Germ Tube Growth Research Articles

Correlation between the intracellular content of glutathione and the formation of germ-tubes induced by human serum in Candida albicans

The physiological role of the tripeptide glutathione (GSH) and its oxidized form (GSSG) was investigated during the initial steps of dimorphism (formation of germ-tubes), which is induced by human serum in exponential yeast-like cells (blastoconidia) of the Candida albicans strain CAI-4 (wild type) and its congenic tps1/tps1 mutant, deficient in trehalose synthesis. The content of glutathione, measured both as GSH and the ratio GSH/GSSG, underwent a moderate drop in parallel with the induction of a significant degree of germ-tube emergence. Whereas the supply of exogenous glutathione did not affect the degree of dimorphic transition, depletion of intracellular glutathione by addition of 1-chloro-2,4 dinitrobenzene (CDNB) caused a clear reduction in the percentage of hyphae formation; although this effect must be due to the severe cell mortality produced by CDNB. Simultaneous measurements of GSH-metabolizing activities revealed a moderate decrease of glutathione reductase concomitant with the activation of glutathione peroxidase. In turn, catalase activity did not show noticeable changes. The putative correlation between the redox status of glutathione and the dimorphic conversion in C. albicans is discussed.

Growth control and polarization

Filamentous fungi and yeasts both undergo polar growth. In Saccharomyces cerevisiae, where the mechanisms for polar growth are well-understood, polarity requires three steps: establishment of cortical markers specifying the site of bud emergence; relaying the bud site information via the Cdc42 Rho GTPase module; and recruitment of the morphogenetic machinery needed to remodel the cell surface to the specified site. Comparison of the genomes of Aspergillus fumigatus, A. nidulans and A. oryzae with that of S. cerevisiae show that the cortical markers are absent or poorly conserved, while the RhoGTPase signaling module and the morphogenetic machinery are highly conserved in the aspergilli. Genetic approaches to polarity using A. nidulans polarity mutants with defects in germ tube emergence (swo mutants) or branching (ahb mutants) will also be discussed.

Effect of some Environmental Factors on In Vitro Germination of Urediniospores and Infection of Lentils by Rust

For accurate lentil (Lens culinaris) rust phenotyping in controlled environments, conditions for infection should be optimized. Therefore, the effects of temperature on germination and germ tube growth of Uromyces viciae-fabae, as well as the effect of different dew periods, were quantified. In all experiments urediniospores of a single-pustule isolate were applied using a previously calibrated settling tower. After 3 h of incubation, a high percentage (≥80%) of spore germination was observed on 1.5% water agar at 10, 15, 20 and 25°C, with an optimum (99%) at 20°C. At this sampling time the length of germ tubes ranged from 66 μm (10°C) to 196 μm (20°C). Growth of germ tubes increased progressively from 10 to 20°C and then declined at 25°C. For minimum infection of lentil cultivar EL-142 at 20°C, a dew period of at least 3 h was required, whereas maximum infection occurred with a dew period of 24 h. Infection efficiency increased linearly as the duration of dew period increased from 0 to 24 h. Regression models that best described the quantitative relationship between the environmental variables and growth of the pathogen and development of rust were derived empirically. Such models are of significance in optimizing studies of the particular pathosystem as well as eventual lentil rust prediction models.

Accumulation of HDMBOA-Glc is induced by biotic stresses prior to the release of MBOA in maize leaves

The effects of biotic stresses on the contents of benzoxazinones (Bxs) were investigated in maize leaves. When the causal agent of southern corn leaf blight, Bipolaris maydis, was inoculated on the third leaf, the amount of 2-hydroxy-4,7-dimethoxy-1,4-benzoxazin-3-one glucoside (HDMBOA-Glc) increased, reaching a maximum level 48 h after inoculation. The inoculation of weakly pathogenic Curvularia lunata and non-pathogenic Alternaria alternata also resulted in accumulation of HDMBOA-Glc, and filtrates of the cultures of B. maydis, C. lunata and A. alternata also showed the accumulation of elicitor-active compounds by the fungi. Furthermore the infection of B. maydis induced formation of dark brown lesions, where most abundant Bx-related compound was 6-methoxy-2-benzoxazolinone (MBOA). The later is formed by degradation of DIMBOA and HDMBOA, whereas HDMBOA-Glc was most abundant in the surrounding green tissues. Among the Bx-related compounds, MBOA exhibited the strongest inhibition of the germination of the conidia and of the growth of germ tubes of B. maydis, C. lunata and A. alternata. In addition to fungal infection, the feeding by rice armyworm larvae resulted in HDMBOA-Glc accumulation. These findings are discussed in relation to the possible ecological relevance of the conversion of DIMBOA-Glc into HDMBOA-Glc.

An immunocytochemical and ultra-structural study of the extracellular matrix produced by germinating spores of Stagonospora nodorum on natural and artificial surfaces

The temporal secretion and ultra-structure of the extracellular matrix (ECM) produced by conidia and germ-tubes of Stagonospora nodorum on wheat and other surfaces during the first 6 h post inoculation was examined. To visualize the ECM, a combination of immunological, histochemical and ultra-structural methods were employed. Lectins and two monoclonal antibodies were used. One antibody specifically recognised a conidial surface protein and the other recognised a carbohydrate epitope present on an antigen in the ECM and the germ-tube cell wall. Three major phases of ECM release on the leaf surface and two on the artificial surfaces were identified: (a) commencing less than 15 min after contact of the conidium with the host, composed of a carbohydrate core and protein halo, (b) coinciding with the emergence and growth of germ-tubes, composed of proteins and carbohydrates, (c) spreading around the conidia after germ-tube production, not seen on the artificial surfaces. Results indicated that the ECM of conidia and germ-tubes differ in thickness and composition and that the intensity of conidial ECM was affected by the surface properties of the substrata in contact. In addition, maintenance of the ECM in a hydrated state was found to be essential for study of its ultra-structure highlighting the importance of using a diversity of methods to visualize the ECM.

Induced resistance toCladosporium cucumerinum in cucumber by pectinases extracted fromPenicillium oxalicum

Pectinases extract (PE) from the fermentation product ofPenicillium oxalicum BZH-2002 was tested for its ability to induce protection against scab caused byCladosporium cucumerinum on cucumber (Cucumis sativus L.) plants. Seedlings with one true leaf were sprayed with various concentrations of PE (10–200 units ml−1) 3 days before inoculation withC. cucumerinum. Results showed that the induced local protection against the pathogen was dose-dependent when the concentrations of PE were between 20 and 120 units ml−1; systemically induced resistance against the pathogen was not observed. Boiled PE had a slight effect on disease reduction. Commercial pectinases prepared fromAspergillus niger showed lower protection against scab compared with PE when they were used at the same concentration of enzyme activity. No inhibitory activity was observed on conidial germination or germ-tube growth ofC. cucumerinum. PE was further evaluated for its enhancement of defense-related enzymes. Peroxidase (PO), polyphenol oxidase (PPO) and phenylalanine ammonia lyase (PAL) increased in cucumber seedlings after treatment with PE. PO and PPO remained at a higher level in PE-pretreated seedlings throughout the experiment period whether pathogen-inoculated or non-inoculated, whereas PAL activity began to decrease 2 days after PE treatment.

Histopatologia da interação Alternaria solani e tomateiros resistente e suscetível

Para esclarecer a natureza da resistência à pinta-preta (Alternaria solani) em tomateiro resistente (Lycopersicon hirsutum var. glabratum) cv. CNPH 417 e suscetível (L. esculentum) cv. Miller, avaliou-se o processo de infecção, através da histopatologia. Às 6, 12, 24, 36, 48 e 72 h após as inoculações (h.a.i.) de suspensões de conídios, coletaram-se amostras de tecidos foliares que foram submetidas ao clareamento, à inclusão em resina para confecção de cortes semifinos e ao processamento para microscopia eletrônica de varredura (MEV). Pela análise das amostras clareadas, observou-se que a germinação de conídios completou-se em 24 h.a.i. O crescimento de tubos germinativos foi similar na superfície de ambos os genótipos. Entretanto, os números de apressórios formados, de penetrações nos tecidos e de lesões foram inferiores no genótipo resistente. Com relação aos eventos pós-penetração, o desenvolvimento inicial de hifas primárias e secundárias, processos posteriores de colonização e desenvolvimento de lesões, bem como a ocorrência de formação de papilas sob apressórios e de reações de hipersensibilidade foram similares em ambos os genótipos. Para a maioria dos aspectos da patogênese de A. solani, portanto, o genótipo resistente CNPH 417 comportou-se de modo similar ao suscetível, cv. Miller, exceto quanto aos números de apressórios, de penetrações e de lesões. Assim, ficou evidenciado que a resistência de L. hirsutum var. glabratum (CNPH 417) a A. solani é expressa na fase de pré-penetração, principalmente pelo baixo número de apressórios formados.

Effect of Temperature on Thekopsora minima Urediniospores and Uredinia.

Thekopsora minima is a heteroecious rust, with spermogonia and aecia occurring on the needles of hemlock (Tsuga spp.) and uredinia, telia, and basidia occurring on the leaves of ericaceous genera, including species of Rhododendron. The effect of temperature was determined for urediniospore germination, germ tube growth, and infection efficiency on Rhododendron 'White Lights'. Percent germination and germ tube growth were assessed at 10, 15, 20, 25, and 30°C after 3 h of incubation on 1.5% water agar in the dark. Polynomial regression analyses revealed a significant effect of temperature on both germination (P < 0.001, R2adj = 0.936) and germ tube growth (P < 0.001, R2adj = 0.933), with predicted optimum temperatures of 21.5 and 22.0°C, respectively. Germination and germ tube growth were reduced greatly at 30°C and below 15°C. Temperature also was found to have a significant effect on infection efficiency, as measured by incubation period (P < 0.001, R2adj = 0.808) and uredinia produced (P < 0.001, R2adj = 0.866). On excised leaf disks of Rhododendron 'White Lights' maintained under a 14-h photoperiod, the shortest mean incubation periods of 10.7 and 10.0 days were at 20 and 25°C, respectively, with a predicted optimum of 23°C. The mean incubation period at 15 and 30°C was approximately 4 and 3 days longer, respectively, than at the predicted optimum temperature. The number of uredinia produced was similar at 15, 20, and 25°C, but was reduced sixfold at 30°C. The predicted optimum temperature for uredinial production was 19.5°C, with a 5% variation in uredinia production between 17.5 and 22°C.

Rapid pregermination and germination responses of Erysiphe pisi conidia to contact and light

In darkness, most Erysiphe pisi conidia responded rapidly to contact with a hydrophobic artificial substratum and released extracellular material (ECM) in the same way as on pea cuticle. On this substratum and barley leaf epidermis, conidia then produced a germ tube that emerged close to the substratum, contacted it, and differentiated an appressorium. By contrast, on a hydrophilic substratum, ECM release and germination were delayed and infrequent, and germ tubes often emerged and faced away from the substratum toward vertical light, thereby failing to make contact and form appressoria. This finding supported the hypothesis that ECM release is involved in both triggering germination and sensing substratum contact. Exposure to white light dramatically affected the germ tube emergence site so most emerged from a site in the conidial wall facing the light. Lateral light did not affect the frequency of germ tubes making substratum contact; but when lit from above, most germ tubes emerged up, facing away from the substratum. The germ tubes formed in light were longer than those formed in darkness, but no phototropism was found for the elongating tubes. Examination of Blumeria graminis indicated that its conidia and germ tubes are insensitive to white light.

An immunological approach to quantifying the saprotrophic growth dynamics of Trichoderma species during antagonistic interactions with Rhizoctonia solani in a soil-less mix.

Studies of the saprotrophic growth dynamics of Trichoderma species and their fungal hosts during antagonistic interactions are severely hampered by the absence of methods that allow the unambiguous identification and quantification of individual genera in complex environments such as soil or compost containing mixed populations of fungi. Furthermore, methods are required that allow discrimination between active hyphal growth and other components of fungal biomass such as quiescent spores that are produced in large numbers by Trichoderma species. This study details the use of monoclonal antibodies to quantify the saprotrophic growth dynamics of the soil-borne plant pathogen Rhizoctonia solani and biological control strains of Trichoderma asperellum and Trichoderma harzianum during antagonistic interactions in peat-based microcosms. Quantification was based on the immunological detection of constitutive, extracellular antigens that are secreted from the growing tip of Rhizoctonia and Trichoderma mycelium and, in the case of Trichoderma harzianum, from quiescent phialoconidia also. The Trichoderma-specific monoclonal antibody (MF2) binds to a protein epitope of the enzyme glucoamylase, which was shown by immunofluorescence and immunogold electron gold microscopy studies of Trichoderma virens in vitro to be produced at the origin of germ tube emergence in phialoconidia and from the growing tip of germ tubes. In addition, a non-destructive immunoblotting technique showed that the enzyme was secreted during active growth of Trichoderma asperellum mycelium in peat. The Rhizoctonia solani-specific monoclonal antibody (EH2) similarly binds to a protein epitope of a glycoprotein that is secreted during active mycelial growth. Extracts derived from lyophilized mycelium were used as a quantifiable and repeatable source of antigens for construction of calibration curves. These curves were used to convert the absorbance values obtained in ELISA tests of peat extracts to biomass equivalents, which allowed comparisons of the saprotrophic growth dynamics of the pathogen and antagonists to be made in single or mixed species microcosms. Trichoderma species were able to compete successfully with R. solani for nutrients and to prevent saprotrophic growth of the pathogen. Specificity of the Trichoderma quantitative assay was tested in non-sterile soil-based microcosms artificially inoculated with T. asperellum. The assay was highly specific and only detected T. asperellum population dynamics. No cross-reactivity was found with extracts from soil samples containing contaminant fungi.

Effect of temperature on conidial germination and growth of the fungus Discosporium populeum (Sacc) Sutton

Dothichiza, poplar canker, due to its significance in the establishment of intensive poplar plantations, has been drawing attention for already a long time. The aim of the test was to study some of the most important physiological characteristics of the fungus, such as conidial germination germ tube growth and growth of mycelium at different temperatures on the isolates from north Potisje and to compare the study results with the domestic and foreign literature sources. The aim of this study was not to identify the different lower systematic categories, but to identify the differences between individual isolates - populations, depending on the most significant ecological factors, such as temperature.

Osmotin and Thaumatin from Grape: A Putative General Defense Mechanism Against Pathogenic Fungi

ABSTRACT Little information is available concerning the expression of pathogenesis-related (PR) proteins in grapevine (Vitis vinifera) and their effect properties on the major fungal pathogens of grape. A systematic study was performed on the effect of total or individual grape proteins on mycelial growth, spore germination, and germ tube growth of Uncinula necator, Phomopsis viticola, and Botrytis cinerea. Two proteins, identified as PR proteins by immunological methods and by N-terminal sequencing as osmotin and thaumatin-like protein, exhibited strong antifungal activities in vitro, blocking the growth of Phomopsis viticola and Botrytis cinerea mycelia. In addition, they inhibited spore germination and germ tube growth of U. necator, Phomopsis viticola, and Botrytis cinerea. The presence of both proteins displayed a synergistic effect. The expression of osmotin and thaumatin-like protein was induced in grapevine leaves and berries infected with U. necator and Phomopsis viticola. Thaumatin previously was thought to occur exclusively in berries. Immunoblot analyses revealed the accumulation of the two PR proteins in infected leaves and berries, supporting a role in vivo in increasing the resistance of grapevine to fungal attack.

The pathogenesis of dermatophyte infections in human skin sections.

A novel ex vivo model for the study of adherence and invasion of dermatophytes to the stratum corneum was developed. A skin of full epidermis thickness was infected by spores of Trichophyton mentagrophytes and examined after various periods of time by scanning and transmission electron microscopy. After 12 h of inoculation a tenacious adherence between the spores and the stratum corneum was observed. There was a time dependent increase in the number of spores adhered to this surface. By 24 h, germination had commenced. The initial growth of germ tubes occurred extracellularly to the corneocytes. Three days after inoculation, the most prominent feature was proliferation of fungal hyphae and penetration of mycelium through the outer keratinocyte layer which is followed by invasion of the outer stratum corneum. The model introduced in the present study may contribute to a better understanding of the nature of the interaction between dermatophytes and skin cells in dermatophytosis process.

Aspergillus nidulans RhoA is involved in polar growth, branching, and cell wall synthesis

Growth of the filamentous fungus Aspergillus nidulans begins when the conidium breaks dormancy and grows isotropically. Eventually a germ tube emerges and the axis of growth remains fixed in the primary hypha while new growth axes are established basally to form secondary germ tubes and lateral branches. Rho1 is a Rho family GTPase that has been shown to be involved in polarity establishment and cell wall deposition in Saccharomyces cerevisiae. A gene predicted to encode a Rho1 homolog was cloned from A. nidulans and named rhoA. Strains carrying ectopic copies of the constitutively active rhoA G14V allele or the dominant rhoA E40I allele were created and characterized. The constitutively active rhoA G14V strain grew slowly relative to wild type and showed an abnormal clustered pattern of branch emergence. The rhoA G14V strain also labeled intensely with calcofluor, showed elevated levels of cell wall N-acetylglucosamine and had unusually thick cell walls. The dominant rhoA E40Istrain was accelerated in the emergence of secondary and tertiary germ tubes, and lateral branches relative to wild type and showed lysis with prolonged incubation. The rhoA E40I strain also was hypersensitive to the cell wall disrupting agents calcofluor and caspofungin acetate and showed an increase in cell wall N-acetylglucosamine levels. Our results suggest that rhoA plays a role in polarity, proper branching pattern, and cell wall deposition.

Effect of Chitosan and Temperature on Spore Germination of Aspergillus niger

AbstractInhibition of radial growth and spore germination of Aspergillus niger in media with added chitosan were detected. The highest radial growth inhibition (73%) was determined at 24 h with 3 g · L−1 of chitosan, and the percent inhibition of spore germination was 40% after 13 h of inoculation. Further, the CC50, that is, the concentration at which spore germination is inhibited by 50%, was estimated by probit analysis (3.5 g · L−1). The activation energies, EA were estimated by an Arrhenius model in control and amended chitosan media, obtaining 35.6 and 36.6 kcal · mol−1, respectively. These values were in the same order of magnitude because chitosan as inhibitor was more effective at low temperature (≤ 18 °C). Hence synergism of temperature and chitosan were only observed at 12 and 18 °C. Therefore, the maximal percentage of germinated spores, Smax was also affected by low temperatures in chitosan‐amended media with estimated values lower than 70% at temperatures &lt; 37 °C whereas in control media Smax reached values close to 100%. Scanning electron micrographs showed that chitosan produced spore aggregation and morphological anomalies affecting swelling, germ tube emergence, and polarization.Germinated spores percentages of Aspergillus niger in Czapeck media at several chitosan concentrations and 30 °C.magnified imageGerminated spores percentages of Aspergillus niger in Czapeck media at several chitosan concentrations and 30 °C.

The Aspergillus nidulans swoC1 mutant shows defects in growth and development.

Previous work identified swoC1 as a single-gene mutant with defects in polarity establishment. In this study swoC1 was shown to have defects in endocytosis, compartmentation, nuclear distribution, and conidiation. Temperature-shift experiments showed that the swoC1 mutant establishes multiple random sites of germ tube emergence. Surprisingly, these experiments also showed that even a slight delay in polarity establishment causes defects in later vegetative growth and asexual reproduction. The swoC gene was mapped to the centromere of chromosome III and cloned by complementation of the temperature-sensitive phenotype. The predicted SwoCp is homologous to rRNA pseudouridine synthases of yeast (Cbf5p) and humans (Dkc1p). However, neither rRNA pseudouridine synthesis nor rRNA processing appears to be affected in the swoC1 mutant. The swoC1 mutation occurs in the putative RNA-binding domain upstream of the C terminus, leaving the N-terminal TRUB catalytic domain intact. Interestingly, while deletion of the swoC gene was lethal in A. nidulans, the C terminus, including NLS, microtubule-binding, and coiled-coil domains, was dispensable for growth. SwoCp likely plays an important role in polar growth and nuclear distribution in A. nidulans, functions not yet described for its homologs.

Confocal microscopy of Spitzenkörper dynamics during growth and differentiation of rust fungi.

The membrane-selective fluorescent dye FM4-64, N-(3-triethylammoniumpropyl)-4-(6-(4-(diethylamino)phenyl)hexatrienyl)pyridium dibromide, was used to stain the apical vesicle cluster within the specialized Spitzenkörper of the germ tube of the rust fungi Uromyces vignae and Puccinia graminis f. sp. tritici grown on glass surfaces. The Spitzenkörper stained within 15 min following addition of the dye. Optical sectioning by confocal microscopy of stained hyphal tips showed that the Spitzenkörper was asymmetrically positioned close to the cell-substratum interface during germ tube growth. The Spitzenkörper showed variations in shape and positioning over short (5 s) time intervals. The movement to a new location in the hyphal dome was followed by new growth in that region, consistent with the view that the Spitzenkörper supplies secretory vesicles for germ tube growth. A pronounced Spitzenkörper disappeared at the onset of appressorium differentiation during swelling of the germ tube. However, a stained structure, similar in appearance to a Spitzenkörper, was again observed during the formation of the highly polarized penetration peg.

A novel improved method for Aspergillus nidulans transformation

We systematically investigated the efficiency of Aspergillus nidulans transformation using protoplasts prepared from different stages of conidiospore germination and young mycelium. Using standard integrative plasmids, increased transformation yields were obtained with protoplasts isolated from a specific stage coincident with germ tube emergence. This increase ranged, on the average, from two- to eightfold depending on different plasmids used. Transformation efficiencies with a replicative plasmid were similar to those obtained using previously described methods. Although this observation suggests that elevated transformation efficiencies might be due to increased efficiency of recombination between plasmid and genomic sequences, we cannot exclude other factors associated with the particular developmental stage used. In the course of this study, we also examined the effect of other parameters that might enhance transformation yields. The method described is also significantly easier and faster than other current methods.

Maximal polar growth potential depends on the polarisome component AgSpa2 in the filamentous fungus Ashbya gossypii.

We used actin staining and videomicroscopy to analyze the development from a spore to a young mycelium in the filamentous ascomycete Ashbya gossypii. The development starts with an initial isotropic growth phase followed by the emergence of germ tubes. The initial tip growth speed of 6-10 microm/h increases during early stages of development. This increase is transiently interrupted in response to the establishment of lateral branches or septa. The hyphal tip growth speed finally reaches a maximum of up to 200 micro/h, and the tips of these mature hyphae have the ability to split into two equally fast-growing hyphae. A search for A. gossypii homologs of polarisome components of the yeast Saccharomyces cerevisiae revealed a remarkable size difference between Spa2p of both organisms, with AgSpa2p being double as long as ScSpa2p due to an extended internal domain. AgSpa2 colocalizes with sites of polarized actin. Using time-lapse videomicroscopy, we show that AgSpa2p-GFP polarization is established at sites of branch initiation and then permanently maintained at hyphal tips. Polarization at sites of septation is transient. During apical branching the existing AgSpa2p-GFP polarization is symmetrically divided. To investigate the function of AgSpa2p, we generated two AgSPA2 mutants, a partial deletion of the internal domain alone, and a complete deletion. The mutations had an impact on the maximal hyphal tip growth speed, on the hyphal diameter, and on the branching pattern. We suggest that AgSpa2p is required for the determination of the area of growth at the hyphal tip and that the extended internal domain plays an important role in this process.

Elemental sulphur is produced by diverse plant families as a component of defence against fungal and bacterial pathogens

Elemental sulphur ( 32S 0 detected as 32S 8) is the only inorganic phytoalexin recorded, first identified as a component of resistance of Theobroma cacao to Verticillium dahliae. Here we report S 0 production in another four, diverse families in response to fungal and bacterial pathogens. S 0 was detected in xylem excised from infected but not control tomato and cotton to V. dahliae, tobacco and French bean to Fusarium oxysporum and tomato to Ralstonia solanacearum. Accumulation was more rapid and greater in disease resistant genotypes than in susceptible lines. No S 0 was detected in strawberry challenged with V. dahliae or maize leaves with Erwinia stewartii. Furthermore, S 0 was absent from leaves of six spp. undergoing hypersensitive reactions to Pseudomonas syringae pathovars. These data suggest that S 0 production may be specific to xylem and does not occur in all plants. Anomalously, high constitutive levels of S 0 occurred in control and hypersensitively responding leaves of Brassica oleracea, irrespective of challenge by an incompatible race of Peronospora parasitica and in uninoculated Arabidopsis thaliana leaves. Scanning electron microscopy–energy dispersive X-ray microanalysis showed sulphur in A. thaliana was present in all leaf cell types. S 8 showed high toxicity (ED 50 0.78–3.125 μg ml −1 to spore germination and germ tube growth) to isolates of V. dahliae and F. oxysporum but had no effect on bacterial pathogens or to a Phytophthora sp. Levels in tomato (10 μg g −1) and Arabidopsis (1.5–6 μg g −1) were potentially inhibitory, but in other spp. (maximum 100–250 ng g −1) were below theoretical inhibitory levels. The highly localised accumulation of other phytoalexins and of S 0 in T. cacao and tomato xylem, suggest S 0 is produced in sufficient amounts, at the right time and place in some interactions to contribute to resistance.