Susan Isaac

Autolysis: A tool for protoplast production from Aspergillus nidulans

The rate and degree of autolysis of Aspergillus nidulans was investigated during a 16 day incubation period. Changes in the activity of chitinase, B -glucanase, α-glucanase, invertase, esterase, acid and alkaline phosphatase and protease liberated into the culture fluid were monitored during growth and autolysis. Protoplasts were released from A. nidulans mycelium by lytic enzymes present in autolytic-phase culture filtrates of the same organism. Maximum protoplast-liberating activity corresponded to both the point of maximum autolysis and maximum lytic enzyme activity. The method described represents a highly efficient and reliable technique for protoplast production from Aspergillus nidulans.

Ultrastructure of infection of excized leaves of celery by Septoria apiicola, causal agent of leaf spot disease

The growth of Septoria apiicola was observed by SEM on excized celery leaves and on agar medium. Initial germ tube growth was random over leaf surfaces and closely appressed to the cuticle. Leaf penetration occurred directly through the epidermis and also via stomata. After infection, hyphal growth was restricted to intercellular spaces at first but became extensive later. Limited intracellular penetration was observed when leaves were necrotic. Development of pycnidia was observed in detail in leaf tissue and on agar.

Isolation and regeneration of protoplasts from Fusarium tricinctum and F. oxysporum

A method is described for the production of protoplasts from the previously untested species Fusarium tricinctum and F. oxysporum. Factors affecting the release of protoplasts from these species were investigated using a commercial enzyme preparation Novozym 234. Maximum yields obtained were higher ( × 50) than yields previously reported for F. culmorum. A number of stabilizer solutions at a range of concentrations, were tested; the most efficient systems were 1.4 M -MgSO4 (F. tricinctum) and 1.2 M -KC1 (F. oxysporum). The effect of mycelial age, mycelial concentration in the lytic mixture and concentration of Novozym 234 were also assessed. Protoplasts liberated using this method possessed the capacity to regenerate new cell wall material and form colonies, although percentage regeneration was low. Up to 60% of protoplasts liberated were enucleate. Few contained 2 or more nuclei. Such protoplast preparations represent a useful method for the bulk production of cell-free extracts.

Electrofusion of protoplasts from celery (Apium graveolens L.) with protoplasts from the filamentous fungus Aspergillus nidulans

A method was developed for electrofusion of higher-plant protoplasts from celery and protoplasts from the filamentous fungus Aspergillus nidulans. Initially, methods for the fusion of protoplasts from ecch species were determined individually and, subsequently, electrical parameters for fusion between the species were determined. Pronase-E treatment and the presence of calcium ions markedly increased celery protoplast stability under the electrical conditions required and increased fusion frequency with A. nidulans protoplasts. A reduction in protoplast viability was observed after electrofusion but the majority of the protoplasts remained viable over a 24-h incubation period. A small decline in protoplast respiration rate occurred during incubation but those celery protoplasts fused with A. nidulans protoplasts showed elevated respiration rates for 3 h after electrofusion.

Use of autolytic enzyme for isolation of protoplasts from Fusarium tricinctum Hyphae

Fusarium tricinctum was grown in shake culture at 25 °C for a 15 day incubation period. The appearance and accumulation of lytic enzymes in the culture fluid were investigated as the culture aged and autolysed. Changes in the activity of chitinase, β-glucanase, α-glucanase, protease, acid and alkaline phosphatase, invertase and esterase were monitored. Although limited autolysis occurred during incubation of F. tricinctum (25 %) and no chitinase activity was detected, culture filtrates containing maximum β-glucanase activity (obtained after 6 days incubation) were effective in liberating protoplasts from young mycelium of the same species. It is suggested that protoplasts liberated by this method were isolated from apical regions of mycelium and the use of autolysis enzyme therefore represents a method for the preferential isolation of apical cytoplasm. Culture filtrate samples from ageing cultures of F. tricinctum also liberated protoplasts from F. oxysporum strains.

Stereological analysis of celery leaves infected by Septoria apiicola

Changes caused by Septoria apiicola during the development of leaf spot disease in celery were quantified stereologically in susceptible cvs, a partially resistant parental wild celery line, fully resistant parsley and lovage. Thick sections, cut in vertical orientation, were stained and using a camera lucida, were superimposed on a lattice grid for stereological analysis. The volume of each component within the leaf space was calculated using the Cavalieri method. In healthy tissue volume fractions of intercellular space, palisade layers and, to a lesser extent, collenchyma, vascular tissue and oil ducts were greater in the more resistant genotypes. This coincided with a reduction in the volume fraction of mesophyll tissue. Estimates of absolute volume fraction were similar. In both resistant and susceptible celery the principal response to infection was a substantial decrease in the volume fractions and absolute volumes of palisade and mesophyll tissue. Volume fractions of mesophyll and palisade tissues declined by 97% in susceptible lesions and by 98% in resistant lesions. The fungus was not observed in xylem or phloem cells. No appreciable changes were observed in either collenchyma or oil duct tissues in response to infection in resistant and susceptible genotypes. Infection had no effect on the epidermis or stomata and these remained intact until late in the infection cycle, when pycnidia erupted through the epidermis of the upper and lower leaf surfaces. No hyphae were found in leaf sections of lovage or parsley, and fungal growth was severely restricted in all resistant material bred from wild celery. In wild celery lines, S. apiicola did not produce any, or produced very few, pycnidia and the volume fraction of vegetative hyphae was lower than that in susceptible cvs, where the fungus proliferated extensively within the lesions and many pycnidia were produced. This accounted for 30–50% of the fungal material in lesions on susceptible celery plants. The amount of fungus (21%) was less in lesions on resistant celery and in this material 96% of the fungus was present as vegetative hyphae. In lesions formed on susceptible celery 26% of the fungal tissue was reproductive and of that 11% was conidia whereas in resistant celery, where pycnidia were present, these structures were morphologically abnormal and did not contain conidia. Stereological analysis demonstrated clear differences in plant and pathogen response during infection of resistant and susceptible celery by S. apiicola.

Uptake of Fungal Protoplasts by Plant Protoplasts

A range of relationships can be formed between fungi and higher plants. Fungi are heterotrophic and grow by absorbing easily assimilable substrates directly or by first breaking down compounds by means of extracellular enzymes. Nutrients may be obtained from nonliving material by saprotrophy; from living tissues symbiotically, conferring mutual nutritional benefit or alternatively by parasitism through biotrophy or necrotrophy, causing disease and often resulting in plant death.

Dual fungal and plant cell culture.

Plant tissue cultures are now well-recognized as valuable experimental systems for use in the study of host-pathogen interactions. These techniques have obvious major advantages for the examination of obligately biotrophic fungi and also those with a necrotrophic life style, and it is in these areas that much research effort has been concentrated (1). Success with combined fungal-plant cultures has been variable, especially in terms of establishing cultures that may be maintained in a balanced state for prolonged periods, but there is no doubt that such systems are useful places for the study of cell-cell interactions.

Uptake of protoplasts from the filamentous fungiAspergillus nidulans andFusarium oxysporum into protoplasts from celery (Apium graveolens L.)

SummaryProtoplasts isolated from celery cell suspension cultures, were mixed with fungal protoplasts, from either the saprophytic speciesAspergillus nidulans or the pathogenic speciesFusarium oxysporum. The incubation of protoplast mixtures with PEG caused close adhesion between plant and fungal protoplasts. Subsequent dilution of PEG resulted in the uptake of protoplasts from either fungal species into the plant protoplast cytoplasm. A range of PEG concentrations, incubation times and dilution rates were tested to maximise adhesion and uptake frequencies. Identification of uptake was achieved either by fluorescent staining of nuclei or by electron-microscopy. A maximum of 10% celery protoplasts had taken upA. nidulans protoplasts after PEG treatment. Fungal protoplasts were taken up into celery protoplast cytoplasm by endocytosis, and were maintained within vesicles; two bounding membranes were observed by electron microscopy. Plant protoplast viability was determined during prolonged incubation following fungal protoplast uptake. The presence ofA. nidulans protoplasts tended to maintain celery protoplast viability and although some morphological disintegration occurred intact celery protoplasts remained for at least 92 h after uptake. The uptake ofF. oxysporum protoplasts markedly depressed celery protoplast viability after 24 h incubation and greater celery protoplast disintegration occurred.

Assessment of fungal growth in liquid cultures and bioassay of toxic products

This article outlines a procedure for the assessment of fungal growth under different cultural conditions; together with the demonstration of the toxic nature of the exudates released from a fungal pathogen during growth in culture, using a simple bioassay.