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.