on adult and young grapevines in a relatively short time, without the prior or concurrent action of other fungi.

Sphaeropsidone and episphaeropsidone, phytotoxic dimedone methylethers produced by Sphaeropsis sapinea f. sp. Cupressi grown in liquid culture

Abstract Two phytotoxic dimedone methyl ethers, named sphaeropsidone and episphaeropsidone, were isolated from Sphaeropsis sapinea f. sp. cupressi , a phytopathogenic fungus causing a canker disease of Italian cypress ( Cupressus sempervirens L.). The same fungus produced the sphaeropsidins A, B and C, which are three phytotoxins recently chemically characterized as pimarane diterpenes. Sphaeropsidone and episphaeropsidone were characterized, using essentially spectroscopic methods, as two new phytotoxic disubstituted 7-oxabicyclo[4.1.0]hept-3-en-2-ones, which are epimers at C-5. Assayed on severed twigs of cypress, sphaeropsidone caused browning and necrosis on Cupressus macrocarpa , no symptoms on C. sempervirens and chlorosis on C. arizonica . Episphaeropsidone caused necrosis on C. macrocarpa , browning and necrosis on C. sempervirens and necrosis on C. arizonica . On the non-host plant tomato, both phytotoxins caused wilting. In a microbial assay, both compounds showed an inhibitory effect on the growth of five fungal species tested. The growth of Verticillium dahliae was enhanced by both dimedone methyl ethers.

Occurrence of fusicoccin in plant tissues infected by Fusicoccum amygdali del

Abstract Green peaches were artificially inoculated with Fusicoccum amygdali Del. and the infected tissues extracted with chloroform. To isolate the phytotoxic substance, extracts were purified on chromatographic columns. About 5 mg pure toxin were obtained from 1 kg infected peach pulp. A similar procedure yielded about 10 mg toxin per kg of tissues from naturally infected almond shoots with cankers of F. amygdali. The physical and biological properties of the purified toxin were found to be identical to those of authentic fusicoccin. The results of this work show that fusicoccin is a vivotoxin sensu Dimond & Waggoner.

New phytotoxic butenolides produced bySeiridium cardinale, the pathogen of cypress canker disease

Two new butenolides, seiridin andiso-seiridin, were isolated from culture filtrates ofSeiridium cardinale, the pathogen of cypress canker, a destructive disease ofCupressus and relatedConiferae These metabolites were characterized as 3-methyl-4-(2-hydroxyheptyl)-2(5H)-furanone and its 4-(3-hydroxyheptyl) isomer, respectively. Chlorotic, and necrotic symptoms were produced on leaves of either host or non-host test plants by absorption of 0.3 mg/ml solutions of either compound. These also showed antibacterial activity.

Phytotoxic extracellular polysaccharide fractions from Cryphonectria parasitica (Murr.) Barr strains

Abstract Two hypovirulent and one virulent strains of Cryphonectria parasitica , the causal pathogen of chestnut blight, produced a large amount of pullulan and a minor polysaccharide fraction containing galactose, mannose and, in one case, also rhamnose. The minor fractions of all strains elicited higher phytotoxicity on chestnut ( Castanea sativa L.) leaves, twigs and seedlings or on tomato cuttings ( Lycopersicon esculentum L.) than that given both by pullulan and native extracellular polysaccharide fractions. One of the components of the minor fraction was identified to be a galactan whose structure, on the basis of chemical and spectroscopic methods consisted of the repeat unit: [→6)-β- d -Gal f -(1→5)-β- d -Gal f -(1→] n . This is the first report on the production of polysaccharides by C. parasitica and their phytotoxic activity.