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Host/Pathogen Interaction

Spatial chemistry of citrus reveals molecules bactericidal

to Candidatus Liberibacter asiaticus

Huanglongbing (HLB), associated with the psyllid-vectored phloem-limited bacterium, Candidatus Liberibacter asiaticus (CLas), is a disease threat to all citrus production worldwide. Currently, there are no sustainable curative or prophylactic treatments available. In this study, we utilized mass spectrometry (MS)-based metabolomics in combination with 3D molecular mapping to visualize complex chemistries within plant tissues to explore how these chemistries change in vivo in HLB- infected trees. We demonstrate how spatial information from molecular maps of branches and single leaves yields insight into the biology not accessible otherwise. In particular, we found evidence that flavonoid biosynthesis is disrupted in HLB-infected trees, and an increase in the polyamine, feruloylputrescine, is highly correlated with an increase in disease severity. Based on mechanistic details revealed by these molecular maps, followed by metabolic modeling, we formulated and tested the hypothesis that CLas infection either directly or indirectly converts the precursor compound, ferulic acid, to feruloylputrescine to suppress the antimicrobial effects of ferulic acid and biosynthetically downstream flavonoids. Using in vitro bioassays, we demonstrated that ferulic acid and bioflavonoids are indeed highly bactericidal to CLas, with the activity on par with a reference antibiotic, oxytetracycline, recently approved for HLB management. We propose these compounds should be evaluated as therapeutics alternatives to the antibiotics for HLB treatment. Overall,

the utilized 3D metabolic mapping approach provides a promising methodological framework to identify pathogen-specific inhibitory compounds in planta for potential prophylactic or therapeutic applications.

Assessment of Pierce’s disease susceptibility in Vitis vinifera cultivars with different pedigrees

Pierce’s disease (PD) of grapevine is caused by the bacterium Xylella fastidiosa. In this study, an integrated approach was applied to assess PD susceptibility among different Vitis vinifera cultivars that incorporated disease severity, bacterial pathogen abundance and loss of stem xylem hydraulic conductivity. It was hypothesized that levels of PD susceptibility in V. vinifera can be attributed in part to the host anatomical features that are shaped by its pedigree background. Two popular wine grape cultivars were initially selected from the occidentalis group, Merlot and Cabernet Sauvignon, and one from the orientalis group, Thompson Seedless. The more recently bred table grape cultivar Scarlet Royal, that has mixed pedigree parentage, was also included. PD susceptibility was compared to the known PD resistant b43-17 V. arizonica/ candicans wild grape species from North America. The data indicated that Thompson Seedless was ranked as the most susceptible to PD because it significantly exhibited the most severe disease symptoms at 12 weeks post-inoculation and hosted the highest X. fastidiosa titre of the cultivars, and lost over 90% of its stem hydraulic conductivity. In contrast, the other three cultivars displayed less susceptibility to PD. The way in which the xylem anatomy could impact PD susceptibility in V. vinifera cultivars is discussed, together with how grape pedigrees and their cognate centre of domestication may have influenced xylem anatomical features. This work provides a reference framework to further test the hypothesis that V. vinifera cultivars with wide xylem vessels may be more prone to PD decline.

Xylem Vessel Diameter Affects the Compartmentalization of the Vascular Pathogen Phaeomoniella chlamydospora in Grapevine

Fungal wilt diseases are a threat to global food safety. Previous studies in perennial crops showed that xylem vessel diameter affects disease susceptibility. We tested the hypothesis that xylem vessel diameter impacts occlusion processes and pathogen compartmentalization in Vitis vinifera L. We studied the interaction between four grape commercial cultivars with the vascular wilt pathogen Phaeomoniella chlamydospora. We used qPCR and wood necrotic lesion length to measure fungal colonization coupled with histological studies to assess differences in xylem morphology, pathogen compartmentalization, and fungal colonization strategy. We provided evidence that grape cultivar with wide xylem vessel diameter showed increased susceptibility to P. chlamydospora. The host response to pathogen included vessel occlusion with tyloses and gels, deposition of non-structural phenolic compounds and suberin in vessel walls and depletion of starch in parenchyma cells. Pathogen compartmentalization was less efficient in wide xylem vessels than in narrow diameter vessels. Large vessels displayed higher number of tyloses and gel pockets, which provided substrate for P. chlamydospora growth and routes to escape occluded vessels. We discuss in which capacity xylem vessel diameter is a key determinant of the compartmentalization process and in turn grape cultivar resistance to disease caused by P. chlamydospora.

Can vessel dimension explain tolerance toward fungal vascular wilt diseases in woody plants? Lessons from Dutch elm disease and esca disease in grapevine

This review illuminates key findings in our understanding of grapevine xylem resistance to fungal vascular wilt diseases. Grapevine (Vitis spp.) vascular diseases such as esca, botryosphaeria dieback, and eutypa dieback, are caused by a set of taxonomically unrelated ascomycete fungi. Fungal colonization of the vascular system leads to a decline of the plant host because of a loss of the xylem function and subsequent decrease in hydraulic conductivity. Fungal vascular pathogens use different colonization strategies to invade and kill their host. Vitis vinifera cultivars display different levels of tolerance toward vascular diseases caused by fungi, but the plant defense mechanisms underlying those observations have not been completely elucidated. In this review, we establish a parallel between two vascular diseases, grapevine esca disease and Dutch elm disease, and argue that the former should be viewed as a vascular wilt disease. Plant genotypes exhibit differences in xylem morphology and resistance to fungal pathogens causing vascular wilt diseases. We provide evidence that the susceptibility of three commercial V. vinifera cultivars to esca disease is correlated to large vessel diameter. Additionally, we explore how xylem morphological traits related to water transport are influenced by abiotic factors, and how these might impact host tolerance of vascular wilt fungi. Finally, we explore the utility of this concept for predicting which V. vinifera cultivars are most vulnerable of fungal vascular wilt diseases and propose new strategies for disease management.

Pathogenesis of Eutypa lata in Grapevine: Identification of Virulence Factors and Biochemical Characterization of Cordon Dieback

Eutypa lata is a vascular pathogen of woody plants. In the present study we (i) determined which component(s) of the cell wall polymers were degraded in naturally infected grapevines and in artificially inoculated grape wood blocks; (ii) compared the pattern of wood decay in the tolerant grape cv. Merlot versus the susceptible cv. Cabernet Sauvignon; and (iii) identified secondary metabolites and hydrolytic enzymes expressed by E. lata during wood degradation. Biochemical analyses and a cytochemical study indicated that glucose-rich polymers were primary targets of E. lata. Structural glucose and xylose of the hemicellulose fraction of the plant cell wall and starch were depleted in infected woods identically in both cultivars. Moreover, the more tolerant cv. Merlot always had more lignin in the wood than the susceptible cv. Cabernet Sauvignon, indicating that this polymer may play a role in disease resistance. In vitro assays demonstrated the production by E. lata of oxidases, glycosidases and starch degrading enzymes. Phytotoxic secondary metabolites were also produced but our data suggest that they may bind to the wood. Finally, we demonstrated that free glucose in liquid cultures repressed primary but not secondary metabolism.

Global transcriptional analysis suggests Lasiodiplodia theobromae pathogenicity factors involved in modulation of grapevine defensive response

Lasiodiplodia theobromae is a fungus of the Botryosphaeriaceae that causes grapevine vascular disease, especially in regions with hot climates. Fungi in this group often remain latent within their host and become virulent under abiotic stress. Transcriptional regulation analysis of L. theobromae exposed to heat stress (HS) was first carried out in vitro in the presence of grapevine wood (GW) to identify potential pathogenicity genes that were later evaluated for in planta expression.

Distinctive expansion of gene families associated with plant cell wall degradation, secondary metabolism, and nutrient uptake in the genomes of grapevine trunk

pathogens

Trunk diseases threaten the longevity and productivity of grapevines in all viticulture production systems. They are caused by distantly-related fungi that form chronic wood infections. Variation in wood-decay abilities and production of phytotoxic compounds are thought to contribute to their unique disease symptoms. We recently released the draft sequences of Eutypa lata, Neofusicoccum parvum and Togninia minima, causal agents of Eutypa dieback, Botryosphaeria dieback and Esca, respectively. In this work, we first expanded genomic resources to three important trunk pathogens, Diaporthe ampelina, Diplodia seriata, and Phaeomoniella chlamydospora, causal agents of Phomopsis dieback, Botryosphaeria dieback, and Esca, respectively. Then we integrated all currently-available information into a genome-wide comparative study to identify gene families potentially associated with host colonization and disease development.

Susceptibility of Cultivated and Wild Vitis spp. to Wood Infection by Fungal Trunk Pathogens

Cultivars of European grapevine, Vitis vinifera, show varying levels of  susceptibility to Eutypa dieback and Esca, in terms of foliar symptoms.  However,  little  is  known  regarding  cultivar  susceptibility  of  their   woody  tissues  to  canker  formation.  Accordingly,  we  evaluated  the   relative susceptibility of V. vinifera cultivars (‘Cabernet Franc’, ‘Cabernet Sauvignon’, ‘Chardonnay’, ‘Merlot’, ‘Riesling’, ‘Petite Syrah’, and  ‘Thompson  Seedless’)  and  species  or  interspecific  hybrids  of  North   American  Vitis (Vitis  hybrid  ‘Concord’,  V.  arizonica  ‘b42-26’,  V. rupestris × V. cinerea  ‘Ill547-1’, and Fennell 6 [V. aestivalis] × Malaga  [V.  vinifera] ‘DVIT0166’)  to  canker  formation  by  seven  trunk  patho- gens (Neofusicoccum parvum, Lasiodiplodia theobromae, Phaeomoniella  chlamydospora,  Togninia  minima,  Phomopsis  viticola,  Eutypa lata, and an  undescribed  Eutypa  sp.).  Susceptibility  was  based  on  the   length  of  wood  discoloration  (LWD)  in  the  woody  stems  of  rooted   plants in duplicate greenhouse experiments. Cultivars of V. vinifera  and Concord did not vary significantly in susceptibility to N. parvum or L.  theobromae  (LWD  of  21  to  88  mm  at  14  weeks  post  inoculation;  P  >   0.16),  suggesting  that  they  are  similarly  susceptible  to  Botryosphaeria   dieback.  The  table-grape  Thompson  Seedless  was  most  susceptible  to P. viticola (mean LWD of 61 mm at 11 months  post  inoculation;  P  <   0.0001). V. vinifera cultivars and Concord showed similar susceptibility  to  the  Esca  pathogens,  Phaeomoniella  chlamydospora  and  T. minima.  Susceptibility  to  E.  lata  was  greatest  in  V.  arizonica  b42-26  (mean   LWD  of  96  mm  at  11  months  post  inoculation; P  <  0.03).  In  fact,  all   four American Vitis spp. were more susceptible to Eutypa dieback than  the  V.  vinifera  cultivars.  Our  findings  suggest  that  no  one  cultivar  is   likely  to  provide  resistance  to  the  range  of  trunk  pathogens  but  that   certain  cultivars  may  be  promising  candidates  for  commercially  relevant  host  resistance  in  grape-production  systems  where  the  dominant   cultivars are very susceptible.

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Dying-Arm Disease in Grapevines: Diagnosis of Infection with Eutypa lata by Metabolite Analysis

Dying-arm disease in grapevines, produced by infection with the ascomycete Eutypa lata, is responsible for major production losses in vineyards. Dieback of the shoots and cordon is believed to be due to acetylenic phenol metabolites produced by the fungus. To identify specific metabolites that could potentially be used for diagnosis of infection, eight E. lata isolates were grown in vitro on hot water extracts from grape varieties with various degrees of tolerance to the foliar symptoms of E. lata dieback. HPLC analysis showed that eutypinol was consistently produced in large amounts, together with smaller amounts of methyleutypinol and eulatachromene; eutypine, the putative toxin, was produced solely on Sauvignon Blanc extract and then in only barely detectable amounts. When E. lata isolates from Cabernet Sauvignon and Merlot were grown on identical media, the amounts of metabolites produced differed significantly between isolates but the pattern of metabolites was quite similar, with eutypinol again predominating. The consistent production of eutypinol indicated that this was the most suitable metabolite for which to analyze in order to diagnose the presence of E. lata. Extraction and analysis of grapevine tissues exhibiting symptoms of dieback failed to show the presence of any metabolites. However, when infected cordon sections were placed in water and cultured for 5 days, eutypinol was readily detected in the aqueous solution; metabolites were not produced from uninfected tissue. This provides a method for detection of infected tissue and indicates that the toxic metabolites react at the point of production, disrupting the vascular structure and inhibiting transport of nutrients, rather than being translocated to tissues that exhibit symptoms.

Behind the curtain of the compartmentalization process: Exploring how xylem vessel diameter impacts vascular pathogen resistance

A key determinant of plant resistance to vascular infections lies in the ability of the host to successfully compartmentalize invaders at the xylem level. Growing evidence supports that the structural properties of the vascular system impact host vulnerability towards vascular pathogens. The aim of this study was to provide further insight into the impact of xylem vessel diameter on compartmentalization efficiency and thus vascular pathogen movement, using the interaction between Vitis and Phaeomoniella chlamydospora as a model system. We showed experimentally that an increased number of xylem vessels above 100 μm of diameter resulted in a higher mean infection level of host tissue. This benchmark was validated within and across Vitis genotypes. Although the ability of genotypes to restore vascular cambium integrity upon infection was highly variable, this trait did not correlate with their ability to impede pathogen movement at the xylem level. The distribution of infection severity of cuttings across the range of genotype's susceptibility suggests that a risk-based mechanism is involved. We used this experimental data to calibrate a mechanistic stochastic model of the pathogen spread and we provide evidence that the efficiency of the compartmentalization process within a given xylem vessel is a function of its diameter.

Contrasting adaptation of xylem to dehydration in two Vitis vinifera L. sub-species

Xylem hydraulic properties of agricultural crop species can be linked to their region of origin, but because crop systems are often irrigated to reach optimum quality and yield, key differences in drought resistance are not often considered. We investigated how hydraulic conductivity and xylem vulnerability to drought-induced cavitation of two grapevine cultivars correspond to their centers of domestication with 'Merlot' (Vitis vinifera subspecies occidentalis) having been domesticated in a temperate forest region, and 'Thompson Seedless' (Vitis vinifera subspecies orientalis) domesticated in a semi-arid region. We used anatomical measurements and xylem vulnerability curves to evaluate hydraulic traits and drought resistance. Our results showed that 'Thompson Seedless' was significantly more vulnerable to drought-induced cavitation than 'Merlot'. Bench dehydration produced significantly different estimations of xylem vulnerability to cavitation in each cultivar. This result was consistent with anatomical measurement, with 'Thompson Seedless' stems having greater maximum stem-specific hydraulic conductivity, more vessels, higher vessel density and a greater lumen fraction than 'Merlot'. The relatively large amount of xylem vessels and lumen area in 'Thompson Seedless' is consistent with domestication in a semi-arid habitat where a greater number and size diversity of xylem vessels would be needed to transport water and meet evaporative demand as opposed to cultivars that were domesticated in temperate forest regions like 'Merlot'. These differences appear to expose 'Thompson Seedless' to high xylem vulnerability to cavitation.

Unravelling the colonization mechanism of Lasiodiplodia brasiliensis in grapevine plants

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, cankers, 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 Botryosphaeria 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 cambium 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 infect-ed tissues were depleted in starch in the ray parenchyma. Cellulose, hemicellulose, and lignin in cell walls were also degraded, supporting in vitro data.

Biochemical and histological insights into the interaction between the canker pathogen Neofusicoccum parvum and Prunus dulcis

The number of reports associated with wood dieback caused by fungi in the Botryosphaeriaceae in numerous perennial crops worldwide has significantly increased in the past years. In this study, we investigated the interactions between the canker pathogen Neofusicoccum parvum and the almond tree host (Prunus dulcis), with an emphasis on varietal resistance and host response at the cell wall biochemical and histological levels. Plant bioassays in a shaded house showed that among the four commonly planted commercial almond cultivars (‘Butte’, ‘Carmel’, ‘Monterey’, and ‘Nonpareil’), there was no significant varietal difference with respect to resistance to the pathogen. Gummosis was triggered only by fungal infection, not by wounding. A two-dimensional nuclear magnetic resonance and liquid chromatography determination of cell wall polymers showed that infected almond trees differed significantly in their glycosyl and lignin composition compared with healthy, noninfected trees. Response to fungal infection involved a significant increase in lignin, a decrease in glucans, and an overall enrichment in other carbohydrates with a profile similar to those observed in gums. Histological observations revealed the presence of guaiacyl-rich cell wall reinforcements. Confocal microscopy suggested that N. parvum colonized mainly the lumina of xylem vessels and parenchyma cells, and to a lesser extent the gum ducts. We discuss the relevance of these findings in the context of the compartmentalization of decay in trees model in almond and its potential involvement in the vulnerability of the host toward fungal wood canker diseases.

The role of melanin in the grapevine trunk disease pathogen Lasiodiplodia gilanensis

Lasiodiplodia (Botryosphaeriaceae) includes fungi that are considered among the most aggressive to grapevine, capable of causing cankers and necrotic lesions which eventually lead to death of host plants. A common characteristic of this genus is the presence of melanin in conidia and mycelium. Melanin is produced by the oxidation of phenolic and/or indolic compounds. For some fungi, this pigment is an essential factor for pathogenicity. This study characterized the types and the roles of melanin produced by Lasiodiplodia gilanensis. Using specific melanin inhibitors, L. gilanensis was shown to synthesize DOPA-melanin, DHN-melanin, and pyomelanin. DOPA-melanin was shown to be involved in production of aerial mycelium and protection against enzymatic lysis and oxidative stress; DHN-melanin to be involved in ramification of mycelium when exposed to nutrient deficiency; and pyomelanin to be related with hyphae development. The fungus used tyrosine as a precursor of DOPA- melanin and as carbon and nitrogen sources, and produced melanin inside the piths of infected plants. Genes involved in melanin synthesis were conserved among the Botryosphaeriaceae, highlighting the importance of melanin in this family.

The role of melanin in the grapevine trunk disease pathogen Lasiodiplodia gilanensis

Lasiodiplodia (Botryosphaeriaceae) includes fungi that are considered among the most aggressive to grapevine, capable of causing cankers and necrotic lesions which eventually lead to death of host plants. A common characteristic of this genus is the presence of melanin in conidia and mycelium. Melanin is produced by the oxidation of phenolic and/or indolic compounds. For some fungi, this pigment is an essential factor for pathogenicity. This study characterized the types and the roles of melanin produced by Lasiodiplodia gilanensis. Using specific melanin inhibitors, L. gilanensis was shown to synthesize DOPA-melanin, DHN-melanin, and pyomelanin. DOPA-melanin was shown to be involved in production of aerial mycelium and protection against enzymatic lysis and oxidative stress; DHN-melanin to be involved in ramification of mycelium when exposed to nutrient deficiency; and pyomelanin to be related with hyphae development. The fungus used tyrosine as a precursor of DOPA- melanin and as carbon and nitrogen sources, and produced melanin inside the piths of infected plants. Genes involved in melanin synthesis were conserved among the Botryosphaeriaceae, highlighting the importance of melanin in this family.

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