4. Discussion
In this study, from a 20-year-old V. vinifera cv. Italia plants
surveyed for “Esca complex of diseases” symptoms development, we
select five vines with brown wood-streaking, five with brown
wood-streaking and white rot and five healthy vines. The isolation
procedure applied on wood cores taken with a Pressler increment borer,
associate Pm. minimum and Pa. chlamydospora to brown
wood-streaking and F. mediterranea to rotted white
tissues. Species of Penicillium and Alternaria but alsoAureobasidium , Chaetomium , Cladosporium ,Epicoccum , Fusarium , Gibberella , Nectria ,Phoma , Paraconiothyrium and Trichoderma have been
reported as endophytic mycota associated with vines as secondary
invaders or saprophytes (Moreno-Sanz, et al., 2013; Jayawardena, et al.,
2018; Elena et al., 2018).
No alterations were viewed on HV, while symptoms were recorded on leaves
and berries of Pm. minimum and Pa. chlamydospora orPm. minimum , Pa. chlamydospora and F.mediterranea infected plants. BWSV and BWSWRV display the
so-called ‘tiger stripes’ pattern on leaves and spots, shrivelling and
wilt of berries. These observations agree with other Authors on this
cultivar (Bruno & Sparapano, 2006b, 2007) and in general for vines
affected by “Esca complex” (Mugnai, Graniti & Surico,1999; Graniti,
Surico & Mugnai, 2000; Surico, 2009; Mondello et al., 2018).
The selected vines allow us to collect xylem sap during the bleeding
period and leaves at the phenological phases of stretched-out leaves,
fruit setting, cluster closing and bunch ripening. Bleeding is a
physiological process that characterizes vine and many perennial plants
as an effect of positive root pressure that upward transport water and
solute. Bleeding occurs in the springtime since soil temperature
stimulates root growth and leads xylem vessels, dissolves and pushes out
air bubbles formed during the winter and restores xylem works (Sperry,
Holbrook & Zimmermann, 1987). During vascular diseases, permanent xylem
blockage is the result of both the fungal presence (conidia, hyphae and
high molecular weight substances secreted by the pathogen) and
development of tyloses and gummosis as plant-barriers that limiting the
fungal invasion. To bypass obstructions, plants respond with a reduction
of leaf potential. Polyphenol-rich zones formation, the accumulation of
pathogenesis-related-proteins and the activation of oxidative burst
contribute to inhibit pathogens progression in the wood. Pm.
minimum and Pa. chlamydospora phaeo-tracheomycosis are
characterized by streaking and necrosis of varying extent in the wood of
the trunk and principal branches of affected vines. The presence ofF. mediterranea results in lignin degradation, the
occurrence of areas consists mainly of cellulose and suggests increases
xylem dysfunction. In these conditions, bleeding sap collected was
reduced. In agreement with previous papers, diseased vines bleed more
abundantly than HV (Bruno & Sparapano, 2006b; Bruno, Sparapano &
Graniti, 2007).
Analysis of the bleeding sap provides useful perceptions of internal
physiological functions. V. vinifera bleeding xylem sap
composition included oxalic, tartaric, malic, glutamic and succinic
acids, chlorides, sulphates, nitrites, nitrates, silicates and
phosphates of sodium, potassium, calcium, iron, magnesium, manganese and
aluminium, diastase, peroxidase and catalase (Wormall, 1924). Plant
hormones (Skene, 1967), phenols, sucrose, glucose, fructose and, on
vines infected by Pa. chlamydospora and Pm. minimum ,
isosclerone, scytalone and pullulans
(Bruno & Sparapano, 2006b, 2007)
were also present.
In this study, BXS was characterized by total ascorbate and glutathione
concentrations, dynamic viscosity coefficients and growth regulators
activity.
Viscosity is the ability of a fluid layer to run with the adjacent one.
Our study reports an increment of the dynamic viscosity coefficient from
HV to BWSV or BWSWRV. These results suggest that substances produced by
the fungal ”endophytes” as well as molecules resulting from
cell-components degradation by the lytic enzymes formed by these
pathogens and vine-response molecules (phenols, tannins, flavonoids, and
other) could influence dynamic viscosity coefficients, and as
consequence xylem flow and potential.
The filter paper disk bioassay (Zhao et al, 1992) correlates the
concentrations of growth regulators substances to their physiological
effects on cucumber cotyledon root formation (auxin) and fresh weight
variations (kinetin). The presences of several plant hormones in the
xylem sap have been displayed on herbaceous and woody plants including
grapevine (Niim & Torikata, 1978). In our research, the higher auxin
and kinetin like activity was detected on diseased vines. Auxin activity
increases when vines show white-rot associate to F.mediterranea .
Plant disease is the result of a constant chemical cross-talk among
pathogen actions and plant defence reactions. Pathogens interfere on the
plant with their pathogenicity or virulence tools, including chemical
weapons (i.e. enzymes, phytotoxins, polysaccharides,
plant-growth-regulators). These substances interfere with the different
physiological function(s), cell organelles and molecules of the plant
and lead to developing morphological changes. Plants react to pathogens
penetration and spread with different weapons. “Esca complex”, and
each of the diseases Esca-associated, can be read in this contest.F. mediterranea , Pm. minimum and Pa.
chlamydospora , closely associated to wood tissues of “Esca complex”
affected grapevines, produce phytotoxic metabolites (Evidente et al.,
2000; Tabacchi et al., 2000; Bruno & Sparapano, 2006a, 2006b; Andolfi
et al., 2011; Luini et al., 2010) and enzymes (Mugnai, Graniti &
Surico, 1999; Andolfi et al., 2011; Chiarappa, 1959; Marchi et al.,
2001; Bruno & Sparapano, 2006c). Vines defence responses included
physical responses or metabolic changes, such as the formation of
tyloses and gels in xylem (Yadeta & Thomma, 2013), cell wall chemical
modifications with suberin deposition (Pouzoulet et al., 2013),
production and accumulation of peroxidases, superoxide dismutases,
glutathione S-transferases, phenolic compounds, stilbenes, and
phytoalexins (del Rio et al., 2004).
Here, differences were recorded on leaves about fresh and dry weight,
leaf surfaces and chlorophyll concentrations. These features varied with
the phenological phases but are significantly affected by the behaviour
of the pathogens inside the woody tissues and, as a consequence, of
physiological function(s) altered. Symptomatic leaves always showed the
lower fresh and dry weight and total chlorophyll concentration. Diseased
plants show a general decline of photosynthetic pigments even with the
absence of visible changes. As reported for Cabernet Sauvignon and
Merlot (Christen et al., 2007), our data suggest that physiological
dysfunctions related to photosynthesis are presents. A decrease of gas
exchange and chlorophyll fluorescence and the repression of
photosynthesis-related genes were registered on pre-symptomatic leaves
of esca affected vines (Magnin-Robert et al., 2011). Chlorophyll decline
leads to a decrease in photosynthesis efficiency, organic carbon
production, growth and, in general, plant health. Symptomatic leaves
show a further reduction on fresh and dry weight associate to lamina
necrosis and wilt. Leaf chlorosis, necrosis and wilting are due to water
stress caused by vascular occlusion, but also to toxic metabolites
produced by pathogens (Van Alfen, 1989; Pennisi & Graniti, 1987). The
main aetiological agents of “Esca complex” produce phytotoxic
metabolites involved, in vitro and in planta, with symptoms development
on leaves (Evidente et al., 2000; Tabacchi et al., 2000; Bruno &
Sparapano, 2006a, 2006b; Luini et al., 2010; Andolfi et al., 2011).
Chlorophyll decline could also explain leaves weight decrement because
of low photosynthesis efficiency. Further, the activation of plant
defence mechanisms modifies sugars metabolism, moving towards the
production of new molecules (Jeandet et al., 2002) and reducing the
carbohydrates used for plant growth and reproduction. In contrast with
these data, we recognize leaf surfaces increase on diseased plants.
These findings agree with the growth regulators activity registered from
bleeding xylem sap by diseased vines. Hormone-like substances with auxin
activity could be produced by Pm. minimum , Pa.
chlamydospora and especially by F. mediterranea and
contribute to cell hyperplasia, hypertrophy and leaf lamina expansion.
It is well-known that grapevine reacts with powerful defence mechanisms
to cope with pathogenic microorganisms. In particular, production and
accumulation of glycolic acid, stilbenes, viniferins and other phenolic
phytoalexins (Jeandet et al., 2002; Amalfitano et al., 2000); rapid and
localized cell death (Chang et al., 2011) synthesis of
pathogenesis-related proteins (Jeandet et al., 2002), and production of
Reactive Oxygen Species (ROS) such as superoxide radical
O2- and hydroxyl radical
HO· (Hung, Yu & Lin, 2005) were reported. The most
prominent features of the plant response to pathogens and, in general,
against stresses, are the ‘oxidative burst’: a rapid increase in the
cellular concentration of ROS and mainly hydrogen peroxide (De Gara, de
Pinto & Tommasi, 2003). This compound works as an antimicrobial and as
an intracellular and intercellular signalling compound to activate
further defence responses as a mechanical and chemical defence. The
modification of cell walls consisting in cross-linking, lignifications
and incorporation of phenolic compounds and the synthesis of secondary
metabolites (phytoalexins) are defence responses activated by hydrogen
peroxide (Torres, Jonathan & Dangl 2006; Almagro et al., 2009). In this
work, leaves collected from HV vines showed the lower
H2O2 concentrations at all considered
phenological phases. The H2O2 production
is a physiological response to light exposure which increased
O2 photo-reduction in photosystem I of chloroplasts and
photorespiration (Asada, 1999; Leshem, 1992). Whereas, in leaves of
diseased plants, H2O2 increases about
3-fold in symptomless leaves independently of phenological phases and
reaches the higher values on symptomatic leaves. This suggests a strong
correlation between pathogens or their metabolic activity and
H2O2 production and accumulation in
leaves. H2O2 in leaves of diseased
plants might have been used to counteract pathogens as antimicrobial, as
a strengthening of cell wall polymers, as a promoter for phytoalexin
synthesis, or in programmed cell death triggering. However, infected
vines failed their defence-upgrade and finally suffer the effects of
oxidative stress. H2O2 by itself
increase oxidative stress and damage the integrity of cell membranes
(Pérez, Villegas & Mejia, 2002). ROS intensify lipid peroxidation of
unsaturated fatty acids of membranes, compromising membrane integrity
and functionality. Analyses of the lipid peroxidation level clearly show
changes in the cell membranes. Levels of MDA, a product of lipid
peroxidation, are correlated to membrane damages (Heath & Packer, 1968;
Pérez, Villegas & Mejia, 2002; Soares et al., 2019).
Plants scavenge ROS are produced under biotic and abiotic stresses.
Enzymes, including superoxide dismutase, catalase, peroxidase, APX and
G-R (Zhang & Kirkham, 1996; Lee & Lee, 2000) and non-enzymatic
antioxidants such as tocopherols, AsA and glutathione (Noctor & Foyer,
1998) work as ROS detoxifiers. AsA is considered as a molecule-key for
H2O2 elimination. AsA reacts with
H2O2 directly or by APX, a Class I
heme-peroxidase which uses AsA as electron donor and is considered to be
the main peroxidase involved in H2O2detoxification (Asada, 1999). Monodehydroascorbate reductase, DHA-R and
GSH regenerate AsA. Glutathione control the redox state in plant cells
under abiotic and biotic stress and it is involved on AsA regeneration
in the AsA-GSH cycle (Hung, Yu & Lin, 2005; De Gara, de Pinto &
Tommasi, 2003; Asada, 1999; Noctor & Foyer, 1998; Mittler, 2002). If
the AsA-GSH cycle works well, an increase in AsA content and APX
activities in leaves of infected vines are expected. However, leaves of
vines with BWS and BWS-WR during all the four phenological phases here
considered, show AsA and GSH concentrations lower than HV. This trend is
also confirmed on of total ascorbate (AsA+DHA) in bleeding xylem sap. On
the contrary, total glutathione (GSH+GSSG) was stimulated by the
presence of pathogens in the trunk.
L-Ascorbic acid (2,3-didehydro L-threo-hexano-1,4-lactone, the
well-known functional form of vitamin C) is a multifunction molecule:
redox buffer in coordination with glutathione, cell photo-protector,
enzyme cofactor, a regulator of cell division and expansion, cell wall
growth and signal transduction (Gallie, 2013). It is involved in
oxalate, tartrate ethylene, gibberellins, anthocyanins and
hydroxyproline synthesis. AsA is an indirect response of plants against
pathogens, changes gene expression in plants and plays a role in
resistance against biotic and abiotic stresses (Khan, Mazid & Mohammad,
2011).
In this study, AsA, DHA, GSH and GSSG concentrations and APX, DHA-R,
AFR-R and G-R activities were quantified in leaves. The findings here
reported signalling that Pm. minimum , Pa. chlamydosporaalone or in association with F. mediterranea affect
antioxidant defences based on the use and recycling of both GSH and AsA
and this could be correlated with an unbalanced oxidative state, damage
to membrane integrity and leaf necrosis appearance.
Leaves of diseased vines show a significant decrease of redox state and
a shift of both AsA and glutathione towards the oxidised forms.
Ascorbate and glutathione redox states providing a reliable estimation
of the extent of oxidative stress in the cell and changes in their
levels are reported during stress conditions (Munné-Bosch & Alegre,
2003). In our studies, diseased vines seem more stressed than healthy
vines.
To explain this physiological status, we invoke the metabolic complex
produced by F. mediterranea , Pm. minimum andPa. chlamydospora (Evidente et al., 2000; Tabacchi et al., 2000;
Bruno & Sparapano, 2006a, 2006b, 2007; Bruno, Sparapano & Graniti
2007) and the accumulation of resveratrol, benzoic acid derivatives and
flavonols as host defence compounds (Bruno & Sparapano, 2006b; Jeandet
et al., 2002; Amalfitano et al 2000). The presences of phenols or
flavonoids contribute to AsA oxidation in the scavenging of
H2O2 in grape leaves (Yamasaki, Sakihama
& Ikehara, 1997): phenoxy or flavonoxy radicals can accept electrons
from AsA that produce the monodehydroascorbate radical (Pérez, Villegas
& Mejia, 2002).
To prevent oxidative stress, following the glutathione-ascorbate
metabolic pathway, APX reduces H2O2 to
water converting AsA to monodehydroascorbate that disproportionates into
AsA and DHA. Using GSH, DHA-R reduces DHA to ascorbate and produces
GSSG. Finally, GSSG is reduced to GSH by G-R using NADPH as the electron
donor (Asada, 1999). In our conditions, the activities of these
AsA-regenerating enzymes, even active in diseased and healthy vines, did
not present marked differences. This implied that APX, DHA-R, AFR-R and
G-R make a non-significant contribution to increasing AsA regeneration
in diseased vines.
Therefore, our data on APX, the key-enzymes of AsA-GSH cycle, are in
contrast with the low concentrations in cv Sultanina protoplasts
(Papadakis, Siminis & Roubelakis-Angelakis, 2001) and the absence in
cv. Sultana leaves (Pérez, Villegas & Mejia, 2002). The difference
could be due to grape varieties, stress considered, and assay procedure
applied.
5. Conclusions
The results of this study suggest that F. mediterranea ,Pm. minimum and Pa. chlamydospora disturb various
morphological, physiological and biochemical functions in cv Italia
grapevine during the vegetative period. Alterations affect both bleeding
xylem sap and leaves. Flux, dynamic viscosity and growth regulators
activity distinguish bleeding xylem sap of vines infected by Pm.
minimum and Pa. chlamydospora or by Pm. minimum ,Pa. chlamydospora and F. mediterranea . Surface,
fresh and dry weight, chlorophyll, hydrogen peroxide, lipid peroxidation
and redox state were altered on leaves of diseased vines. The presence
of F. mediterranea in wood tissues of infected vines
further worsens the physiological status. These alterations were
detected on symptomatic leaves and, with low intensity, on symptomless
leaves of diseased vines. Probably, these damages mark a pre-symptomatic
stage that, over time, will cause irreversible alterations which induce
symptoms appearance. In diseased vine, low concentrations of AsA, GHS
and moderate levels of DHA and GSSG are also associated with higher
H2O2 and MDA values and remarkable
oxidative stress status. On these conditions, the scavenging enzymes are
not enough able to restore the balance between ROS and antioxidants
level managing stress conditions. The oxidative unbalance stress
enhances lipid peroxidation of unsaturated fatty acids of membranes,
damages membrane integrity and contributes to cell death and leaf
symptoms development. As it is obvious from the present data, AsA-GSH
cycle might be involved in grapevine susceptibility of “Esca
complex”-associated fungi.