A. Ragazzi

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.

Use of RFLP and SSCP analysis to differentiate the pine rusts Cronartium flaccidum and Peridermium pini

Two rapid, independent molecular assays have been developed for discriminating between the closely related rusts of hard pines, Cronartium flaccidum and Peridermium pini . A portion of the ITS region and a portion of the IGS region from the ribosomal RNA operon of the two organisms were amplified with the PCR. Amplifications were made using DNA extracted from aeciospores collected from unopened aecia, taking a small number of spores as a source of template DNA. Amplified fragments were subjected to RFLP or to SSCP analysis. Digests of the amplified products from the IGS1 region were electrophoresed on polyacrylamide gel and stained with ethidium bromide. Hinf I digestion of these fragments created polymorphic restriction profiles which allowed differentiation of the autoecious P. pini from the heteroecious C. flaccidum . Similar-sized DNA fragments representing the ITS2 region of the two rusts were denatured, subjected to electrophoresis as single strands on polyacrylamide gel under non-denaturing conditions and ‘silver stained’. The different mobility displayed by these short fragments revealed sequence polymorphism in the examined portion of the ITS region. This technique therefore represents an accurate and sensitive method for detection of base changes in given sequences of genomic DNA. A high level of homology was found between the two biotroph organisms in the loci screened. Results obtained in this trial indicate that PCR-RFLP and PCR-SSCP can be used as simple, speedy taxonomic tools for elucidating relationships among related organisms.

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.

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.

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.

In vitro growth of the aspen rust Melampsora larici-tremulae

Axenic cultures of the aspen rust Melampsora larici-tremulae were established for the first time by seeding aeciospores artificially produced on larch onto three chemically defined media. Cultures were maintained for 9 months and evaluated for their appearance, growth rates and sporulation ability. Two types of colonies were observed. One, white and fluffy tending to felty, did not sporulate in culture. The other, white-orange with a more compact aerial mycelium, sporulated profusely producing various spore like bodies and other unusual structures. An initial lag phase, with reduced growth, characterised primary rust cultures in the first 4–5 weeks. After about one month, cultures seemed adapted to utilising exogenous nutrients for sustained growth and their development rate increased substantially. As a result of selection under unusual growing conditions, fast-growing genetic variants were also observed in the nutrient agar. Growth on all the three test media, containing different organic and inorganic constituents, suggested that nutritional requirements were non-specific. The importance of axenic cultures in the study of the nutritional requirements of aspen rust and the potential of these investigations to elucidate some physiological and genetic aspects of the host-parasite relationship are discussed.

Establishment of single-genotype axenic cultures from the haploid stage of the pine blister rust Cronartium flaccidum

The haplophase of the pine blister rust Cronartium flaccidum was used to grow mycelial clones axenically from single genotypes. Single telia were suspended over the media in order to obtain a direct cast of uninucleate basidiospores, providing nurse cultures from a mass basidiospore inoculum. Nurse culture and medium composition were crucial factors in successful germination and establishment of single-spore colonies. Significant differences in percentage germination between treatments and controls suggested that an extracellular matrix secreted by high density seeded basidiospores could be involved in the germination of single propagules. Proteins present in this matrix could exert a surface contact stimulus triggering germination of newly seeded basidiospores. Pre-production of extracellular enzymes in the medium could also initiate early substrate catabolism and thus create a favourable environment to support subsequent colony establishment. Growth was good on various media, and excellent on HG1Y + BSA medium. Primary mother colonies were divided into several smaller ones, re-grown axenically, then macerated. Aliquots of the resulting mycelial fragments were dispensed with a micropipette into fresh agar media. Clones derived from each pure culture were in general morphologically similar in texture, margin, compactness, and surface topography. These genetically pure lines grew at a faster rate and rapidly produced a considerable amount of mycelium. The significance of culturing rust fungi from single genotypes is discussed in relation to potential applications and new directions of research.

Epidemiological Investigations Shed Light on the Ecological Role of the Endophyte Phomopsis quercina in Mediterranean Oak Forests

Findings of a study extending over a number of years on some key aspects of the biology and ecology of Phomopsis quercina in Mediterranean oak forests are reported. The main biometric parameters and physiological requirements of this significant endophytic fungus were determined in the laboratory. The microorganism was also studied in the field to explore its lifestyle in natural oak stands. The isolation frequencies of the fungus in various oak species were also related to the vitality of the trees (healthy or declining) and this showed that the fungus is involved in the widespread phenomenon of oak decline. In planta studies have proved the pathogenic activity of P. quercina in oak, and have found that it remains latent for a long time in the inner wood of oaks, but may turn into an aggressive and harmful coloniser of the tree if this is weakened by some stress factors. An important part of the study concerns an examination of the antagonism that some other endophytes, which colonise the same oak tissues and organs as P. quercina, display against this fungus. The increasing presence and abundance of P. quercina in declining (and especially drought-stressed) oaks suggests that climate change is having a significant role in destabilising the precarious balance between the host tree and the fungus, to the benefit of the latter. Lastly, some practical suggestions are offered on ways to counter the epidemic spread of this latent pathogen.