Dipartimento di Biologia Animale e Genetica, Universitd di Firenze;htituto Agronomico per I'Oltremare, Firenze, Italy
In vitro Analysis of Defence Mechanismin the System Solatium tuberosum-Alternaria alternata
M. G. PELLEGRINI, A. R. GARBUGLIA, S. GUERRAZZI, P. BOGANI, P. BETTINI,
E. STORTI, C . SIMETI, M . BUIATTI, M . BROGGIO and G. NASCARI
Authors' addresses: M. G. PELLEGRINI, A. R. GARBUGEIA, S. GUERRAZZIS, P. BOGANI, P. BETTINI,
E. STORTI, C . SIMETI, M . BUIATTI, Dipartimento di Biologia Animale e Genetica, Universita diFirenze, Firenze, Italy. M. BROGGIO, G . NASCARI, Istituto Agronomico per I'Oltremare, Firenze,
Italy.
Wltb 4 figures
Received August 7, 1989; accepted fehruary 13, 1990
Abstract
In a preliminary study on the interaction between an Altemaria alternata pathotype andSolanttm tuherosum a series of parameters, indicative of active and passive defence processes, wasinvestigated in vitro using tolerant (Chiquita) and susceptible (Superior) cultivars. Ion leakageexperiments along with growth of suspension cultures and plating efficiencies in the presence of fungalculture filtrates suggest that toxin tolerance is a valuable character in distinguishing between resistantand susceptible genotypes. As far as active defence was concerned, although TLC patterns suggestedthe synthesis of different fluorescent compounds, probably phenolic acid, when fungal elicitors wereapplied to Chiquita or susceptible tissue cultures, no consistent pattern suggesting a relationship withdefence could be observed. On the other hand, peroxidase isozyme analysis after different dual cultureperiods showed the activation of ionically and covalently bound peroxidases in the presence of thepathogen only in cv. Chiquita.
Zusamtnenfassung
In vj'tro-Analyse des Abwehrraechanismusim System Solanum tuberosum-Alterniria akernsCs
In einer vorherigen Untersuchung uber die Interaktion zwischen einem Altemaria alternata-Pathotyp und Solanum tuberosum, wurde in vitro eine Reihe von Parametern, die fur aktive undpassive Abwehrprozesse indikativ sind, an den toleranten (Chiquita) und anfaJligen (Superior) Sortenuntersucht. Ionenverlustversuche sowie das Wachstum von Suspensionsktilturen und in Platten im
Beisein von pilziidien Kulturfiltraten deuteten darauf hin, dafi die Toxintoleranz ein wichtigesMerkmal bei der Unterscheidung zwischen resistenten und anfaJJigen Genotypen ist. Im Hinblick aufdie verschiedenen fluoreszierenden Verbindungen, wahrscheinlich Phenolsaure, bei Gewebekulturenvon Chjquita oder anfalligen Sorten nach einer Applikation von pilzJichen EJicitoren hindeuteten,konnte kein einheidiches Muster festgestellt werden, das einen Zusammenhang mit der Abwehrvermuten liefi, Andererseits zeigten Peroxidaseisoenzymanalysen nach unterschiedlichen Dualkultur-perioden, dafi ionisch- und kovalent gebundene Peroxidasen nur bei der Sorte Chiquita im Beisein desPathogens aktiviert wurden.
Altemaria spp. are often called "saprophytic pathogens" (KOHOMOTO et al.1989) in the sense that their saprophytic behaviour may turn to pathogenicitythrough an activation of virulence mechanisms. As in Helminthosporium, thisprocess is often mediated by the production of host-specific toxins like thoseknown to be synthesized in different formae speciales oiAltemaria alternata (Fr.)Keissler. This is the case, for instance, for the AAL toxins oi Altemaria alternata.i. sp. lycopersid (GiLCHRiST and HARADE 1989) whose host specificity and interac-tion with the plant genotype have been studied in great detail and proposed as atool for screening pollen earring genes for resistance (BiNO et al. 1988). Toleranceto these and a series of other compounds like AM-toxin, AK-toxin, ACR-toxin,AF-toxin, ACT-toxin (KoHOMOTO et al. 1989) was found to be correlated withresistance. They have therefore been proposed as a tool for discriminatingresistant from susceptible germinating pollen grains. In view of the importance oftoxins in A. alternata pathogenesis the use of culture filtrates as a tool for direct invitro selection of resistant genotypes (THANUTONG et al. 1983) has been suggested.On the other hand, very little is known about possible active defence mechanismswhich may be involved in the complex interaction between A. alternata and hostplants.
The aim of this paper is to analyse several possible mechanisms of resistanceto an isolate of A. alternata which was found to be pathogenic to the potato(Solarium tuberosum). For this purpose, through screening of potato cultivars asusceptible (Superior) and a resistant (Chiquita) genotype were identified. Theirresponses to the pathogen were then analyzed in vitro in terms of active defence(peroxidase induction in dual cultures, phytoalexin production after treatmentswith heat-released cell wall components, called elicitors), and tolerance to culturefiltrate (growth on toxic media and ion leakage in the presence of toxins).
Material and Methods
Pathogenicity tests
Leaf disks of different cultivars ^vere inoculated with A. alternata conidia (20—50 conidia Xdisk) and maintained in a moist chamber at 27 ° ± 1 °C, 12 h photoperiod. A quantitative diseaseindex was obtained after 3 days by measuring chlorotic areas. After a broad cultivar screening,Chiquita and Superior were chosen as being representative, respectively, of resistant and susceptiblegenotypes, the average chlorotic areas being 4.5 ± 1.06 mm in Chiquita and 37.7 ± 4.46 mm inSuperior.
In vitro Analysis of Defence Mechanism 139
Plant material
Tubers from cvs Chiquita and Superior were sterilized in hypochlorite solution, 1 % for20 min, cut into discs (20 mm diameter, 1.2 mm thick) and placed on Faludi medium (Faiudi 1966).Explants were placed in a growth chamber at 25 ° ± I °C (14 h photoperiod). After about 2 weeks,the explants began to develop callus, which was transferred on the same medium every 4 weeks.
Fungal culture
A. alternata was grown on potato dextrose broth (PDB) in Petri dishes and maintained at28 ° ± ! °C under continuous light. To reactivate virulence attenuated by growth in culture, detachedpotato leaves were inoculated with cultures in a tnoist chamber every 2 months. After a few days,monosporic cultures were prepared on "V 8 medium" (10 % V 8 juice; 1.4 % agar) from conidiaisolated from leaves, and maintained in the dark at 18 ° ± 1 "C for 7 or 10 days. Conidia wereobtained from these cultures, suspended in distilled water and utilized.
Production of cukure filtrate
A mycelial fragment of the pathogen, maintained as described above, was transferred inErlenmeyer flasks containing 200 mi of liquid PDB and maintained in superficial standing culture at28 ° ± 1 °C under continuous light. Culture fluids were drawn at different times, filtered through a0.45/J nylon mesh and maintained at —80 °C; the mycehum was rinsed, weighted and frozen at- 8 0 °C.
Toxicity test of culture filtrates on tomato plantlets
Toxicity tests of culture filtrates were carried out prior to treatment of potato cells or tissueswith the aim of establishing a standard collection time for culture fluids. For this purpose tomatoplantlets grown from seeds under glasshouse conditions, were cut off and placed in test tubescontaining increasing percentages of A. alternata culture filtrates, drawn at different times afterinoculation of PDB. After 24 h the wilting was evaluated using an arbitrary index scaled from 0 to 10according to the degree of wijting. Toxic activity of the culture filtrate from A. aiternata, obtained 17and 28 days after inoculation, was assayed. Tests, carried out on 5 tomato plantlets for eachconcentration, showed a progressive increase of symptoms depending on the concentration used andhigher toxicity of the 28-day-old culture filtrate. In all further experiments culture filtrates collected2S days after inoculation were used.
Plating on toxic medium
Ten to 14 day-old suspension cultures were filtered through nylon mesh (900 ^ pore size),centrifuged at 800 X g for 5 min and resuspended in fresh medium at a concentration of 40,000 U/ml.Plating was carried out adding 1 ml of the cellular suspension to 4 ml of agarized medium contaitiingincreasing percentages of J4. alternata culture filtrate. Culture filtrate was added after sterile filtrationto obtain 4;8;16 and 32 % of the final concentration. Five Petri dishes were plated for eachconcentration of toxic filtrate. Sur\'iving colonies were counted after about 40 days and transferred tofresh toxic media in selection experiments.
Ion leakage
One g of tuber callus was placed in 30 ml of the culture medium containing 50 % A. alternataculture filtrate and 37 g/i sucrose or sucrose only. The increase in conductance was measured with aconductimeter (Toptronic X74174 Pabisch). Values were taken every minute after the addition ofculture filtrate. Changes in conductance due to the substance present in the filtrate were calculatedfrom the diference between the values registered in the presence and in the absence of filtrate at thesame times. All values were expressed in micro Siemens.
l O - '
140 PELLEGRINI et al.
Elicitor extraction
The extraction of possible elicitors of phytoalexin production was conducted according to themethods of GARAS et al. (1979). Frozen tnycelium was sonicated at 100 W for 10 ttjin at 4 °C for threeconsecutive titnes, and centrifuged at 20,000 X g for 40 min at 4 °C. The pellet was resuspended inborate buffer 0.05 M, pH 8.8, (v/w; 1.5 : 1), homogenized for 1 min and autoclaved for 10 min.Alternatively, the extraction was carried out at room temperature. The autociaved suspension wascooled in an ice bath and centrifuged at 1,500 X g for 40 min at 4 °C. The supernatant containingcrude elicitors was filter sterilized (0.45 ,u pore size) and stored at —80 °C. Elicitor concentration wasexpressed as mg glucose equivalent, and determined by the phenol-sulphuric acid method of Hodgeand Hofreiter (1962). Extraction was also perfortned without thermic shock, eliminating denaturationin the autoclave and proceeding with the other steps as described above.
Analysis of the response after elicitor treatment
Seven to 10 day-old tuber calluses were treated with A. altemata elicitor (1—2 drops/ctn tissue)and maintained at 25 ^ ± 1 °C in the dark for 96 h. The extraction of the substances induced by thetreatment was performed according to the methods described by BuiATTI et al. (1985) and by LYON(1984) tnodified as folloivs. Tissues were freezer dried for 36 h, ground with sand and suspended incold methanol (v/w 10 : 0.1 dry weight); the suspension centrifuged twice at 10,000 X g for 10 min at4 °C. The supernatants were then combined and dried under pressure in a rotary dryer at 40 °C; drymaterial was suspended in ethylacetate (v/w i : 0.1 dry weight) and stored at —20 °C. The extractswere developed on TLC (Silica Gel plates, 0.25 mm thick — Merck) with the elution mixture :chloroform: methanol (96 : 4). As a standard, 1 |Ug of pure rishitin and phytuberin were used. Plateswere visualized both under UV light (300 nm) and after spraying with sulphuric acid 98 % (BDH)followed by heating for a few minutes.
Dual culture and peroxidase extraction and assay
Dual culture experiments were carried out by s o ^ n g A. altemata conidia on filter paper disks(6.10* X disk), placed in Petri dishes in medium described by MuRASHlGE and SKOOG (1962) butsupplemented with 7.7 mg/1 PDB and 18mg/l 1-naphthalenacetic acid (NAA). Potato callus wasplaced at 1 cm from the filterpaper and collected at different times for peroxidase analysis. Soluble andcovalently bound peroxidases were extracted from tissues collected after different periods of dualculture according to AMPOMAH (1983). For the ionically bound fraction, pellets from the previouscentrifugation step were treated three times with 1 % Tritoti X-100. The extracts were desaltedthrough a G-25 Sephadex column (Pharmacia PD-10) and equilibrated with phosphate buffer 0.05 M,pH 6, before analysis. Protein content was determined by the Bradford reagent method (BRADFORD1976J. Peroxidase lsoenzymes were revealed using 7.5 % polyacrylamide gel slab electrophoresis withhigh pH discontinuous buffer system of DAVIS (1964). The sample concentration was 10/jg of totalprotein, the standard consisting of 2 units of horseradish peroxidase (Sigtna). Peroxidase staining wasperformed with guaiacol and H2O2 according to KAVand BASILE(1987). The gels were read at 546 nmwith a densitometer (SEAC minidensit).
Results
Response to toxic filtrates
To test a possible differential response of the two cultivars to toxic filtrates,suspension cultures from the two genotypes were plated on Faludi mediumsupplemented with culture filtrate from the pathogen at increasing concentrations(4, 8, 16, 32 %). The results reported in Table 1 show a higher tolerance of cv.Chiquita to toxic filtrates. Only cells from this cultivar were able to form colonies
In vitro Analysis of Defence Mechanism 141
Table 1Plating efficiencies of Chiquita and Superior cells plated on media
containing different concentrations of culture filtrate
Cultivar
Treatment ControlConcentration of culture filtrate
4 % 8 % 15 % 32
SuperiorChiquita
28.0 •
29.8 •3.4-
28.4 • 10-" 28.0 • 5.0 •
at concentrations higher than 4 %. Similar results were obtained when toxintolerance was measured by testing the effect of culture filtrate on cell membranepermeability through ion leakage analysis. As shown in Fig. 1 challenge withtoxic filtrates induced ion release only in cv. Superior but did not have any effecton Chiquita.
Fig. i. Ion leakage from cellsof the CVS Superior (=1-) andChiquita (O) in the presenceof A. alternata culture filtrateas judged from increases in
Fig. 2. Electropherograms of soluble peroxidases of cv. Chiquita (left) and Superior (right);1) Chiquita control, 2) Chiquita 4 days dual cultures, 3) Chiquita 6 days dual cultures, 4) Superior
control, 5) Superior 4 days dual cultures, 6) Superior 6 days dual cultures
"Active" response to the pathogen
Plant "active" defence mechanisms involve the activation of a series ofmetabolic pathways eventually leading to the inhibition of pathogen growth.Induction of peroxidase and the synthesis of antibiotic compounds have beenshown to be involved in defence processes in a number of cases (KALSA andSHARMA 1988, VANCE et al. 1976, SMITH and HAMMERSCHMIDT 1988), and weretaken as possible indicators of resistance in our system. The presence oi A. alter-nata in dual cultures preferentially elicited the synthesis of peroxidases in
Fig. 3. Electropherograms of ionically bound peroxidases of cv. Chiquita (left) and Superior (right);1) Chiquita control, 2) Chiquita 4 days dual cultures, 3) Chiquita 6 days dual cultures, 4) Superiorcontrol, 5) Superior 4 days dual cultures, 6) Superior 6 days dual cultures. The arrows point to specificbands (isozymes) which are shown to change with the periods of host-pathogen interaction (0, 4, 6
days). Vertical lines arbitrarily divide the peaks from one another
Chiquita as shown by in vivo staining obtained by transferring dual cultures onmedia containing guaiacol. The response to the pathogen was qualitativelydifferent for the two cultivars as can be seen from analysis of electropherogramsfor soluble, ionically bound and covalently bound peroxidases (Figs 2, 3, 4). Thesoluble peroxidase pattern after 6 days of dual culture did not seem to be differentfrom the controls in either of the two cultivars (Fig. 2), although late fractionsseems to decrease more in Chiquita at later times. When ionically (Fig. 3) or
Fig. 4. Electropherograms of covalently bound peroxidases of cv. Chiquita (left) and Superior (nght);1) Chiquita control, 2) Ghiquita 4 days dual cultures, 3) Chiquita 6 days dual cultures, 4) Superior
control, 5) Superior 4 days dual cultures, 6) Superior 6 days dual cultures. Arrows as in Fig. 3
covalently (Fig. 4) bound peroxidases were considered only Superior tended toremain stable. In Chiquita at least four new ionically bound peroxidase bandsappeared in the presence of the pathogen and increased with time. Moreover, onecovalently bound peroxidase band, constantly present in Superior seemed todisappear in Chiquita. The possible synthesis of phytoalexins was tested throughTLC of extracts of calluses of the two cultivars which had been pre-treated withelicitors extracted from A. alternata mycelium, both at room temperature and
In vitro Analysis of Defence Mechanism 145
after heat shock treatments, as described in Material and Methods. As shown inTable 2 UV sensitive patterns changed according to the treatments (biotic andabiotic elicitors). Moreover, differences were observed between patterns inducedby different elicitors, the difference being more evident in both cultivars whenHgCl2 effects were compared with those oi A. ahernata cell wall components.However, in no case could the production if rishitin, the most abundantterpenoid phytoalexin synthesized by the potato, be shown to occur.
Table 1TLC analysis (Rf values) of unknown compounds extracted from calli of a susceptibie (Superior) and atolerant (Chiquita) potato cv. treated for 96 h with A. altemata cell wall components extracted atroom temperature or after heat-shock, or with 5 mM HgCl2 (elution solvent: Chloroform : methanol,96 : 4 v/v.). Rf values correspond to UV-Huorescent compounds (300 ntn wave-length) and colours
were visualized after reaction to cone. H2SO4 and heating
Colour reaction to cone.1 2 3 4 5 6 7 8 HjSO, and heating
1) Superior calii control; 2) Superior calli treated with A. altemata room tetnperature released cell wailcomponents; 3) Superior calli treated with ̂ 4. ahernata heat released cell wall components; 4) Superiorcaili treated with HgClj; 5) Chiquita calli treated with HgCl2; 6) Chiquita calii treated with A.altemata heat released cell wall components; 7) Chiquita calli treated with A. altemata roomtemperature released cell wall components; 8) Chiquita calli control
Discussion
The results reported in this paper, although by no means conclusive, haveshown differences in in vitro response to A. altemata between a resistant cultivar(Chiquita) and a susceptible one (Superior) which seem to be consistent with theirbehaviour in vivo. This was particularly true when tolerances to toxic A. alter-naria filtrates were compared in the two genotypes, Chiquita being more resistantthan Superior both as judged from cell plating and ion leakage experiments. Dualculture experiments, showing specific patterns of ionically bound peroxidasesonly in Chiquita in the presence of the pathogen, suggest the possible existence ofan active defence mechanism in the resistant cultivar based on the production ofoxidised compounds as already hypothesized for the potnto-Phytophthora corre-lation. However, the synthesis of rishitin, the most abundant potato phytoalexin,was not observed in calluses pre-treated with fungal cell wall components.
146 PELLEGRINI et al. In vitro Analysis of Defence Mechanism
Therefore, although differences in the production of UV sensitive compoundswere shown to occur after elicitor treatment, no conclusive evidence of phytoale-xin involvement in defence mechanisms could be obtained in our experiments.
This work was supported by contracts EEC-BAP-0088-I and M.A.F. 16/7240/89.We thank Dr. ssa. R. MATTIOLI for the use of SEAC minidensit (SEAC, Via Carlo Del Prete,
139 Firenze, Italy).
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