Unravelling the colonization mechanism of Lasiodiplodia brasiliensis in grapevine plants

Summary. Botryosphaeriaceae cause the degenerative disease Botryosphaeria dieback in many woody hosts, including grapevine. These pathogens penetrate host plants through pruning wounds, and colonize vascular tissues causing necrotic lesions, can-kers, and eventually plant death. Colonization processes by Botryosphaeriaceae and their interactions with their hosts has been understudied. The colonization mechanisms were examined for Lasiodiplodia brasiliensis , a common pathogen causing Botry-osphaeria dieback in Mexican vineyards. Lasiodiplodia brasiliensis MXBCL28 was inoculated onto grapevine ‘Cabernet Sauvignon’ plants, and after 2 months, infected tissues were observed with microscopy using histological techniques. Lasiodiplodia brasiliensis was also cultured on different carbon sources representing cell walls and non-structural plant components, to complement histology data. The host responded to wounding by developing xylem vessel occlusions with tyloses and deposition of suberin in cambi-um and ray parenchyma. Infection response also included deposition of suberin in pith tissues, reinforcement of cell walls with phenolic compounds, and lignin deposition in xylem vessels and ray parenchyma. The pathogen could overcome host compartmentalization mechanisms and colonize wood tissue causing extensive necrosis. The fungus was visualized in host cambium, vascular bundles, xylem vessels, and pith, and infected tissues were depleted in starch in the ray parenchyma. Cellulose, hemicellulose, and lignin in cell walls were also degraded, supporting in vitro data.


INTRODUCTION
Grapevine is threatened by many pathogenic microorganisms that reduce yields and fruit quality (Armijo et al., 2016).Grapevine Trunk Diseases (GTDs) are considered the most destructive diseases causing significant economic losses to the grape industry (Gubler et al., 2005;Kaplan et al., 2016;Gispert et al., 2020).These diseases are caused by a complex of wood-inhabiting fungi in Lasiodiplodia, Neofusicoccum, Diplodia, Eutypa, Eutypella, Phaeomoniella, and Phaeoacremonium, among others (Bertsch et et al., 2013;Fon-taine et al., 2016;Claverie et al., 2020).These fungi form sexual and/or asexual fruiting bodies on infected woody tissues.Spores produced within these structures are released and spread in vineyards by wind and water, and enter plants through pruning wounds.Management of GTDs is mostly achieved by preventative measures that consist of protecting pruning wounds against infections (Rolshausen et al., 2010;Kaplan et al., 2016;Martínez-Diz et al., 2021;Blundell and Eskalen, 2022).
Following wound infection, the GTD pathogens colonize host vascular tissues after spore germination.Most fungi causing GTDs are necrotrophs, degrading structural and non-structural host cell wall components (lignin, cellulose, hemicellulose, starch) for metabolic processes.Imaging data has indicated that GTDs initially invade host xylem vessels before spreading to neighbouring tissues (Amponsah et al., 2012;Obrador-Sánchez and Hernandez-Martinez, 2020).The qualitative and quantitative virulence traits (cell wall degrading enzymes, toxins, degradation of anti-fungal plant compounds) are key determinants of the wood colonizing capabilities, symptom types, and aggressiveness of the pathogen (Morales-Cruz et al., 2015;Garcia et al., 2021).For example, Eutypa lata and Neofusicoccum parvum develop characteristic wedge-shaped cankers in host woody tissues, and both pathogens share similar hydrolytic enzyme profiles that can break down glucose-rich polymers in secondary cell walls.In contrast, Phaeomoniella chlamydospora has limited enzymatic capabilities primarily targeting pectin-rich components, and infections are restricted to xylem elements leading to symptoms concentric rings of dark spots in wood (Rolshausen et al., 2008;Morales-Cruz et al., 2015;Pouzoulet et al., 2017).In addition, the most aggressive Botryosphaeriaceae (Lasiodiplodia and Neofusicoccum) associated with the largest plant lesions also possessed the greatest numbers of protein-coding genes involved in wood degradation and host colonization (Garcia et al., 2021).
Host plants respond to invasions by pathogens with constitutive and induced defense mechanisms (Freeman and Beattie 2008;Mithöfer and Boland 2012).Compartmentalization is a general defense response to wounding and pathogen infection to isolate injured tissues (Shigo and Tippett, 1981;Shigo, 1984).The model of compartmentalization of decay in trees (CODIT) describes formation and host reinforcement of anatomical walls to spatially restrict the movement of pathogens.The host impedes longitudinal movement of the pathogen by producing pectin-rich tyloses and gels in the xylem vessels, whereas vertical, radial and tangential movement are restricted by depositing lignin, suberin, and phenolic compounds in ray parenchyma, growth rings and vascu-lar cambium (Shigo and Marx, 1977;Tippett and Shigo 1981;Pearce, 1996;Pearce, 2000).Histological studies have shown reinforcement of cell walls against wood colonizing agents in several pathosystems including those in grapevine (Fleurat-Lessard et al., 2013;Pouzoulet et al., 2013;Pouzoulet et al., 2017;Pouzoulet et al., 2022).
Induced defense mechanisms are triggered by effector molecules produced by pathogens, and by host plant cell wall degradation products, and compounds include reactive oxygen species (ROS), enzymes, tannins and phytoalexins (Joshi et al., 2021;Kaur et al., 2022).Production of phytoalexins (stilbene, resveratrol, flavonoid, viniferin) plays important roles in grapevine defense mechanisms, including plant cell wall reinforcement against biotrophic and necrotrophic pathogens (Adrian et al., 2012), and these compounds have been reported in vines infected by GTD pathogens (Spagnolo et al., 2014;Magnin-Robert et al., 2016;Rusjan et al., 2017).Together, this information indicates that phenolic compounds have essential physical and chemical properties that contribute to limiting GTD pathogen colonization, and could affect disease tolerance amongst grapevine cultivars (Rolshausen et al., 2008;Gómez et al., 2016).
GTD infections lead to host decline and death, because they affect host hydraulic functions by obstructing xylem and phloem translocation of water and nutrients.Infections also affect carbohydrate metabolism by decreasing photosynthesis and depleting carbon storage (Rudelle et al., 2005;Fontaine et al., 2016;Pouzoulet et al., 2017;Batista et al., 2021).Some pathogens of the GTD complex have also been reported to live within hosts without causing symptoms (Slippers and Wingfield, 2007;Zhang et al., 2021).The transition from commensal to pathogenic phase has been related to stress factors such as drought or heat, which predispose plants and increase their susceptibility (Hrycan et al., 2020).The number of reports of diseases caused by Botryosphaeriaceae has increased in the past decade, possibly because of climate change (Mehl et al., 2017;Batista et al., 2021).Transcriptomic and genomic analyses have revealed gene families of cell wall degrading enzymes (CWDE), and carbohydrate metabolism, were induced under heat stress conditions (Paolinelli et al., 2016;Yan et al., 2018;Félix et al., 2019;Gonçalves et al., 2019;Garcia et al., 2021;Nagel et al., 2021).
Despite their agricultural importance, studies of Botryosphaeriaceae designed to increase understanding of host colonization mechanisms have been limited.Lasiodiplodia spp.are among the causal agents of Botryosphaeria dieback, and are aggressive pathogens of grapevine (Úrbez-Torres, 2011).A total of 749 hosts of these fungi have been reported (Batista et al., 2021).
In Mexico, in the states of Baja California and Sonora, Lasiodiplodia brasiliensis has been frequently isolated from grapevine and has been reported as highly virulent (Rangel-Montoya et al., 2021).
The present study aimed to increase understanding of the colonization processes of L. brasiliensis in grapevine, and to broaden knowledge of its pathogenesis and subsequent symptom development.

Fungus and plant materials
Lasiodiplodia brasiliensis MXBCL28 was isolated from a diseased 'Cabernet Sauvignon' grapevine growing in Baja California (Rangel-Montoya et al., 2021).This isolate was preserved in 20% glycerol at 4°C, and has been routinely cultivated in Potato Dextrose Agar (PDA; Difco) at 30°C.
One-year-old 'Cabernet Sauvignon' grapevines (the main cultivar in Baja California) were obtained from a local nursery.Plants were inoculated with L. brasiliensis MXBCL28 through wounds made in the woody tissues with a drill bit (2 mm diam.)(Úrbez-Torres et al., 2010).For each inoculation, a mycelial plug of the fungus was placed inside the wound and was the covered with Para-film®.Plugs of sterile PDA were used for inoculation control plants.Plants were kept in a greenhouse under semicontrolled conditions at an average temperature of 35°C day and 25°C night, for 2 months during summertime.Ten plants were used per treatment.Subsequently, samples were taken from necrotic lesions.For each sample, a 4 cm long section, including 2 cm above and 2 cm below the inoculation site, was obtained, and was then fixed in FAA solution (Formaldehyde (SIGMA), Acetic acid (Faga Lab), Ethyl Alcohol (Jalmek); 5:5:9) for 24 h at 4°C, and then rinsed and preserved in 80% ethanol at 4°C until used.Part of each sample close to the necrotic tissue was used to re-isolate the inoculated fungus onto PDA to confirm the fungus colonization.
All stained sections were observed using a Nikon Eclipse E200 microscope with an AxioCam HRc camera (Zeiss), and with an AxioVert200 with a RisingCam® U3CMOS camera.Epifluorescence microscopy was carried out using an AxioVert200 microscope supplied with a HBO100 100W mercury lamp with ebq100 power.Cellulose and suberin in the plant tissues were observed using a DAPI filter (excitation at 330-380 nm, emission at 420 nm).A TEXAS RED filter (excitation at 542-595 nm, emission at 644 nm) was used to analyze hemicellulose and suberin.Images were analyzed using AxioVision 4.8.2,RisingView, and ImageJ 1.49v software packages.
Fungal biomass was measured by inoculating test tubes containing MM9-liquid supplemented with the different carbon sources listed above, each with a mycelium disk of L. brasiliensis.All cultures were incubated at 30°C and 120 rpm for 7 d.Subsequently, the mycelium produced for each growth condition was recovered by filtration on a previously weighed Whatman® filter grade 1, and was dried at 50°C for 3-5 d.The biomass was determined by weighing the dry mycelium obtained from each growth condition, and subtracting the weight of the filter and the mycelium disk used for inoculation.For treatments with grapevine wood, ground wood contained in each sample was carefully removed.The experiment was carried out in triplicate.

Statistical analyses
Assumptions of normality of data from the biomass, growth rate, and area of the mycelium experiments from different carbon sources were confirmed using the Kolmogorov-Smirnov test (P ≥ 0.05).One-way ANOVA followed by post hoc Fisher LSD analyses (α < 0.05 for significance) were then carried out for all variables, using STATISTICA 8.0 software.

Colonization of grapevine plants by Lasiodiplodia brasiliensis MXBCL28
Two months after inoculation, grapevine plants infected with L. brasiliensis showed necrotic lesions along the xylem of mean length 5.5 (+/-1.2cm), that were not observed in the control plants (Figure 1A).Fontana-Masson stain allowed observation of melanized mycelium in the inoculation zones, in the vascular cambium of the plants, as well as in the vascular bundles, including those with occlusions (Figure 1 C, D, E and  F).No hyphae were observed in the wounded areas or in the pith of the control plants (Figure 1 A).
Cross-sections of control plants stained with iodinepotassium iodide had strong dark colour, indicating the presence of starch in the ray parenchyma and in the wound areas (Figure 2 A).In contrast, cross-sections of infected plants each showed a light brown stain, indicating starch depletion in the ray parenchyma and the pith (Figure 2 B).Under epifluorescence microscopy, control plant tissues stained with Calcofluor White M2R had brownish areas at the wounds, indicating the presence of cellulose (Figure 2 C).Tissues of plants infected with L. brasiliensis showed dark zones, indicating depletion of cellulose in the necrotic tissues (Figure 2 D).In sections stained with Congo Red, dark zones were observed in the infected tissues (Figure 2 F) were observed, but not in the control plants (Figure 2 E), indicating the lack of hemicellulose in that area.
Staining with Toluidine Blue O allowed the visualization of phenolic compounds and tyloses in the xylem vessels in both control (Figure 3, A, B and C) and infected plants (Figure 3, D, E and F).In control plants, pectic compounds were observed in the regenerated tissue in reddish-orange and greenish-blue colors (Figure 3A), tyloses in blue-purple dark (Figure 3B), and less abundance of phenolic compounds in the pith (Figure 3C).Observed in purple and greenish-blue colors, in plants inoculated with L. brasiliensis, there was also pectin deposition in the occluded xylem vessels (Figure 3E), and phenolic compounds breaking down in the pith (Figure 3F) and in the vascular cambium (Figure 3D).
Infected plants showed lighter pink colour in the lesion areas and in the vascular cambium compared with non-infected tissues (Figure 4, A and B), indicating that the fungus could break down phenolic polymers.Comparison of infected and non-infected wood tissues using the Mäule stain showed that L. brasiliensis was able to break down lignin, as indicated by a discolouration in xylem tissues, the ray parenchyma, and the fibres surrounding the occluded vascular bundles (Figure 4 D).Staining with Sudan IV showed red suberin deposits in the control plants, in the cambium cork in the wound areas (Figure 4 E) and in the ray parenchyma (Figure 4 H).In contrast, red suberin was observed in the cambium cork (Figure 4 F), vascular cambium, ray parenchyma (Figure 4 G), and in the pith (Figure 4J), in response to fungal infection.Suberin was not observed in the necrotic rays (Figure 4 K).

Evaluation of carbon sources used for Lasiodiplodia brasiliensis MXBCL28
Growth of L. brasiliensis MXBCL28 without a carbon source was weak with stunted mycelium (Figure 5 A), and biomass was reduced (Table 1).The fungus was able to utilize pectin, xylan, xylose, and starch as carbon sources (Figure 5 C to F), and glucose (Figure 5 B), producing dense dark gray aerial mycelium (Table 1).Lasiodiplodia brasiliensis had a significantly increased growth rate and biomass production using pectin as a carbon source (Figure 5C) or ground grapevine wood (Figure 5 K), since the fungus completely covered the media in Petri dishes in 4 d for these carbon sources.The fungus produced less aerial mycelium with cellulose, lignin and tannic acid as carbon sources, as indicated by the calculations of mycelium area (Figure 5, H to J and Table 1).The reduced biomass and mycelium areas were observed with lignin and tannic acid (Figure 5 H and J) as carbon sources.For tannic acid, although production of aerial mycelium and biomass were low, the fungus covered almost the entire medium surfaces in Petri dishes, with sparse mycelium after 7 days.

DISCUSSION
This study used histology to outline the pathogenesis of L. brasiliensis in grapevine, by comparing mechanically wounded plants with those wounded and infected with L. brasiliensis.The data obtained have indicated that the physical, constitutive and induced defense responses enacted by the host did not successfully compartmentalize the pathogen.
Wood decay in infected grapevine plants was extensive after 2 months of incubation, indicating that species of Lasiodiplodia are highly virulent, as previously shown by Úrbez-Torres and Gubler ( 2009   Melanin is considered to be a virulence factor in several phytopathogenic fungi (Eisenman et al., 2020), and is commonly produced by Botryosphaeriaceae (Phillips et al., 2013;Rangel-Montoya et al., 2020).Several L. gilanensis genes involved in the synthesis of different melanin pathways were found differentially expressed in the presence of grapevine wood and under heat shock conditions (genes involved in DHN-melanin and pyomelanin pathways), or without heat shock (genes involved in DOPA-melanin pathway) (Paolinelli- Alfonso et al., 2016).Melanin protects against UV radiation, enzymatic lysis, and ROS, and L. gilanensis can metabolize tyrosine as carbon and nitrogen sources, and uses this amino acid as a DOPA-melanin precursor (Rangel-Montoya et al., 2020).Phenylalanine is a precursor of plant lignin and suberin syntheses (Lewis et al., 1987), and could be a precursor of DOPA-melanin and pyomelanin pathways, and tyrosine catabolism (Schmaler-Ripcke et al., 2009;Eisenman and Casadevall, 2012).We speculate that Lasiodiplodia could derail host plant  metabolic pathways and use phenylalanine or tyrosine as precursors for melanin biosynthesis.
Grapevine responded to L. brasiliensis infection by occluding xylem vessels with tyloses and gels, and reinforcing cell walls with phenolic compounds in tyloses, vascular bundles, cambium, and pith.Infections were also accompanied by starch depletion in the ray parenchyma.These results support previous observations from histological studies conducted on several pathogens causing GTDs (Fleurat-Lessard et al., 2013;Pouzoulet et al., 2013;Gómez et al., 2016;Pouzoulet et al., 2017;Obrador-Sánchez and Hernandez-Martinez, 2020), indicating that these are constitutive responses that are enacted by host plants regardless of the type of GTD fungal infection.
In woody plants, non-structural carbohydrate reserves stored mainly as starch in ray parenchyma are  remobilized to fulfill several biological processes, including refilling embolized xylem vessels caused by freezethaw cycles or drought, supporting plant vegetative growth after dormancy, and activating host defense systems (Nardini et al., 2011;Fontaine et al., 2016;Noronha et al., 2018;Călugăr et al., 2019).Depletion of starch in wood has been observed in many pathogen and grapevine interactions (Rudelle et al., 2005;Rolshausen et al., 2008;Pouzoulet et al., 2017).The present study in vitro data also indicated that L. brasiliensis can use starch as a carbon source.Genomic and transcriptomic analyses indicated that a putative amylase was induced by L. gilanensis in the presence of grapevine wood (Paolinelli-Alfonso et al., 2016), and that genes involved in starch metabolism were induced in L. theobromae during infection (Yan et al., 2018).Together, these results suggest that starch plays a pivotal role as an energy pool accessible for GTD fungi for wood colonization and for grapevine to activate its defense system.Several fungi causing GTDs can also metabolize structural carbohydrates located in plant cell walls.The hemicellulosic fraction has been shown to be especially degraded during infection by E. lata, and aggressive fungi such as Neofusicoccum can break down cellulosic material (Rolshausen et al., 2008;Stempien et al., 2017).Results from the present study showed that cellulose and hemicellulose were depleted in necrotic tissues, and were metabolized in vitro by L. brasiliensis.Cellulose and hemicellulose are carbohydrate-rich compounds that each constitute 30% of grapevine cells (Rolshausen et al., 2008).Genomic information from virulent Botryosphaeriaceae (Neofusicoccum and Lasiodiplodia) demonstrated the high numbers of genes encoding carbohydrateactive enzymes (Garcia et al., 2021), indicating that these compounds embedded in cell walls are primary targets for fungal metabolism.
Vessel occlusion was observed in control and inoculated (infected) plants, but the response was much stronger in L. brasiliensis infected plants, as was shown in grapevine responses to P. chlamydospora inoculations (Pouzoulet et al., 2017).Vessel occlusions, such as tyloses, gums, and gels, are formed in vascular bundles to limit the longitudinal spread of the fungus (De Micco et al., 2016;Pouzoulet et al., 2017).Several Botryosphaeriaceae have been shown to first invade xylem vessels before spreading to neighbouring woody tissues (Amponsah et al., 2012;Obrador-Sánchez and Hernandez-Martinez, 2020).Tyloses and gels are pectin-rich, and the present study results indicated that L. brasiliensis growth was stimulated in the presence of pectin in comparison to other substrates.Expression of a pectate lyase was upregulated during the first stages of L. gilanensis infection in grapevine (Paolinelli-Alfonso, et al., 2016).Occlusion of xylem vessels results in loss of hydraulic conductivity, which eventually leads to decreased host physiological functions (Zhao et al., 2014).
The present study has shown that suberin accumulated in tyloses walls, and that lignin was deposited near ray parenchyma next to occluded xylem vessels, probably as responses to pathogen tracheid invasion.Suberin deposition was observed in other tissues, including vascular cambium, vascular cork, xylem fibre of vascular bundles, and ray parenchyma.Pouzoulet et al. (2013) also noted that grapevine accumulates suberin over lignin, in response to P. chlamydospora infections in those tissues.Qualitative and quantitative accumulation of phenolic compounds as physical and chemical barriers has been shown to be a common compartmentalizing mechanism for wood-decay fungi in other pathosystems (Skyba et al., 2013;Mounguengui et al., 2016).In grapevine, increased cell wall phenolics were found in the wood of the Eutypa lata tolerant cultivar Merlot compared with susceptible Cabernet Sauvignon (Rolshausen et al., 2008).Increased resveratrol induction was also measured in N. parvum tolerant Vitis sylvestris compared to V. vinifera (Labois et al., 2020).
Phenolic compounds in grapevine have also been shown to inhibit the growth of several GTD fungi (Lambert et al., 2012;Gómez et al., 2016).However, accumulation of phenolics (flavonoids, and stilbenoids) did not effectively wall-off aggressive pathogens, and these compounds are fungistatic rather than fungicidal (Lambert et al., 2012;Galarneau et al., 2021).The present study did not measure accumulation of phytoalexins.Lasiodiplodia, like Neofusicoccum, has been reported to display similar virulence factor patterns with respect to detoxification of antimicrobial compounds (Garcia et al.,2021).These fungi can degrade phenolic compounds that restrict in planta movement.Several genes involved in phenolic metabolism have been found in Lasiodiplodia spp.(Paolinelli-Alfonso et al., 2016;Yan et al., 2018;Gonçalves et al., 2019;Garcia et al, 2021).These could assist metabolization of phenolic compounds such as salicylic acid and phenylpropanoids produced by plants, to avoid the host defense responses.In addition, results from the present in vitro studies showed that L. brasiliensis was able to grow in culture media supplemented with glycerol, tannic acid, and lignin, supporting its ability to use these substrates as carbon sources.
Based on the information obtained in this study, and from previously published reports, we propose that once Lasiodiplodia conidia penetrate grapevines through wounds, the fungus initially colonizes xylem vessels following germination.The proposed model of the colonization process is illustrated in Figure 6.Upon recognition of the pathogen, the host remobilizes starch resources to trigger defense responses, with rapid xylem occlusion.The pathogen initially metabolizes the readily available starch stored in parenchyma cells and pectin-rich tyloses walls to weaken the intensity of host response and further colonize xylem vessels.Subsequently, the pathogen spreads to neighbouring tissues degrading cell wall structures to gain access to more complex sugars for metabolic processes.Host plant production of suberin, lignin and phytoalexins to compartmentalize the pathogen is probably inadequate against Lasiodiplodia, as the pathogen melanin can deactivate their antimicrobial effects and further breaks down phenolics.The pathogen then colonizes host cambium to initiate development of canker symptoms.When growing within the host, the melanized mycelium protects the fungus from the host defenses.At the same time, the fungus produces cell wall degrading enzymes, and continues the colonization process using hemicellulose, starch and pectin as carbon sources.This induces vessel occlusions and the production of phenolic compounds.Over time, the fungus degrades lignin and suberin, moving to further xylem rays.This movement induces formation of typical Botryosphaeria cankers.
),Garcia et al. (2021) andRangel-Montoya et al. (2021), at least under the local environmental conditions.The high maximum temperatures encountered during the summer months in Mexico are conducive to L. brasiliensis pathogenesis(Rangel-Montoya et al., 2021).Melanized hyphae were observed in all infected host woody tissues, including

Figure 1 .
Figure 1.Grapevine plants 'Cabernet Sauvignon' 2 months after inoculation with Lasiodiplodia brasiliensis MXLBC28.A, Tissue regeneration in control plants (left), and necrotic lesion caused by L. brasiliensis (right); white arrows indicate the wound area.Mycelia of Lasiodiplodia brasiliensis growing in grapevine plants 'Cabernet Sauvignon' stained using the Masson-Fontana method.B, Control plants non-inoculated; red arrows indicate the wound area and occlusions.C, Melanized fungus colonizing the vascular cambium and vascular bundles.D, melanized fungus colonizing the plant piths.E, melanized hyphae covering the xylem and ray parenchyma.F, melanized hypha growing in a vascular bundle.Yellow arrows indicate melanized hyphae.

Figure 2 .
Figure 2. Starch and wood degradation by Lasiodiplodia brasiliensis in grapevine plants 'Cabernet Sauvignon' 2 months after inoculation.A, C, and E, non-inoculated plants.B, D, and F, plants inoculated with L. brasiliensis.A and B, cross sections stained with iodine-potassium iodide for starch content.A, starch deposition is observed as black zones in the ray parenchyma in control plants; white arrows indicate the wound area and the tissue regeneration, while hollow arrows indicate starch in the newly formed tissue.B, infected plants showing starch depletion in the ray parenchyma of the necrotic tissue; arrows indicate the ray parenchyma in the wound area and necrotic tissue.C, D, E and F, epifluorescence microscopy to observe the presence of cellulose and hemicellulose.C and D, sections stained with Calcofluor White M2R, or E and F, Congo Red.Control plants (C and E) show brownish areas in the wound areas.Plants infected with L. brasiliensis (D and F) show dark zones without cellulose in the necrotic tissues.White arrows indicate zones without fluorescence where there is no cellulose or hemicellulose.

Figure 3 .
Figure 3. Responses of grapevine plants 'Cabernet Sauvignon' to wounding and infection, producing phenolic and pectic compounds.Cross-sections stained with toluidine blue O. A, B and C, Control plants showing production of phenolic and pectic compounds in the vascular cambium, and in the regenerated tissue.D, E and F, Plants infected with Lasiodiplodia brasiliensis showing phenolic compounds in the vascular cambium, phloem (D), vessel occlusions with pectic compounds (E), and the pith (F).Red arrows indicate phenolic compounds stained purple, greenish-blue and blue; and pectic compounds stained purple and reddish-orange in the regenerated tissue and occlusions.

Figure 4 .
Figure 4. Grapevine plant 'Cabernet Sauvignon' responses of phenolic compounds, lignin, and suberin to Lasiodiplodia brasiliensis infections.A and B, cross sections of plant stems stained with Phloroglucinol-HCl show lignin in control plants as intense pink in all tissues (A), while in infected plants lighter pink colouration occurred in the lesion areas and vascular cambium, and phenolic deposits in the fibres surrounding the vascular bundles were observed (B).C to D, cross sections stained with the Mäule method to observe the presence of lignin (red colour).Control plants showed slight discolouration in the tissue surrounding occluded vascular bundles near the wound sites (C), while infected plants showed discolouration in the xylem, ray parenchyma, and fibres surrounding occluded vascular bundles (D).Green arrows indicate zones without lignin, and black arrows indicate lignin surrounding vessels.E to K, Epifluorescence microscopy for suberin localization of grapevine plants 'Cabernet Sauvignon' .Samples were stained with Sudan black IV, and observed under TEXAS RED and DAPI filters.Control plants presenting red suberin in cambium cork (E) and ray parenchyma (H) near the wound area, and without suberin in the pith (I).Infected plants 2 months after inoculation with L. brasiliensis showed red suberin deposits in the cork (F), vascular cambium, occlude vessels, and ray parenchyma (G and K), and in the pith (J).White arrows indicate the presence of suberin.The hollow arrow indicates ray parenchyma in the necrotic area without suberin.

11150 Figure 6 .
Figure6.Proposed model of the colonization of grapevine by Lasiodiplodia brasiliensis.Spores of L. brasiliensis enter a plant wound, germinate and penetrate through xylem vessels.When growing within the host, the melanized mycelium protects the fungus from the host defenses.At the same time, the fungus produces cell wall degrading enzymes, and continues the colonization process using hemicellulose, starch and pectin as carbon sources.This induces vessel occlusions and the production of phenolic compounds.Over time, the fungus degrades lignin and suberin, moving to further xylem rays.This movement induces formation of typical Botryosphaeria cankers.

Table 1 .
Mean weights of biomass, growth rates, and mycelium areas of Lasiodiplodia brasiliensis MXBCL28 colonies growing in Petri plates containing PDA amended with different carbon sources.