Phenotypic characterisation of Phaeoacremonium and

Phaeomoniella strains isolated from grapevines: enzyme production

and virulence of extra-cellular filtrate on grapevine calluses

Conceicao Santos a,*, Sılvia Fragoeiro a, Helena Valentim a, Alan Phillips b

a Department of Biology, University of Aveiro, Centre of Cell Biology, Aveiro 3800, Portugalb Universidade Nova de Lisboa, Monte da Caparica, Portugal

Received 16 March 2004; accepted 7 April 2005

Abstract

Extra-cellular enzyme production of different Phaeoacremonium spp. and Phaemoniella chlamydospora isolates were used to assay the

possibility of inter-specific characterisation. Isolates of Phaeoacremonium aleophilum, Phaeoacremonium angustius, P. viticola and Ph.

chlamydospora were grown on solid media and the activities of extra-cellular amylases, lipases, proteases, cellulases, xylanases, laccase,

polygalacturonase, pectate lyase, lignin peroxidase, manganese peroxidase, urease and chitinase were assayed. Phaeoacremonium species showed

activities of a larger number of enzymes and also enzyme activity was frequently higher suggesting that Phaeoacremonium can be more virulent.

To assay if the produced extra-cellular enzymes could reflect the virulence capacity of the two genera, calluses of Vitis vinifera L. (cvs. Baga and

Maria Gomes) and of a rootstock (R3309) were inoculated with filtrated culture liquid medium of three isolates of Ph. chlamydospora and one of

P. angustius. Filtrates from all strains decreased callus growth and membrane integrity, while soluble protein content of calluses decreased with the

strains CAP 054 and 1AS. P. angustius (CAP 054) induced the more severe symptoms in all genotypes. Water content decreased together with an

increase of osmolality in both cultivars but not in rootstock suggesting that osmorregulatory capacity is more affected in cultivars. Data show that:

(1) Phaeoacremonium and Phaeomoniella genera have different patterns of extra- cellular enzymatic production; (2) these fungi produce extra-

cellular compound(s) that induce(s) senescence symptoms in plant cells inhibiting callus proliferation; (3) among the strains tested in plant calluses

the most virulent isolate (CAP 054) also produced higher amounts of some extra-cellular enzymes; (5) rootstock calluses were less sensitive to

inoculation than grapevine calluses.

# 2005 Elsevier B.V. All rights reserved.

Keywords: Esca; Extracellular enzymes; Phaeoacremonium; Phaeomoniella; Virulence; Vitis vinifera

www.elsevier.com/locate/scihorti

Scientia Horticulturae 107 (2006) 123–130

1. Introduction

Esca is a highly destructive disease of Vitis vinifera L.

that is probably caused by a sequence or a combination of

microorganisms. Fungi of the genera Phaeoacremonium and

Phaeomoniella have been consistently isolated from grapevines

showing esca symptoms in several countries as USA, Portugal,

Spain, France, Italy, South Africa, Australia and Greece

(Calzarano and Marco, 1997). Besides being associated with

esca, Phaeomoniella chlamydospora (Ph. chlamydospora) and

Phaeoacremonium spp. have also been stated to be related with

DOI of related article: 10.1016/j.scienta.2005.04.015.

Abbreviations: BAP, benzylaminopurine; IAA, indolacetic acid; MDA,

malondialdehyde; MEA, malt extract agar; MS, Murashige and Skoog

* Corresponding author. Tel.: +351 234 370780; fax: +351 234 426408.

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

0304-4238/$ – see front matter # 2005 Elsevier B.V. All rights reserved.

doi:10.1016/j.scienta.2005.04.014

other vine affections as Petri disease (Zanzotto et al., 2001) and

the ‘‘hoja de malvon’’ decline (Gatica et al., 2000).

Some aspects of the interaction between the host and the

pathogen in these vine diseases are not clear yet. For example, it

is still unknown how the fungus enters the plant, being

considered different hypothesis that focus on soil (Sawyer,

1997), air or through the graft union (Bertelli et al., 1998;

Surico et al., 1998; Mugnai et al., 1999). Larignon and Dubos

(1997) studied the mode of action of fungi thought to be

involved in the primary (Cephalosporium sp. and Eutypa lata)

and secondary (Phellinus sp. and Stereum hirsutum) stages

of esca evolution. These authors found that the first group

of fungi degraded preferably cellulose and hemicellulose of

the secondary wall while the second group degraded all

components of cell wall (Larignon and Dubos, 1997). Also

Sparapano and collaborators studied the interactions between

grapevine plants and some fungi associated with esca as

C. Santos et al. / Scientia Horticulturae 107 (2006) 123–130124

Table 1

Phaeoacremonium and Phaeomoniella strains used in this work

Strain Observations

P. aleophilum

CBS 631.94 Isolated from Vitis vinifera L.; Italy

P. angustius

CBS 249.95T Isolated from Vitis vinifera L.; California, USA

CBS 100397 Isolated from Vitis vinifera L.; Italy

CAP 054 Isolated from Vitis vinifera L. cv. Tinta Barroca;

Montemor-o-Novo, Portugal

P. viticola

CBS 101738T Isolated from Vitis vinifera; France

CBS 101739 Isolated from Vitis vinifera; France

Phaeomoniella chlamydospora

CBS229.95T Isolated from Vitis vinifera L.; Italy

CBS 161.90 Isolated from Vitis vinifera L.; South Africa

CAP 052 Isolated from Vitis vinifera L.; Douro region, Portugal

CAP 053 Isolated from Vitis vinifera L. cv. Tinta Barroca;

Montemor-o-Novo, Portugal

CAP 072 Isolated from Vitis vinifera L.;

Senhora da Hora (Matosinhos), Portugal

CAP 080 Isolated from Vitis ‘‘Rootstock 99R’’;

Sobral de Monte Agraco, Portugal

CAP 081 Isolated from Vitis vinifera L. cv. Boal Ratinho;

Carcavelos, Portugal

1AS Isolated from Vitis vinifera L.; Bairrada, Portugal

CBS 249.95 and CBS 100397 are P. viticola according to Pedro Crouss (2002,

personal communication).

Fomitiporia punctata, Phaeoacremonium clhamydosporum

(presently Ph. chlamydospora) and P. aleophilum (Sparapano

et al., 2000a,b, 2001a,b). The same group also found that

phytotoxic extra-cellular compounds may play an important

role in the development of the disease (e.g. Sparapano et al.,

2000b, 2001b). Some of these phytopathogenic fungi produce

phytotoxins that may affect cell membrane, cellular transport

system or interfere with enzymatic reactions (Tabacchi et al.,

2000). Some phytotoxins were already isolated from fungi

associated with esca as E. lata, S. hirsutum, P. aleophilum and

Ph. chlamydospora (Evidente et al., 2000; Sparapano et al.,

2000b, 2001b; Tabacchi et al., 2000). Also it was demonstrated

that P. aleophilum produces phytotoxic exopolysaccharides

(pullulans) in vitro (Sparapano et al., 2000b). Besides

phytotoxins, many phytopathogenic fungi produce extra-

cellular enzymes that are able to degrade plant cell components

as, for example, xylanases, cellulases and pectinases, or

enzymes degrading carbohydrates of the host cell (St. Leger

et al., 1997). On the other hand, the relative activity of one or

more of these enzymes by the isolates may determine their

virulence degree. For example Erwinia carotovora ssp.

carotovora degrades plant tissues by the action of extra-

cellular enzymes and a mutant line lacking cellulases is less

virulent than the wild strains (Walker et al., 1994; Chatterjeen

et al., 1995). Recently, Marchi et al. (2001) reported the

production of some pectic extra-cellular enzymes by italian

Ph. chlamydospora isolates but no correlation with their

virulence was assessed.

The virulence of some Ph. chlamydospora and P. aleophilum

isolates was assayed in calluses and in vitro plants with

different responses among the vine cultivars (Fragoeiro et al.,

2000; Sparapano et al., 2001a). The use of callus cultures in

pathogen-host studies may present several advantages: first, as

callus formation occurs in vivo due to cuts in plant stem as a

way for scaring it is important to study how the pathogens affect

callus formation. If Phaeoacremonium comes into the plant

through the graft union and inhibits callus formation, this

inhibition can lead to a permanent wound in the stem from

where other pathogenic microorganisms can enter. Some

studies showed that infection of grapevine cuttings or calluses

reduced callus formation and/or growth (Khan et al., 2000;

Sparapano et al., 2001a). This reduction may be due to the

action of an extra-cellular compound released by the fungus, as

it was referred for other fungi (Deacon, 1997). Besides the

importance of callus formation during a grapevine life cycle,

the use of in vitro assays in studies preceding field assays also

has some advantages as defended by Smalley and Guries

(1993). In fact, these authors recommend the combination of

short term assays (e.g. in vitro) with field ones in breeding

programmes (e.g. screening resistant lines), as the first are

generally performed under extremely controlled conditions and

are, therefore, excellent tools to study host-pathogen interac-

tions. 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 spp.

or Phaeomoniella spp. infection in axenic grapevine plants or

cells. For example, Sparapano et al. (2001a) used callus culture

to assay resistance to these fungi. Recently our group studied

the response of both calluses and plants to different

Phaeoacremonium and Phaeomoniella isolates and to fungi-

cides (e.g. Fragoeiro et al., 2000; Oliveira et al., 2001; Santos

et al., 2005a,b) and agree with Sparapano group when they

proposed that calluses may be a mean of select grapevines for

resistance to esca (Sparapano et al., 2001a).

It was our goal to verify if Phaeoacremonium spp. and

Phaeomoniela chlamydospora isolates produce the same type

of extra-cellular enzymes and check if they release any

substance to the culture medium that causes toxicity in plant

cell, and in particular in the cicatrising tissue (callus).

Parameters used here to measure the effects of fungus

extracellular compounds in callus cells were already reported

to be reliable indicators of the general status of stressed plant

cells (e.g. Santos et al., 2001; Brito et al., 2003).

2. Material and methods

2.1. Fungus collection and growth

Several isolates obtained from CBS (CBS, The Netherlands)

culture collection and Portuguese field isolates (Dr. Alan

Phillips private collection) belonging to the species Ph.

chlamydospora, Phaeoacremonium angustius, P. aleophilum

and P. viticola were used in this work (Table 1). All isolates

were routinely grown on MEA (Malt Extract Agar) at 25 8C.

C. Santos et al. / Scientia Horticulturae 107 (2006) 123–130 125

2.2. Fungus extracellular enzyme production

Extra-cellular enzyme production was assayed by growing

the fungi on solid media containing the appropriate substrate:

amylase, lipase (Hankin and Anagnostakis, 1977), proteases,

polygalacturonase, pectate lyase and chitinase (adapted from

Hankin and Anagnostakis, 1977), cellulase and xylanase (St.

Leger et al., 1997), urease (Hankin and Anagnostakis, 1977),

lignin and manganese peroxidase (Conesa et al., 2000) and

laccase (Rigling, 1995). The assays allow the detection of the

enzymatic activities by measuring the area of transparent or

coloured halos produced in the culture medium or in the

mycelium. The plates were incubated at 25 8C in the dark for

2–4 weeks. All tests were repeated al least three times (with

three replicates each).

2.3. Preparation of fungus culture filtrate

P. angustius (CAP 054) and Phaeoacremonium chlamydos-

pora (1AS, CAP 053 and CAP 080) strains were grown in

250 ml liquid Czapeks medium, with constant stirring at 25 8C.After 15 days, 50 ml of the culture medium were centrifuged at

10,000 � g at 4 8C and the supernatant was filtered through a

0.2 mm pore filter.

2.4. Grapevine callus growth and inoculation

The grapevine cultivars (V. vinifera L. cvs. Baga and Maria

Gomes) and the rootstock (R3309, i.e. Vitis riparia var

tomentosa x V. rupestris) were kindly provided by Estacao

Vitivinıcola da Bairrada, Portugal. For callus induction,

cuttings were disinfected according to Santos et al. (2005a)

Table 2

Extra-cellular enzymes produced in vitro by Phaeoacremonium spp. and Phaeomo

Strain Enzyme

Protease

(cm2)

Lipase

(cm2)

Amylase

(cm2)

Cellulase

(cm2)

Xylanase

(cm2)

Poligalac

(cm2)

Ph. chamydospora

CBS 229.95 16 � 4 a 11 � 4 a 12 � 3 a 10 � 3 a 7 � 3 a 11 � 3 a

CBS 161.90 13 � 2 a 15 � 5 a I5 � 5 a 14 � 2 ab 6 � 2 a 10 � 2 a

CAP 052 13 � 4 a 12 � 3 a 11 � 4 a 9 � 4 a 8 � 2 a 12 � 4 a

CAP 053 28 � 2 b 11 � 3 a 15 � 2 a 11 � 2 a 14 � 4 b 11 � 5 a

CAP 072 23 � 4 b 20 � 6 a 12 � 5 a 8 � 3 a 12 � 2 ab 6 � 4 a

CAP 080 21 � 6 ab 16 � 5 a 15 � 4 a 5 � 2 a 15 � 2 b 11 � 3 a

CAP 081 12 � 3 a 11 � 4 a 11 � 4 a 10 � 4 a 9 � 3 a 16 � 5 a

1AS 14 � 2 a 13 � 6 a 18 � 3 a 15 � 2 b 14 � 3 b 17 � 4 b

P. angustius

CBS 249.95 33 � 4 c 14 � 5 a nt 19 � 2 bc 18 � 2 bc 25 � 5 b

CBS 100397 32 � 3 c 13 � 3 a nt 22 � 3 c 23 � 4 c 27 � 6 b

CAP 054 38 � 5 c 11 � 3 a 20 � 4 a 21 � 3 c 25 � 8 c 24 � 5 b

P. aleophilum

CBS 631.94 35 � 5 c 12 � 4 a 10 � 3 a 18 � 3 bc 27 � 4 c 22 � 4 b

P. viticola

CBS 101738 31 � 4 c 14 � 3 a 13 � 4 a 15 � 3 bc 25 � 2 c 25 � 6 b

CBS 101739 33 � 2 c 14 � 4 a 12 � 3 a 21 � 4 c 18 � 3 bc 21 � 4 b

Quantification is evaluated by determining the area (mean � S.D.) of the halos in the

significantly different means among the strains within the same enzymatic assay (P �

and grown on half strength Murashige and Skoog (1962)

medium (1/2MS) supplemented with 4.4 mM BAP (6-benzy-

laminopurine), 30 g/l of sucrose and pH adjusted to 5.8, at a

light intensity of 90 mmol m�2 s�1 and a photoperiod of 12 h

for shoot proliferation. Explants were obtained from in vitro

petioles and were grown on half strength MS medium (1/2 MS)

supplemented with 2 mM BAP and 1 mM IAA (indole-3-acetic

acid), under the same conditions described above. One month-

old calluses were inoculated with 10 ml of fungus culture filtrate.

Controlwas inoculatedwith sterileCzapecksmedium. Initial and

final callus weights were determined at 0, 10 and 20 days.

The effect of filtrate on plant cell proliferation and

senescencewere determined by (a) callus fresh weight increase;

(b) callus osmolality and water content, as described by

Brito et al. (2003); (c) membrane integrity, by quantifying

malondyaldehyde (MDA, correlated with lipid peroxidation)

production according to Dhinsa andMatowe (1981); (d) soluble

protein content using Bradford (1976) method.

2.5. Statistical analysis

Values are given as mean � S.D. as calculated from data

from three independent experiments in which samples were

performed in triplicate. Values were statistically tested using

the one-way and two-way ANOVA analysis for significance

between the means of the control and the means of inoculated

samples, assuming a significance of P � 0.05.

3. Results

The results obtained for the phenotypic characterisation

(extra-cellular enzyme production) of the strains are presented

niella spp.

turonase Pectate

lyase

(cm2)

Chitinase

(cm2)

Urease

(cm2)

Laccase

(cm2)

Lignin

peroxidase

Managanese

peroxidase

(cm2)

– – 13 � 3 a – – –

– – 10 � 4 a – – –

– – 15 � 5 a – – –

– – 11 � 5 a – – –

– – 15 � 2 a – – –

– – 18 � 6 a – – –

b – – 10 � 3 a – – –

– – 11 � 4 a – – –

6 � 2 a – 15 � 3 a 7 � 1 a – –

7 � 3 a – 14 � 5 a 8 � 2 a – –

11 � 4 a – 16 � 3 a 7 � 3 a – –

10 � 3 a – 10 � 2 a 12 � 4 a – –

13 � 4 a – 13 � 5 a 10 � 2 a – –

10 � 3 a – 15 � 6 a 13 � 6 a – –

mediumwith the specific substrate. In the same column, different letters indicate

0.05; with three independent assays with three replicates each). nt: not tested.

C. Santos et al. / Scientia Horticulturae 107 (2006) 123–130126

Fig. 1. Example of plate assays for detection of extra-cellular enzyme production: (A) proteases (positive); (B) amylase (positive); (C) laccase (positive); D-cellulase

(positive); (E) lipases (positive); (F) phosfatases (positive); (G) pectinase (poligalacturonase; positive); (D) pectin lyase (negative).

in Table 2 and Fig. 1. By the biochemical methods used it was

not possible to detect chitinase, lignin and manganese

peroxidase activities in all strains tested. All strains produced

protease, lipase, amylase, cellulase, xylanase, pectinase

(polygalacturonase) and urease activities. Also, it should be

noted that strains belonging to Phaeoacremonium genus (P.

aleophilum, P. angustius and P. viticola) had, for some of these

enzymes, higher enzymatic activities than Ph. chlamydospora

(Table 2), revealed by the production of bigger halos in the

culture media, and only Phaeoacremonium species showed

pectate lyase and laccase activities. On the other hand, there

was some heterogeneity, mostly among Ph. chlamydospora

strains in protease, xylanase and cellulase activities. For

example, for pectinase (poligalacturonase), CAP 081 and 1AS

strains showed a higher activity than the remaining strains of

Ph. chlamydospora (Table 2). Contrarily, Phaeoacremonium

species showed some homogeneity in what concerns enzymatic

activities, as the halos dimensions did not differ significantly

among the strains of the three Phaeoacremonium species. All

strains showed no ability to degrade chitin.

3.1. Effect of extracellular filtrates on in vitro cell

proliferation

The influence of extracellular compounds released by the

fungus in the production of cicatrising tissue was simulated in

vitro by cultivating grapevine calluses (cvs. Baga and Maria

Gomes and the rootstock R3309) in the presence of fungus

filtrate (CAP 054 and CAP 053, 1AS and CAP 080 strains) and

the effects on calluses growth, membrane degradation and

soluble protein content were determined.

Calluses inoculated with the filtrate developed a brownish

colour and showed a reduction in growth. This reduction was

more evident in Maria Gomes cultivar while the rootstock was

less affected. Fig. 2 shows growth curves of Baga,Maria Gomes

and R3309 calluses (inoculated with CAP 054 and with CAP

053, CAP 080 and 1AS filtrates). CAP 054 filtrate causes the

worst effect in relation to growth rate in all grapevine

genotypes.

Respectively to callus senescence parameters, the CAP 054

extra-cellular filtrate induced not only the highest decrease of

callus growth but also the highest degree of lipid peroxidation

(Fig. 3a), together with a decrease of protein content (Fig. 3b).

Also, calluses inoculated with this strain showed less water

content (Fig. 4a) and higher osmolality (Fig. 4b).

4. Discussion

The study of the enzymatic machinery produced by a

pathogen may be a precious tool in the understanding of

virulence factors. Among the enzymes produced by pathogenic

microorganisms, those capable of degrading polysaccharides

are of particular importance (see Warren, 1996). According to

St. Leger et al. (1997) the enzymatic machinery of a fungus

reflects evolutive mechanisms that lead to a pathogenic

adaptation to a given host or group of hosts. So, these fungi

are clearly adapted to live in plants as hosts.

Among the tested enzymes, data show that all strains have no

ability to degrade chitin. Extra-cellular chitinases are produced

preferentially by entomopathogenic fungi and others whose

hosts are chitinous (Hodge et al., 1995; Gooday, 1999).

Phytopathogenic fungi produce several enzymes capable of

hydrolysing these macromolecules in plant tissues, namely

cellulose, pectin, xylan and lignin (e.g. Binz and Canevascini,

1996; St. Leger et al., 1997). Among the Phaeoacremonium and

Phaeomoniella strains studied, all produced several polysac-

charides (amylase, xylanase, cellulase and pectinase). No

differences were found in amylase activity between the two

genera. In the case of esca, the fungi associated with the disease

exhibit several capabilities in using nutrients available in the

tissues, such as starch stored abundantly in medullar

parenchyma.

With respect to xylanase, although all isolates presented this

cell wall degrading enzyme, Phaeoacremonium species had a

higher activity suggesting a higher ability to degrade xylans.

Xylans are predominantly found in the secondary walls of

mature plant cells in woody tissue and represent a minor

fraction in primary walls; they are, however, the major

C. Santos et al. / Scientia Horticulturae 107 (2006) 123–130 127

Fig. 2. Effect of extra-cellular filtrate on grapevines callus growth of Baga (a), Maria Gomes (b) and the rootstock R3309 (c): control (^); CAP 080 (*); CAP 053

( ); 1AS (~); CAP 054 (&). Table below shows statistically significant differences among means of different assays (P � 0.05; with three independent assays with

three replicates each).

Fig. 3. Effect of extra-cellular filtrate on grapevines callus malondialdehyde (MDA) production (a) and soluble protein content (b). In each graphic, same letter

indicates significantly not different means (P < 0.05; with three independent assays with three replicates each).

C. Santos et al. / Scientia Horticulturae 107 (2006) 123–130128

Fig. 4. Effect of extra-cellular filtrate on grapevines callus water content (a) and osmolality (b). In each graphic, same letter indicates significantly not different means

(P � 0.05; with three independent assays with three replicates each).

hemicellulose found in angiosperms (Binz and Canevascini,

1996). Extracellular xylanases may be involved in the

interaction between plants and pathogens: firstly, xylanases

are produced by a wide range of fungal pathogens; secondly, as

hemicellulose contributes to the rigidity of cell wall it

represents a mechanical barrier to penetration, and finally,

xylans of mature cell walls are potential source of nutrient to

invading microorganisms (Binz and Canevascini, 1996), Data

presented here suggest that Phaeoacremonium species have

higher abilities to degrade hemicellulose than Ph. chlamydos-

pora but also that, as reported for other pathogenic fungi (e.g.

Binz and Canevascini, 1996), this cell wall enzymemay play an

important role in the evolution of esca disease, enabling the

fungus to colonize the woody tissues.

Respectively to cellulose, also the higher activity of

cellulose found in Phaeoacremonium species may suggest

that species from this genus may attack more easily plant

cells, and therefore may be more virulent. Additionally, P.

aleophilum, P. angustius and P. viticola strains produce another

polysaccharidase (pectate lyase). Polygalacturonase and pec-

tate lyase have as substrate polygalacturonate and low

methylated pectin but they differ in their cleavage mechanism

and in their optimum pH (Marchi et al., 2001). From these

results it can be seen that Ph. chlamydopora uses the hydrolase

polygalacturonase to degrade pectic substances, while Phaeoa-

cremonium species use both pectate lyase and polygalactur-

onase to obtain carbon sources. Recently, Marchi et al. (2001)

reported that Ph. chlamydospora italian isolates also produced

polygalacturonase in vitro and some intraspecific heterogeneity

was found, corroborating the findings reported here. We show

here that Phaeoacremonium produces a larger battery of pectin

degrading enzymes than Ph. chlamydospora.

The polysaccharidases produced by these fungi are

apparently of great importance in the degradation of plant

cell wall components, and may also be associated with the

development of symptoms such as the spots in grapes that may

be the result of the oxidation or polymerization of phenolic

compounds (Mugnai et al., 1999). According to these results we

suggest that the genus Phaeoacremonium may have higher

potential to be more adaptable and to act differently according

to host conditions, and it may be, therefore, a pioneer fungus

due to its larger battery of cell wall degrading enzymes.

All Phaeoacremonium species (P. aleophilum, P. angustius

and P. viticola) produce laccase, a poliphenoloxidase involved

directly or indirectly in lignin degradation (Reid, 1995), while

other enzymes involved in this process, such as lignin and

manganese peroxidases (Reid, 1995) were not found. The well-

known ligninolytic activity of white rot basidiomycetes, that

confers them the ability to completely degrade lignin, suggests

that F. punctata has a key role in the development of white rot in

grapevines affected by esca (Chiarappa, 1959; Mugnai et al.,

1997). In the work of Mugnai et al. (1997) only F. punctata

strains produced laccase. However, our results also show that

Phaeoacremonium species have this enzyme suggesting that

P. aleophilum, P. angustius and P. viticola may also have

some part in the process. Therefore the ability to degrade lignin

due to laccase activity (that has previously been attributed to

F. punctata) may, with this report, be extended to Phaeoacre-

monium species. This enzyme may also be important in the

pioneer function attributed to these fungi, participating in the

detoxification of phenolic compounds like resveratrol synthe-

sized by the plant in response to infection (Mugnai et al., 1997)

and that was shown to have an efficient effect in limiting in vitro

growth of these fungus species (Fragoeiro et al., 2000; Santos

et al., 2005b). The effect of laccase in phenolic detoxification

has already been proposed for other fiingi like B. cinerea

(Amalfltano et al., 2000).

In some white rot fungi two heme peroxidases (lignin and

manganese peroxidases) are the major components of the

lignin degradation system (Conesa et al., 2000) and should

also be expected to be present in fungi involved in esca

development. However, they were not detected in the strains

of the species tested. Peroxidase may however be present

in at least some strains as Mugnai et al. (1997) detected

peroxidase in only one strain of P. aleophilum, contrarily to

the majority of F. punctata strains that showed this enzyme

activity.

Some of the enzymes mentioned above are recognized as

virulence factors of several fungi, namely phytopathogenic

(e.g. Rigling, 1995; Binz and Canevascini, 1996; St. Leger

et al., 1997). These data for phenotipic characterisation also

correlate, in general, with the genetic characterisation of these

isolates that was already done in order to establish genotypic

differences among these different species (Alves et al., 2001,

C. Santos et al. / Scientia Horticulturae 107 (2006) 123–130 129

2004). Using genomic sequences, these authors showed that

there was a clear distinction between Ph chlamydospora and the

other Phaeoacremonium species, and that P. aleophilum and P.

angustius are grouped closely supporting other reports that

these two species may represent only one, while P. viticola

seems to be amore distinct group within this genus (Alves et al.,

2001, 2004).

4.1. Effect of extracellular filtrates on in vitro cell

proliferation

The reduction of growth, the higher levels of lipid

peoxidation, the decrease of soluble protein content, together

with the higher osmolality and decrease of water content

suggest that infected tissues were unable to osmoregulate as a

probable consequence of membrane degradation, induced by

the fungus filtrate. These data show that, similarly to what was

reported for in vitro Baga andMaria Gomes plants infected with

spores of these two fungus species (Santos et al., 2005a),

protein content, MDA production and osmoregulation capacity

may be used as reliable senescence parameters to evaluate plant

cell status after inoculation with culture medium filtrate.

This report shows a clear correlation between the higher

production of some extra-cellular enzymes and virulence;

it is evident that the species P. angustius has a characteristic

production of some enzymes and shows higher virulence to

grapevine cells than other Phaemoniella sp. isolates. Data also

demonstrate that both P. angustius and P. chlamydospora

produce extra-cellular substances that are intervenients in the

infection process and that extra- cellular filtrate of P. angustius

causes more severe damages in plant cells than Ph.

chlamydospora filtrate. This data is in accordance with

previous data that showed that P. angustius spores are generally

more virulent than those of Phaeomoniella spp. to grapevine

plants (Fragoeiro et al., 2000; Santos et al., 2005a). Some recent

experiments also demonstrated that introduction in grapevine

plants of phytopathogenic metabolites (pollulans and naphta-

lone pentaketides) produced by Ph. chlamydospora and/or P.

aleophilum induced some symptoms similar to those shown by

esca-affected vines (Sparapano et al., 2000b). It is in course the

analysis and purification of some of these extra-cellular

enzymes for application independently in axenic plants in order

to evaluate the development of esca-like symptoms.

Finally, this report also shows that, for grapevine studies,

tissue cultures are a convenient means for use in bioassays to

determine the susceptibility/resistance of a plant/host when

infected by the pathogen or by one of its phytotoxins supporting

findings for other strains and cultivars of Sparapano et al.

(2001b). Recently, our group found some similarity in the

behaviour of in vitro grapevine plants and calluses when

infected with spores of P. angustius (CAP 054) or of Ph.

chlamydospora (1AS) isolates (Santos et al., 2005a) and to a

combination of infection and pesticides (including resveratrol

that is produced by vines, Santos et al., 2005b) which suggests

that calluses may be a mean of study this grapevine disease and

select genotypes for resistance to esca or to screen fungus

strains virulence.

Acknowledgment

This work was supported by FCT, Project PRAXIS/PANAT/

11142/AGR/98.

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