Salvatore Moricca

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.

Heterogeneity in intergenic regions of the ribosomal repeat of the pine-blister rusts Cronartium flaccidum and Peridermium pini

Mixed aeciospore isolates ofCronartium flaccidum andPeridermium pini were obtained from single-tree infections in Britain, Italy and Greece. The 5.8s ribosomal RNA gene and flanking intergenic transcribed spacer regions ITS 1 and ITS2 were found to be highly similar betweenC. flaccidum andP. pini. Within samples heterogeneity was detected at three nucleotide loci in the ITS1 and at four loci in the ITS2 suggesting that several fungal genotypes may occur at a single infection court. The heterogeneity was confirmed by heteroduplex polymorphism analysis of mixed aeciospore products. RFLP of the ribosomal intergenic spacer region 1 (IGSI) amplified from the same templates indicated limited sequence polymorphism in some copies of this repeated locus. Both the sexual and asexual forms ofC. flaccidum show evidence of sequence polymorphism in two independent, non-coding regions of the ribosomal gene array. Variation appears to be greater in the sexual formC. flaccidum, than in the monoaecious formP. pini.

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.

Axenic culture of the aecial state of Cronartium flaccidum from Italy

Axenic cultures of Cronartium flaccidum were obtained for the first time by seeding aeciospores on modified Schenk and Hildebrandts and Harvey and Grashams media. Aeciospores came from foci at four locations in Italy. Colonies developed more readily on thickly seeded plates. High variation was observed in hyphal length and morphology, and in colony appearance, margin and morphology. The appearance and morphology of individual colonies also varied greatly over the five-month incubation period.

Analyses of genetic variation suggest that pine rusts Cronartium flaccidum and Peridermium pini belong to the same species.

Cronartium flaccidum and Peridermium pini are rust fungi occurring on two-needle hard pines. According to previous molecular and morphological analyses, they are very closely related despite differences in their life-cycles. In this study we showed that although a low level of genetic differentiation occurs among populations of both P. pini and C. flaccidum, there is no overall differentiation between the two rusts, and in this respect they resemble a single taxon. We also observed evidence for linkage disequilibrium occurring between different alleles of separate loci of Peridermium pini suggesting that its population structure would be clonal. This suggests that strains of P. pini would have originated as asexual or self-fertilizing host range mutants of C. flaccidum.

The Holomorph Apiognomonia quercina/Discula quercina as a Pathogen/Endophyte in Oak

This chapter summarises research carried out on the biology, ecology and the impact of the holomorph Apiognomonia quercina/Discula quercina in oak forests. The major life-history traits and aspects of the epidemiology (isolation, survival, reproduction, dispersal, host selectivity) and control of the oak anthracnose agent are elucidated. The role of weather patterns in disrupting the delicate interaction between the host tree and the microorganism is outlined. The evidence suggests that changes in the climate profoundly alter the plant-endophyte symbiosis, generating conflicts of interest between the partners in the interaction. When such competing interests arise, the survival and reproduction of one member of the interaction do not conform with that of the partner. The interaction becomes thus disadvantageous and harmful to one of the organisms and the symbiosis from mutualistic or neutral turns antagonistic. The fungal partner, which under normal conditions survives in quiescence, with a low biomass, resumes growth but now switches from a latent, asymptomatic occupier of inner oak tissues to an aggressive coloniser that sporulates profusely over the tree surface. The importance of investigating the functioning and the role of the plant-endophyte symbiosis in perennial host trees in natural forests is stressed.

Cladosporol A, a new peroxisome proliferator-activated receptor γ (PPARγ) ligand, inhibits colorectal cancer cells proliferation through β-catenin/TCF pathway inactivation.

BACKGROUND Cladosporol A, a secondary metabolite from Cladosporium tenuissimum, exhibits antiproliferative properties in human colorectal cancer cells by modulating the expression of some cell cycle genes (p21(waf1/cip1), cyclin D1). METHODS PPARγ activation by cladosporol A was studied by overexpression and RNA interference assays. The interactions between PPARγ and Sp1 were investigated by co-immunoprecipitation and ChIp assays. β-Catenin subcellular distribution and β-catenin/TCF pathway inactivation were analyzed by western blot and RTqPCR, respectively. Cladosporol A-induced β-catenin proteasomal degradation was examined in the presence of the specific inhibitor MG132. RESULTS Cladosporol A inhibits cell growth through upregulation of p21(waf1/cip1) gene expression mediated by Sp1-PPARγ interaction. Exposure of HT-29 cells to cladosporol A causes β-catenin nuclear export, proteasome degradation and reduced expression of its target genes. Upon treatment, PPARγ also activates E-cadherin gene at the mRNA and protein levels. CONCLUSION In this work we provide evidence that PPARγ mediates the anti-proliferative action of cladosporol A in colorectal cancer cells. Upon ligand activation, PPARγ interacts with Sp1 and stimulates p21(waf1/cip1) gene transcription. PPARγ activation causes degradation of β-catenin and inactivation of the downstream target pathway and, in addition, upregulates E-cadherin expression reinforcing cell-cell interactions and a differentiated phenotype. GENERAL SIGNIFICANCE We elucidated the molecular mechanisms by which PPARγ mediates the anticancer activity of cladosporol A.

Infection of cotton byfusarium oxysporum f.sp.vasinfectum as affected by water stress

Since virulence ofFusarium oxysporum f.sp.vasinfectum (FOV) on cotton (Gossypium hirsutum) is enhanced when the fungus is cultivated in a saline environment, excessively saline water must not be used for the irrigation of cotton. However, the limitations thus placed on the available water resources may lead to conditions of enforced water stress for the plant. The present study investigated whether water stress affects the susceptibility of cotton to FOV. Groups of 2-month-old cotton plants of theFusarium-susceptible Coker 304 and the moderately resistant GSC 20 varieties were maintained without watering for varying periods immediately before or after being inoculated with FOV (15 plants per group, two replications). Watering was suspended for 3, 6, 12 or 24 days before inoculation, and for 3, 6, 12 or 15 days after inoculation. After inoculation the plants were maintained in a controlled environment with a 15,000 lux, 12-h photoperiod, at 28°/24°C D/N, 20% r.h. Xylem water potential was determined in a pressure chamber. Percent infected leaf area and date of onset of wilt were the parameters used to define severity of FOV infection. There was a consistent relation between low water potential in the xylem (-7 and -20 MPa) and severity of infection, particularly when the dry period occurred after inoculation. After exposure to the lowest post-inoculation water potentials, even variety GSC 20, which is normally moderately resistant, exhibited a fairly high percent infected leaf area. This should be taken into account when the cotton grower is faced with water shortages, especially during the period from branching to flower bud break.

Biological And Integrated Means to Control Rust Diseases

This chapter reviews strategies in rust control, with a special emphasis on biological control, in the light of evidence produced in recent years showing that plant disease control is most effective when an integrated management approach is followed. A survey of the fungal antagonists (hyperparasites) most effective against rust pathogens is given. The mode of action of these antagonists is described, and the main problems concerning biological control are discussed, on the basis of the optimal characteristics of an antagonist or biocontrol agent. The value and limitations of other control measures besides biological control (eradication, definition of hazard areas, quarantine, cultural practices, chemical treatments, and plant breeding for disease resistance) are also outlined. A consideration of all control measures suggests that crop protection requires a holistic approach integrating a broad range of control techniques.

The antiproliferative and proapoptotic effects of cladosporols A and B are related to their different binding mode as PPARγ ligands

Cladosporols are secondary metabolites from Cladosporium tenuissimum characterized for their ability to control cell proliferation. We previously showed that cladosporol A inhibits proliferation of human colon cancer cells through a PPARγ-mediated modulation of gene expression. In this work, we investigated cladosporol B, an oxidate form of cladosporol A, and demonstrate that it is more efficient in inhibiting HT-29 cell proliferation due to a robust G0/G1-phase arrest and p21(waf1/cip1) overexpression. Cladosporol B acts as a PPARγ partial agonist with lower affinity and reduced transactivation potential in transient transfections as compared to the full agonists cladosporol A and rosiglitazone. Site-specific PPARγ mutants and surface plasmon resonance (SPR) experiments confirm these conclusions. Cladosporol B in addition displays a sustained proapoptotic activity also validated by p21(waf1/cip1) expression analysis in the presence of the selective PPARγ inhibitor GW9662. In the DMSO/H2O system, cladosporols A and B are unstable and convert to the ring-opened compounds 2A and 2B. Finally, docking experiments provide the structural basis for full and partial PPARγ agonism of 2A and 2B, respectively. In summary, we report here, for the first time, the structural characteristics of the binding of cladosporols, two natural molecules, to PPARγ. The binding of compound 2B is endowed with a lower transactivation potential, higher antiproliferative and proapoptotic activity than the two full agonists as compound 2A and rosiglitazone (RGZ).