Physiological response of grapevine cultivars and

a rootstock to infection with Phaeoacremonium and

Phaeomoniella isolates: an in vitro approach using

plants and calluses

Conceicao Santosa,*, Silvia Fragoeiroa, Alan Phillipsb

aDepartment of Biology, University of Aveiro, 3810 Aveiro, PortugalbFaculdade de Ciencias e Tecnologia, Universidade Nova de Lisboa, Quinta da Torre,

2829-516, Caparica, Portugal

Accepted 16 April 2004

Abstract

In vitro plants and callus culture of two Vitis vinifera cultivars (cv. Baga and Maria Gomes) and

one rootstock (R3309, i.e. Vitis riparia var tomentosa � Vitis rupestris) were inoculated with conidia

of Phaeoacremonium angustius and Phaeomoniella chlamydospora. Response to infection was

determined in plants grown in vitro by assaying growth rates, malondialdehyde (MDA) production

(lipid peroxidation) and chlorophyll content and fluorescence. Growth rate and malondialdehyde

production were also used to determine resistance of calluses to infection. Infection reduced growth

and increased MDA in infected plants and calluses, and reduced chlorophyll content and fluorescence

in infected leaves. Symptoms were more evident in plants infected with P. angustius, showing that

this species is more virulent to plants and calluses than Ph. chlamydospora. Differences in virulence

among strains of Ph. chlamydospora were also found, as 1AS and CAP053 were more virulent

(induced more severe decreases of growth and chlorophyll fluorescence, together with higher MDA

production in both cultivars) then CAP080. Growth of rootstock plants and calluses was less affected

by infection than growth of other cultivars. Contrarily to Baga and Maria Gomes, chlorophyll content

and fluorescence of rootstock plants were only affected by P. angustius. Also Baga plants and calluses

were more resistant than those of Maria Gomes. These data show different degrees of resistance

among genotypes. Reduction of callus production by infection supports the idea that fungus infection

may reduce cicatrisation by inhibiting callus formation during grafting or wounding; and therefore,

www.elsevier.com/locate/scihorti

Scientia Horticulturae 103 (2005) 187–198

* Corresponding author. Tel.: +351 234 370 780; fax: +351 234 426 408.

E-mail address: csantos@bio.ua.pt (C. Santos).

0304-4238/$ – see front matter # 2004 Published by Elsevier B.V.

doi:10.1016/j.scienta.2004.04.023

contribute to the entrance of opportunist pathogens. Implications of using in vitro cultures to assay

host/pathogen relationship and virulence/resistance degrees among the different genotypes of fungus

and grapevines are discussed.

# 2004 Published by Elsevier B.V.

Keywords: Esca disease; Phaeomoniella chlamydospora; Phaeoacremonium angustius; Vitis vinifera;

Resistance

1. Introduction

Esca is a serious disease of grapevines, with a wide variety of symptoms, affecting

vineyards all over the world (e.g. Italy, France, Portugal, USA and South Africa). This

complex disease is probably caused by a sequence of fungi, as proposed by other authors

Maurin (1986) and Larignon and Dubos (1997). Several fungi including Phaeoacre-

monium aleophilum, Phaeomoniella chlamydospora (formerly Phaeoacremonium

chlamydosporum), Phellinus punctatus, Stereum hirsutum (Larignon and Dubos,

1997), Eutypa lata, Phomopsis viticola and Phellinus ignarius (Stamp, 1999) have

been isolated from diseased grapevines. Due to the large variety of microorganisms that

have been found in diseased grapevines, it is uncertain which are really involved in the

development of the disease. According to the hypothesis of Larignon and Dubos (1997),

tissue colonisation may be done initially by Ph. chlamydospora and Phaeoacremonium

sp., followed by the colonisation of S. hirsutum and P. ignarius. The importance of

Phaeoacremonium in the development of this disease is also supported by the fact that

Phaeoacremonium sp. is almost always present in esca diseased trunks in many

countries, such as France (e.g. Larignon and Dubos, 1997), South Africa (Ferreira et al.,

1994), etc. These fungi are also associated with the Petri disease, detected in young

grapevine plants (Zanzotto et al., 2001) or with other vine declines as the ‘‘hoja mavon’’

(Gatica et al., 2000).

Several studies have been done on the pathogenicity/virulence of fungi that are

probably involved in esca disease. For example, Scheck et al. (1998) reported that 67%

and 71% of grapevine (cv. Carignane) plants died when infected, respectively, with P.

chlamydospora and Phaeoacremonium inflatipes. Khan et al. (2000) found that the

capacity for callus formation in grapevine (cv. Chardonnay) cuttings infected with P.

inflatipes, P. aleophilum and with Ph. chlamydospora was reduced by 72%, 22% and

62%, respectively. Also, infected cuttings suffered reductions in the number of

internodes, roots formed and dry weight with respect to healthy plants. Ferreira et al.

(1994) suggested that infection reduced the capacity to form callus tissue in cuttings

growing in vitro. More recently, Sparapano et al. (2000) reported that grapevine calluses

and in vitro plantlets from three cultivars responded differently to Ph. chlamydospora and

Phaeacremonium sp. infection.

The understanding of the infection process and the mechanisms involved in the

resistance may be an opportunity to develop strategies to combat this disease if, for

example, resistant genotypes and genes involved in resistance process are found.

Although this disease is relatively well documented in the field, host–pathogen field

trials pose some problems: (a) field results can be misleading and they are frequently

C. Santos et al. / Scientia Horticulturae 103 (2005) 187–198188

severely affected by seasonal influence; (b) they do not separate the effect of the pathogen

from effects induced by other biotic and/or abiotic agents present in the environment;

and (c) many field trial assays and breeding programs of woody plants are frequently

time consuming and selection of resistant lines may take years (Smalley and Guries,

1993).

Smalley and Guries (1993) recommend the use of short term assays in controlled

conditions for screening resistant lines, where in vitro cultures may be combined with

conventional ones in breeding programmes. Also, in vitro cultures are excellent tools to

study host–pathogen interactions as individuals are grown in extremely controlled

conditions. This methodology also allows a screening of a large number of genotypes in

short period and small areas. The more resistant lines could then be used for in vivo assays

in the field. Although the opportunities offered by in vitro culture, there are still few studies

reporting the effect of Phaeoacremonium sp. or Phaeomoniella sp. infection in axenic

grapevine plants or cells.

We report here the use of senescence parameters to evaluate the response of in vitro

grapevine plants to Phaeoacremonium sp. or Phaeomoniella infection. The use of in vitro

cultures in these kinds of studies is also discussed.

2. Methods

2.1. Fungi growth

Three isolates of Ph. chlamydospora (1AS, CAP053 and CAP080) and one of P.

angustius (CAP054) were collected from diseased grapevine plants in Portugal. Isolates

were maintained on solid Malt Extract Agar (MEA) at 25 8C in the dark. Spore suspensions

(107 spores/ml) were prepared in sterile distilled water and were used to inoculate axenic

grapevine plants and calluses.

2.2. Plant material and growth conditions

Cuttings of Vitis vinifera L. (cvs. Baga, Maria Gomes and rootstock R3309 (Vitis riparia

var tomentosa x Vitis rupestris) were supplied by Estacao Vitivinicola da Bairrada,

Portugal. Cuttings were rooted and maintained for two years in a greenhouse at 22 � 2 8C,

with Osram 18 W lamps emitting a light intensity of 458 � 3 mmol/m2/s and a photoperiod

of 16 h.

For micropropagation, explants consisting of nodal segments from greenhouse plants

were disinfected by rinsing in ethanol for 10 s and then by immersing in commercial bleach

for 15 min. After washing in sterile distilled water, explants were grown axenically on half

strength Murashige and Skoog (1962) medium (1/2MS) with 30 g/l sucrose, 0.6% (w/v)

agar, pH adjusted to 5.8 and supplemented with 4.4 mM BAP (6-benzylaminopurine) for in

vitro shoot proliferation. Shoots were rooted and maintained on the same 1/2MS medium

but supplemented with 0.1 mM IBA (indole-3-butyric acid). Cultures took place in a

growth chamber at a light intensity of 90 mmol/m2/s and a photoperiod of 12 h. Every four

weeks, plants were transferred to fresh medium.

C. Santos et al. / Scientia Horticulturae 103 (2005) 187–198 189

For callus induction, petiole fragments from axenic plants were grown on 1/2MS

medium with 30 g/l sucrose, 0.6% (w/v) agar, pH adjusted to 5.8 and supplemented with

2 mM BAP and 1 mM IAA (indole-3-acetic acid). Cultures took place in a growth chamber

under the conditions described above for in vitro plants. Every three weeks, calluses were

transferred to fresh medium.

2.3. Plant and callus inoculation

Two-month-old in vitro plants with approximately the same size and growing in

vessels (800 ml volume) containing 100 ml of rooting medium were used for infection

experiments. Each vessel contained two plants. Calluses with approximately the same size,

and growing in petri dishes containing 15 ml of 1/2MS medium with 2 mM BAP and

1 mM IAA, were used for callus infection experiments. Each petri dish contained two

calluses.

Plants were inoculated by applying 10 ml of a spore suspension at the base of the stem of

the in vitro plants. Control plants were treated in the same way, but the suspension of spores

was replaced by distilled water. Plants were grown under the conditions described above.

Samples were collected for analysis at days 3, 9, 15 and 21.

One-month-old calluses of each cultivar were injected with 5 ml of spore suspension of

each isolate. In control calluses, spore suspension was replaced by the same volume of

distilled water. Growth rates of calluses and plants were evaluated by determining fresh

weight every 8 days.

2.4. Determination of MDA production

Lipid peroxidation was determined by malondialdeyde (MDA) content according to

Dhinsa and Matowe (1981) in which 0.25 g of tissue samples were homogenized in 5 ml

trichloroacetic acid 0.1% (w/v) and centrifuged at 10000 � g for 10 min. The supernatant

was collected and 1 ml was mixed with 4 ml 20% (w/v) trichloroacetic acid and 0.5% (w/v)

thiobarbituric acid. The mixture was heated at 95 8C (30 min), quickly cooled and

centrifuged at 10000 � g for 10 min. MDA concentration in the supernatant was

determined from the absorbance at 532 and 600 nm according to Dhinsa and Matowe

(1981).

2.5. Chlorophyll content and fluorescence in infected plants

Leaves of in vitro plants were extracted with 80% acetone and chlorophyll content was

determined according to Arnon (1949). For chlorophyll fluorescence determination, leaves

were adapted to darkness for 20 min in a growth chamber at 22 � 2 8C. Fluorescence was

monitored in expanded leaves using a Plant Efficiency Analyser (Hansatech Instruments

Ltd., UK). Leaves were illuminated with a peak wavelength of 650 nm and a saturating

light intensity of 3000 mmol/m2/s. Basal fluorescence (F0), maximum fluorescence (Fm),

variable fluorescence (Fv = Fm-F0) and the ratio Fv/Fm were estimated (Maxwell and

Johnson, 2000).

C. Santos et al. / Scientia Horticulturae 103 (2005) 187–198190

2.6. Statistical analysis

Data were performed in triplicate with at least nine replicates in each independent

analysis. The significances of the differences between the means of the treatments were

compared by one-way and two-way ANOVA analysis (SigmaStat Program).

3. Results

3.1. In vitro plant and callus growth responses

The two grapevine cultivar plants and the rootstock plants showed similar in vitro

growth increments after 21 days and under control conditions (Fig. 1a–c). Growth of plants

was reduced by infection in both cultivars Baga and Maria Gomes. This reduction was

evident by the decrease of the number of internodes and ramification of shoots (Fig. 2) and

by the decrease of fresh weight (Fig. 1a–c). P. angustius induced more severe effects in

plants of both cultivars than the other fungus species (with a more drastic reduction of fresh

weight), but Baga plants were less affected than those of Maria Gomes (Fig. 1a and b).

Rootstock plants were more tolerant to infection than cultivars, as only P. angustius

reduced growth (at day 21, rootstock-infected plants had 42% the fresh weight of control

plants, Fig. 1c). Also, in all genotypes, senescence symptoms (e.g. chlorosis and growth

reduction) were detected sooner in plants infected with P. angustius (Fig. 1a–c) than in

plants infected with Ph. chlamydospora isolates. Leaf area of infected plants was also less

than in healthy plants (data not shown) and leaves showed dehydration and chlorosis with a

yellow or red colour.

Rootstock control calluses grew more slowly than those of Baga and Maria Gomes

(Fig. 1d–f). Calluses of the cultivars Baga and Maria Gomes developed a dark red-

brownish colour and reduced growth rates when infected with all strains (Fig. 1d and e)

while rootstock calluses became brownish but maintained growth showing a more severe

growth reduction with P. angustius (at day 21 these calluses had 40% of the fresh weight of

control rootstock calluses, Fig. 1f).

Similarly to what was observed in the in vitro plants, CAP054 (P. angustius) was the

strain that caused more severe damages in calluses, with calluses showing a reduction of

fresh weight (and other senescence symptoms) soon during the first days of infection

(Fig. 1d–f). Also some differences of virulence among isolates of the same species could be

observed. In fact, for both cultivars, the strains 1AS and CAP053 induced more severe

effects on plants and calluses growth than CAP080.

3.2. Senescence parameters

During the first days of assay, some leaves of infected plants showed intervascular

chlorosis. This chlorosis was confirmed by a decrease of chlorophyll a and b contents in all

infected Maria Gomes plants with the greatest effects in plants inoculated with CAP054

(where chl a and chl b decreased, respectively, 89% and 85%, Fig. 3a and b). Reductions of

84%, 77% and 43% were observed in chl a contents in Baga plants inoculated with

CAP054, 1AS and CAP053, respectively, while chlorophyll b only decreased in plants

C. Santos et al. / Scientia Horticulturae 103 (2005) 187–198 191

C. Santos et al. / Scientia Horticulturae 103 (2005) 187–198192

Fig. 1. Effect of spore infection on grapevine plant and callus growth: (a) Maria Gomes (MG) plant growth; (b)

Baga plant growth; (c) Rootstock R3309 growth; (d) Maria Gomes (MG) callus growth; (e) Baga callus

growth;and (f) Rootstock R3309 callus growth. Vertical bars mean the STDEV of three independent assays

inoculated with 1AS (64%) and CAP054 (87%) (Fig. 3a and b) resulting in an increase of

the ratio chla/chlb. The decrease of chlorophyll content was less evident in infected

rootstock plants than in infected Baga or Maria Gomes plants. In fact, in rootstocks, chl a

content only decreased significantly (72%) in plants infected with CAP054 isolate, while

chl b decreased 82% and 70% in plants infected with CAP054 and 1AS strains, respectively

(Fig. 3a and b). P. angustius (strain CAP054) affected more severely chlorophyll

fluorescence in grapevine genotypes than Ph. chlamydospora. Among Ph. chlamydospora

strains, 1AS and CAP053 caused more severe damage than the other isolate (CAP080). In

Baga and Maria Gomes plants, chlorophyll fluorescence variation was due to decreases of

Fm and often a decrease of F0, leading to a decrease of Fv/Fm ratio (Table 1). In contrast,

chlorophyll fluorescence of the rootstock was less affected by infection and F0, Fm and Fv/

Fm only decreased in plants infected with P. angustius (Table 1).

Leaves from infected plants had an increase of lipid peroxidation (and consequently a

decrease of membrane integrity) shown by the increase of malondialdehyde (MDA)

production (Fig. 4a). Infected Maria Gomes plants had highest values of MDA suggesting

that this cultivar is more sensitive to infection while the rootstock had the lowest values of

lipid peroxidation. Also, in contrast to what was observed in the genotypes Baga and Maria

Gomes (where all isolates induced an increase of MDA content), only CAP054 caused an

increase of MDA production from 18 nmol/gfw in control to 52 nmol/gfw in infected

rootstock plants (Fig. 4a). However, this increase was lower than the one observed in Baga

C. Santos et al. / Scientia Horticulturae 103 (2005) 187–198 193

Fig. 2. Aspect of grapevine (cv. Baga) plants infected with Phaeomoniella chlamydospora: (A) control at day 15;

(B) infected with Ph. chlamydospora (1AS strain) at day 15; (C) infected with Ph. chlamydospora (1AS strain) at

day 30; arrows: chlorotic regions. Bars: 5 cm.

(with at least three replicates each). Control (^); infection with CAP080 (*); infection with CAP053 ( );

infection with 1AS (~); infection with CAP054 (&). Table below shows statistically significant differences

among means of different assays (P < 0.05 with three independent assays with three replicates each).

C. Santos et al. / Scientia Horticulturae 103 (2005) 187–198194

Table 1

Chlorophyll fluorescence in Vitis vinifera leaves of cultivar Baga, Maria Gomes and Rootstock R3309.

Infection strain cultivar F0 Fm Fv/Fm

Control

Baga 687.2 � 33.2a 3811.5 � 143.2a 0.8197 � 0.1035a

Maria Gomes 734.1 � 48.1a 3977.2 � 186.5a 0.8154 � 0.0675a

R3309 710.2 � 54.3a 3614.5 � 268.3a 0.8035 � 0.0793a

Phaeomoniella chlamydospora

1AS

Baga 613.3 � 18.3b 1912.5 � 135.4c 0.6636 � 0.0253b

Maria Gomes 651.3 � 23.2b 1798.5 � 126.4c 0.6378 � 0.0111b

R3309 702.3 � 23.2a 2965.6 � 499.7a 0.7631 � 0.0953a

CAP080

Baga 785.4 � 94.3a 2765.6 � 45.6b 0.7160 � 0.1024ab

Maria Gomes 845.4 � 83.2a 2132.4 � 392.8b 0.6035 � 0.0023b

R3309 743.2 � 38.5a 3879.7 � 184.3a 0.8084 � 0.0263a

CAP053

Baga 602.4 � 45.3b 1876.5 � 104.3c 0.6789 � 0.0173b

Maria Gomes 598.3 � 52.4c 1236.4 � 154.7c 0.5160 � 0.0938b

R3309 724.4 � 33.6a 3875.3 � 201.7a 0.8130 � 0.1002a

Phaeoacremonium angustius

CAP054

Baga 403.7 � 36.5c 534.5 � 48.4d 0.2447 � 0.0045c

Maria Gomes 567 � 26.4d 723.2 � 38.1d 0.2159 � 0.0240c

R3309 508.3 � 42.1b 1335.6 � 108.5b 0.6294 � 0.0409c

Average � STDEV of three independent assays (with nine replicates each). F0: basal fluorescence; Fv: variable

fluorescence (Fv = Fm�Fo); Fm: maximum fluorescence. In the same column: same letter indicates significantly

not different means within the same cultivar (P < 0.05).

Fig. 3. Effect of spore infection on grapevine plant (a) chlorophyll a and (b) chlorophyll b contents (mg/pfw).

Vertical bars mean the STDEV of three independent assays (with at least nine replicates each): Baga ( ); Maria

Gomes (&); rootstock R3309 (&). Same letter indicates significantly not different means within the same cultivar

(P < 0.05).

and Maria Gomes plants showing that this isolate is less virulent to the rootstock. Infected

Baga and Maria Gomes calluses also produced more MDA than the rootstock (Fig. 4b).

MDA production was higher in all calluses infected with CAP054, and this was the only

fungus that induced MDA production in rootstock calluses (Fig. 4b).

4. Discussion

The role played by Ph. chlamydospora (CAP080, CAP053 and 1AS) and P. angustius

(CAP054) on the development of esca and other vine decays is still unknown but some

authors suggest that these are pioneer fungi (e.g. Larignon and Dubos, 1997). Most of the

studies available at the moment were not done axenically; and therefore, there was no

clear information on the real effect at the plant level of these fungi acting solely, without

the interference of other microorganisms or other uncontrolled conditions. In this work,

we analyzed some of the effects of infection of two species Ph. chlamydospora and

P. angustius in grapevine cells under extremely controlled conditions.

Besides growth analysis, the senescence of infected plants and calluses was expressed

by parameters that are frequently used to evaluate senescence in stressed plant cell systems

(e.g. Lutts et al., 1996; Santos et al., 2001). The increase of membrane degradation (MDA

production) in both plants and calluses together with a decrease of chlorophyll content

and fluorescence in plants show that these parameters are reliable and may be used as

bio-markers in these kind of phytopatological studies.

Chlorophyll content decreased significantly in infected plants, corroborating the observed

chlorosis and the development of a yellow-reddish color in leaves. This may be associated

with the fact that chloroplasts are one of the first places of catabolism when leaf senescence

starts (Quirino et al., 2000) with a concomitant decrease of photosynthetic efficiency; and

C. Santos et al. / Scientia Horticulturae 103 (2005) 187–198 195

Fig. 4. Effect of spore infection on grapevine (a) plant and (b) callus malondialdehyde production. Vertical bars

mean the STDEV of three independent assays (with at least nine replicates each). Baga ( ); Maria Gomes (&);

rootstock R3309 (&). Same letter indicates significantly not different means within the same cultivar (P < 0.05).

therefore, leading to decreased growth. Photosynthetic parameters, such as chlorophyll

content and photosynthetic efficiency are reported as senescence parameters (Buchanan-

Wollaston, 1997; Lutts et al., 1996; Santos et al., 2001). In particular, infection with CAP054

severely affected chlorophyll concentration and fluorescence. The stability of F0 found in

some Ph. chlamydospora-infected plants (e.g. Baga and rootstock) indicates that in these

cultivars infection with those isolates does not affect significantly the Photosystem II reaction

centres. The change found in plants infected with P. angustius (CAP054) indicates losses of

energy transference from pigments to the reaction centre associated with Photosystem II.

Changes in (Fm�F0)/Fm indicate changes in the photochemical efficiency of this

photosystem, mostly due to a reduction of the maximum fluorescence (Fm) and reflecting,

therefore, an increase of energy dissipation and deterioration of the photosynthetic apparatus.

This degradation of the photosynthetic apparatus may be closely related with the degradation

of plastid membranes that can be measured by the solute and/or electrolyte leakage and,

indirectly, by production of malondialdehyde.

P. angustius induced higher levels of MDA production, which shows that this fungus

strain caused more cell degradation than the other fungus species tested (Ph.

chlamydospora). The increase of lipid peroxidation is often related to a decrease of

membrane integrity that may lead not only to osmotic imbalances, but also to changes of

the photosynthetic apparatus (with thylakoid membrane degradation). On the other hand, if

enzymes involved in photosynthesis (e.g. rubisco) are affected, a decrease of the soluble

protein contents should also be expected and this was observed in leaves of grapevine

plants infected with CAP054 (Fragoeiro, 2001). A reduction of plant growth and

development of leaf chlorosis were described in in vitro italian grapevine cultivars infected

with some fungi associated with esca (Sparapano et al., 2001), but no further studies were

published up to the moment.

From this work, it can be concluded that the infection of in vitro plants induces

symptoms of senescence. In vitro plants seem to be a valuable tool in these kind of studies

as fast and reliable results can be obtained in a large number of plants. On the other hand,

these in vitro plants showed the same pattern of response showed by plants in a greenhouse

when infected with Phaeoacremonium, namely, growth rate reduction, chlorosis (mainly

intervascular) and necrosis (Chiarappa, 1959; Pascoe, 1999; Scheck et al., 1998a) and a

reduction of the root system (Khan et al., 2000). This similarity of results of in vitro and ex

vitro plants confirms that in vitro assays may replace, for some approaches, the more time

consuming and laborious assays with ex vitro plants.

In a similar way, the infection with callus tissue was also succeeded. Fungus infection

induced reduction of callus growth and an increase of membrane degradation. A reduction

of callus growth was also described by Dai et al. (1995) who inoculated grapevine calluses

with Phaeoacremonium viticola and by Khan et al. (2000) who infected grapevine cuttings

(cv. Chardonnay) and observed a reduction of callus formation. More recently, Sparapano

et al. (2001) observed decreases of callus growth when infected with some fungi associated

with esca. These studies on the interaction of pathogenic fungi and plant hosts grown in

vitro show that the use of callus tissue has advantages not only because it allows a large

number of samples in a short period and space, but also because senescence symptoms in

response to infection develop more quickly. Also in our study it was evident that the strain

that was more virulent to plants was also the most virulent to calluses, showing a similarity

C. Santos et al. / Scientia Horticulturae 103 (2005) 187–198196

in the pattern of response between calluses and plants in these grapevine genotypes. Using

in vitro plants and calluses, Sparapano et al. (2001) also found that some grapevine

cultivars were more tolerant to infection than others, suggesting that calluses and in vitro

plants may be a mean to select grapevines for resistance to esca.

Besides showing that calluses may be reliable culture systems for screening resistance

in grapevine cultivars to these two fungus species, the use of calluses in this study also

allowed to evaluate that callus proliferation is affected by infection confirming other

reports with in vitro cultures (Khan et al., 2000; Sparapano et al., 2001). Also, the rootstock

R3309 showed to be more tolerant than Baga and Maria Gomes genotypes with calluses

still dividing after infection. This higher tolerance may support the hypothesis proposed by

Morton (1997) that one way of transmission of esca disease may be by grafting with

infected rootstocks.

In conclusion, data show that among the senescence parameters studied here, plant/

growth, MDA production and chlorophyll content and fluorescence are reliable

parameters to distinguish infected from healthy plants, supporting previous suggestions

that these parameters may be used to indicate the general status of the plants exposed to

other stresses. Data also show different degrees of virulence between the two fungus

species and different resistance abilities among grapevine genotypes. Also the celerity of

this in vitro screening technique leads us to suggest that this technique may be used to

evaluate urgently the largest number of cultivars as possible in order to select those more

resistant to these fungi and include resistant cultivars in breeding programs of infected

areas all over the world. Another potentiality will be to find genes involved in resistance

to these fungi and to introduce them in susceptible but highly economically important

grapevine cultivars.

Acknowledgement

This work was supported by FCT/Proj PANAT/11142/AGR/98. Authors thank Eng.

Armando Costa, Miss Marta Costa, Miss Helena Valentim for technical support, and

Estacao Vitivinicola of Bairrada for providing plant material.

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