M. G. Pellegrini

Isolation of tomato cell lines with altered response to Fusarium cell wall components

SummaryTo obtain Tomato cell lines with an altered capacity to respond to heat-released cell wall components (elicitor) of a tomato pathogen (Fusarium oxysporum f. sp. lycopersici), positive and negative selection experiments, using BUdR enrichment techniques, were carried out on suspension cultures of the susceptible, low phytoalexin producer cultivar Red River. Both high and low phytoalexin producing clones were isolated. Further tests demonstrated that not all phytoalexin-producing clones were more susceptible to the elicitor toxic effect, and that they were altered also in the speed of response to fungal cell wall components. Cells selected with Fusarium elicitor showed the same behaviour when challenged by Phytophthora infestans elicitor, thus suggesting in this case lack of specificity. The results are finally discussed with a view to using the technique both as a tool to study the genetics and physiology of hostparasite interactions and as a possible new method for the selection of pathogen resistant genotypes.

Modification of competence for in vitro response to Fusarium oxysporum in tomato cells. II. Effect of the integration of Agrobacterium tumefaciens genes for auxin and cytokinin synthesis.

We have studied the effect of a change in the endogenous hormone equilibria on the competence of tomato (Lycopersicon esculentum) cells to defend themselves against the fungal pathogen Fusarium oxysporum f. sp. lycopersici. Calluses from cvs ‘Davis’ and ‘Red River’, respectively resistant and susceptible to Fusarium and transgenic for an auxin- or cytokinin-synthesizing gene from Agrobacterium tumefaciens, were used. The integration of Agrobacterium hormone-related genes into susceptible cv ‘Red River’ can bring the activation of defense processes to a stable competence as assessed by the inhibition of mycelial growth in dual culture and gem-tube elongation of Fusarium conidia, the determination of callose contents, peroxidase induction and ion leakage in the presence of fusaric acid. This is particularly true when the transformation results in a change of phytohormone equilibria towards an higher cytokin in concentration. On the contrary, in resistant cv ‘Davis’ the inhibition of both fungal growth in dual culture and conidia germination is higher when the hormone balance is modified in favour of the auxins. No significant effect was observed for ion leakage and peroxidase induction, probably because of a constitutive overproduction of cytokinins in ‘Davis’ cells.

The in vitro physiological phenotype of tomato resistance to Fusarium oxysporum f. sp. lycopersici

SummaryWith the aim of dissecting host-parasite interaction processes in the system Lycopersicon aesculentum-Fusarium oxysporum f. sp. lycopersici we have isolated plant cell mutants having single-step alterations in their defense response. A previous analysis of the physiological phenotypes of mutant cell clones suggested that recognition is the crucial event for active defence, and that polysaccharide content, fungal growth inhibition, peroxidase induction in in vitro dual culture and ion leakage induced by cultural filtrates of the pathogen can be markers of resistance. In this paper we present the results of a similar analysis carried out on cell cultures from one susceptible (‘Red River’), one tolerant (‘UC 105’) and three resistant (‘Davis UC 82’, ‘Heinz’, ‘UC 90’) tomato cultivars. Our data confirm that the differences in the parameters considered are correlated with resistance versus susceptibility in vivo. Therefore, these parameters can be used for early screening in selection programmes. These data, together with those obtained on isolated cell mutants, suggest that the selection in vitro for altered fungal recognition and/or polysaccharide or callose content may lead to in vivo — resistant genotypes. The data are thoroughly discussed with particular attention paid to the importance of polysaccharides in active defense initiation.

The pleiotropic phenotype of tomato cells selected for altered response to Fusarium oxysporium f. sp. lycopersici cell wall components

SummaryWith the aim of better understanding in vitro host-parasite interactions, tomato cell lines selected for altered response to Fusarium oxysporum f. sp. lycopersici cell wall components were further characterized. Particularly, their behaviour in dual culture in regard to both fungal inhibition and peroxidase activation was analysed and selected, and control cell clones were screened for esopolysaccharide content and toxin tolerance. Interclonal differences in growth response to 2,4-D and DMSO and the capacity to grow on a medium devoid of hormones (habituation) were taken as parameters representative of physiological variability not directly correlated with the response to pathogens. Significant differences between clones selected for increased (F+) and decreased (F−) response to fungal elicitors were found for pathogen inhibition, peroxidase and esopolysaccharide content, toxin tolerance being reduced in F but not significantly different from the control in F+. As expected, clonal variability for the response to 2,4-D and DMSO, although significant, was not connected with hostparasite interactions. The data reported thus show that selection for a character (response to elicitors), probably critical for the response to pathogens, may lead to the recovery of genotypes showing a set of modifications suggestive of a cascade of events leading to active defense.

Modification of competence for in vitro response to Fusarium oxysporum in tomato cells. I. Selection from a susceptible cultivar for high and low polysaccharide content

Plant cell walls play a major role in the outcome of host-parasite interactions. Wall fragments released from the plant, and/or the fungal pathogen, can act respectively as endogenous and exogenous elicitors of the defence response, and other wall components, such as callose, lignin, or hydroxyproline-rich glycoproteins, can inhibit pathogen penetration and/or spreading. We have previously demonstrated that calli from tomato cultivars resistant in vivo to Fusarium oxysporum f.sp. lycopersici show a high amount of polysaccharides in vitro. The aim of the present work was to assess the possible role of polysaccharide content and/or synthetic capacity in determining the competence of plant cells for active defence. For this purpose, tomato cell clones with increased and decreased polysaccharide (FL+, FL-) and callose (A+, A-) content have been selected by means of specific stains as visual markers and tested for the effect of these changes on the extent of response to Fusarium. The analysis of several parameters known to be indicative of active defence (cell browning after elicitor treatment, peroxidase and β-glucanase induction and inhibition of fungal growth in dual culture) clearly shows that FL+ and A+ clones have acquired an increased competence for the activation of defence response. The results are thoroughly discussed in terms of an evaluation of the relative importance of constitutive and/or inducible polysaccharide synthetic capacity for plant response to pathogens, and their possible regulation by plant physiological backgrounds.

Induction or Enhancement of Competence for Active Defense by Genes Involved in the Synthesis of Phytohormones from Agrobacterium tumefaciens in Transgenic Tomato Cells Susceptible or Resistant to Fusarium

Host — pathogen interactions in plants are controlled by a complex network of processes, probably induced by recognition events, whose quantitative modulation is critical for the choice between susceptibility and resistance. Although a gene-for-gene system probably has a trigger effect on the timing and the intensity of the active response, the modulation by exogenous and endogenous factors may affect the outcome of the interaction.

Toxin Tolerance as a Marker for Resistance Breeding

Although the relevance of toxins in plant pathogenesis is still under discussion, purified toxins or fungal culture filtrates have been used to select cells and plants resistant to diseases, and toxin tolerance has been suggested as a promising marker for resistance in long range breeding programs. The aim of our work was to investigate the reliability of correlations between toxin tolerance and resistance in susceptible and resistant cultivars. The systems analyzed were Dianthus caryophyllus-Fusarium oxysporum f.sp. dianthi and Solanum tuberosum — Alternaria solani. Toxin tolerance was assayed using the two screening methods: plant cell and callus growth on media containing fungal culture filtrate, and ion leakage from callus cells or leaf cuttings challenged with culture filtrate (or fusaric acid).