Multilocus Genotyping of 'Candidatus Phytoplasma Solani' Associated with Grapevine Bois Noir in Iran.

Grapevine Bois noir (BN) is associated with 'Candidatus Phytoplasma solani'. It has been recorded in vineyards throughout Europe as well as in different countries in Asia, where it now constitutes a threat to Iranian viticulture. BN is strictly dependent on 'Ca. P. solani' strains, wild host plants, and insect vectors. The molecular typing of 'Ca. P. solani', based on the nonribosomal gene tuf and the two hypervariable markers vmp1 and stamp, is valuable for the reconstruction and clarification of the pathways of BN spread. In this study, an RFLP analysis was performed on the vmp1 gene, and a single-nucleotide polymorphism analysis confirmed new vmp types in 'Ca. P. solani'. A stamp gene phylogenetic analysis allowed us to distinguish between the new genotype infections in the grapevines and the 'weeds' Convolvulus arvensis and Erigeron bonariensis in Iranian vineyards, highlighting the close genetic relatedness of the strains of 'Ca. P. solani' found in Iran and Azerbaijan. The most common genotype in the grapevines was tuf b/V24/stamp III, which was associated with C. arvensis. This information contributes toward the identification of further routes of introduction of 'Ca. P. solani' in Iran to sustain the control measures for the management of BN.

The Mycorrizal Status in Vineyards Affected by Esca.

In this work we analyzed the relationship among native arbuscular mycorrhizal fungi (AMF) and vine roots affected by esca, a serious grapevine trunk disease. The AMF symbiosis was analyzed on the roots of neighboring plants (symptomatic and asymptomatic to esca) in 14 sites of three vineyards in Marche region (central-eastern Italy). The AMF colonization intensity, identified by non-vital staining, showed higher value in all esca symptomatic plants (ranging from 24.6% to 61.3%) than neighboring asymptomatic plants (from 17.4% to 57.6%). The same trend of Glomeromycota phylum abundance was detected by analyzing fungal operational taxonomic units (OTUs) linked to the AMF community, obtained by amplicon high throughput analysis of ITS 1 region. Overall, the highest amount of OTUs was detected on roots from symptomatic plants (0.42%), compared to asymptomatic roots (0.29%). Specific primer pairs for native Rhizophagus irregularis and Funneliformis mosseae AMF species, were designed in 28S rRNA and large subunit (LSU) ribosomal RNA, respectively, and droplet digital PCR protocol for absolute quantification was set up. A higher number of DNA copies of both fungal species were detected more frequently in symptomatic than asymptomatic vines. Our study suggests a relationship between esca and native AMF in grapevine. These results underline the importance of native rhizosphere microbial communities for a better knowledge of grapevine esca disease.

Detection and Quantification of Stagonosporopsis cucurbitacearum in Seeds of Cucurbita maxima Using Droplet Digital Polymerase Chain Reaction.

Stagonosporopsis cucurbitacearum is an important seedborne pathogen of squash (Cucurbita maxima). The aim of our work was to develop a rapid and sensitive diagnostic tool for detection and quantification of S. cucurbitacearum in squash seed samples, to be compared with blotter analysis, that is the current official seed test. In blotter analysis, 29 of 31 seed samples were identified as infected, with contamination from 1.5 to 65.4%. A new set of primers (DB1F/R) was validated in silico and in conventional, quantitative real-time PCR (qPCR) and droplet digital (dd) PCR. The limit of detection of S. cucurbitacearum DNA for conventional PCR was ∼1.82 × 10-2 ng, with 17 of 19 seed samples positive. The limit of detection for ddPCR was 3.6 × 10-3 ng, which corresponded to 0.2 copies/µl. Detection carried out with artificial samples revealed no interference in the absolute quantification when the seed samples were diluted to 20 ng. All seed samples that showed S. cucurbitacearum contamination in the blotter analysis were highly correlated with the absolute quantification of S. cucurbitacearum DNA (copies/µl) in ddPCR (R 2 = 0.986; p ≤ 0.01). Our ddPCR protocol provided rapid detection and absolute quantification of S. cucurbitacearum, offering a useful support to the standard procedure.

Sixty Years from the First Disease Description, a Novel Badnavirus Associated with Chestnut Mosaic Disease.

Although chestnut mosaic disease (ChMD) was described several decades ago, its etiology is still not clear. Using classical approaches and high-throughput sequencing (HTS) techniques, we identified a novel Badnavirus that is a strong etiological candidate for ChMD. Two disease sources from Italy and France were submitted to HTS-based viral indexing. Total RNAs were extracted, ribodepleted, and sequenced on an Illumina NextSeq500 (2 × 150 nt or 2 × 75 nt). In each source, we identified a single contig of ≈7.2 kb that corresponds to a complete circular viral genome and shares homologies with various badnaviruses. The genomes of the two isolates have an average nucleotide identity of 90.5%, with a typical badnaviral genome organization comprising three open reading frames. Phylogenetic analyses and sequence comparisons showed that this virus is a novel species; we propose the name Chestnut mosaic virus (ChMV). Using a newly developed molecular detection test, we systematically detected the virus in symptomatic graft-inoculated indicator plants (chestnut and American oak) as well in chestnut trees presenting typical ChMD symptoms in the field (100 and 87% in France and Italy surveys, respectively). Datamining of publicly available chestnut sequence read archive transcriptomic data allowed the reconstruction of two additional complete ChMV genomes from two Castanea mollissima sources from the United States as well as ChMV detection in C. dentata from the United States. Preliminary epidemiological studies performed in France and central eastern Italy showed that ChMV has a high incidence in some commercial orchards and low within-orchard genetic diversity.

Endophytic Fungi as Potential Biological Control Agents against Grapevine Trunk Diseases in Alentejo Region.

Grapevine trunk diseases (GTDs) are the most widespread fungal diseases, affecting grapevines in all the major growing regions of the world, and their complete eradication is still not possible. Aiming to search alternatives to avoid the spread and high incidence of these diseases, the present work intended to molecularly identify the grapevine endophytic community, the phytopathogenic fungi associated with GTDs in vineyards within the Alentejo region, and to test potential antagonist microorganisms as biological control candidates against GTDs-associated fungi. Grapevine endophytic community showed a wide variety of fungi in GTDs' asymptomatic and symptomatic plants, nine of them previously described as GTDs-associated fungi. GTDs prevalent fungi identified in symptomatic plants were Diaporthe sp., Neofusicoccum sp., and H. viticola. Almost all these fungi were also detected in asymptomatic plants, which shows the importance of investigating the interactions of fungal communities and confirms the need for early diagnosis of these diseases. Direct inhibition antagonism tests were performed among identified endophytes and GTDs phytopathogenic fungi, and all the endophyte fungi showed potential as biocontrol agents. Our findings suggest that endophytes are promising candidates for their use in biological control due to their antagonistic activity against the mycelia growth of some GTDs-associated fungi.

Multilocus Genotyping Reveals New Molecular Markers for Differentiating Distinct Genetic Lineages among "Candidatus Phytoplasma Solani" Strains Associated with Grapevine Bois Noir.

Grapevine Bois noir (BN) is associated with infection by "Candidatus Phytoplasma solani" (CaPsol). In this study, an array of CaPsol strains was identified from 142 symptomatic grapevines in vineyards of northern, central, and southern Italy and North Macedonia. Molecular typing of the CaPsol strains was carried out by analysis of genes encoding 16S rRNA and translation elongation factor EF-Tu, as well as eight other previously uncharacterized genomic fragments. Strains of tuf-type a and b were found to be differentially distributed in the examined geographic regions in correlation with the prevalence of nettle and bindweed. Two sequence variants were identified in each of the four genomic segments harboring hlyC, cbiQ-glyA, trxA-truB-rsuA, and rplS-tyrS-csdB, respectively. Fifteen CaPsol lineages were identified based on distinct combinations of sequence variations within these genetic loci. Each CaPsol lineage exhibited a unique collective restriction fragment length polymorphism (RFLP) pattern and differed from each other in geographic distribution, probably in relation to the diverse ecological complexity of vineyards and their surroundings. This RFLP-based typing method could be a useful tool for investigating the ecology of CaPsol and the epidemiology of its associated diseases. Phylogenetic analyses highlighted that the sequence variants of the gene hlyC, which encodes a hemolysin III-like protein, separated into two clusters consistent with the separation of two distinct lineages on the basis of tufB gene sequences. Alignments of deduced full protein sequences of elongation factor-Tu (tufB gene) and hemolysin III-like protein (hlyC gene) revealed the presence of critical amino acid substitutions distinguishing CaPsol strains of tuf-type a and b. Findings from the present study provide new insights into the genetic diversity and ecology of CaPsol populations in vineyards.

Epidemiological Investigations and Molecular Characterization of 'Candidatus Phytoplasma solani' in Grapevines, Weeds, Vectors and Putative Vectors in Western Sicily, (Southern Italy).

Bois noir is caused by 'Candidatus Phytoplasma solani', and it is one of the most important and widespread diseases in the Euro-Mediterranean region. There are complex interactions between phytoplasma and grapevines, weeds, and vectors. These ecological relationships can be tracked according to molecular epidemiology. The aims of the 2-year study (2014-2015) were to describe incidence and spatial distribution of Bois noir in a vineyard with three grapevine varieties in Sicily, and to identify the molecular types of the tuf and vmp1 genes in these naturally infected grapevines, according to the potential reservoir plants and vectors. Disease incidence in 2015 was significantly higher in 'Chardonnay' (up to 35%) than for 'Nero d'Avola' and 'Pinot noir' (<5%). All grapevine, weed, and insect samples were infected by 'Ca. P. solani' tuf-type b. Most of the collected insects were strictly related to Vitis spp. and belonged to Neoaliturus fenestratus, Empoasca spp., and Zygina rhamni. The characterization of the vmp1 gene revealed six different vmp types in grapevines (V1, V4, V9, V11, V12, V24), three in weeds (V4, V9, V11), and four in insects (V4, V9, V11, V24). Notably, V4, V9, appear both in hosts and vectors, with V9 predominant. Virtual restriction fragment length polymorphism (RFLP) analysis based on the nucleotide sequences supported the data of the conventional RFLP. Connections between the molecular data recorded in the vineyard ecosystems and the application of innovative tools based on the geostatistical analysis will contribute to further clarification of the specific ecological and epidemiological aspects of 'Ca. P. solani' in Sicily.

Hypobaric Packaging Prolongs the Shelf Life of Refrigerated Black Truffles (Tuber melanosporum).

Black truffle (Tuber melanosporum Vitt.) is a fine agro-food product known for its unique aroma and very limited shelf life (maximum of 5-7 days, room temperature). Hypobaric packaging at 30 kPa, a mix of 1% O2/99% N2, and 40% CO2/60% N2 were studied to prolong the shelf life of black truffle at 4 °C in sealed polypropylene vessels, compared to normal atmosphere. Epiphytic microbial population, firmness, weight loss, CO2 formation, and sensory properties were monitored weekly up to 35 days of storage and were related to the volatile profile. Principal components analysis revealed good correlation between the storage time and the decrease of firmness, and the increase of the microbial count and CO2 production. Only truffles stored under hypobaric conditions showed an acceptable quality after 14 days storage. Hypobaric packaging is a cheap strategy to prevent the swelling of vessels caused by respiration and can reduce the deviation from the high-quality level of the fresh product from one to at least two weeks.

Sequence Analysis of New Tuf Molecular Types of 'Candidatus Phytoplasma Solani' in Iranian Vineyards.

Grapevine Bois noir (BN) is caused by 'Candidatus Phytoplasma solani' ('Ca. P. solani') and is one of the most important phytoplasma diseases in the Euro-Mediterranean viticultural areas. The epidemiology of BN can include grapevine as a plant host and is usually transmitted via sap-sucking insects that inhabit herbaceous host plants. Tracking the spread of 'Ca. P. solani' strains is of great help for the identification of plant reservoirs and insect vectors involved in local BN outbreaks. The molecular epidemiology of 'Ca. P. solani' is primarily based on sequence analysis of the tuf housekeeping gene (which encodes elongation factor Tu). In this study, molecular typing of tuf, through restriction fragment length polymorphism and sequencing, was carried out on grapevine samples from Iranian vineyards. According to the molecular characterization, three molecular types-tuf b1, tuf b5 and tuf b6-were found, with tuf b1 being the most prominent. These data provide further knowledge of tuf gene diversity and question the ecological role of such "minor" tuf types in Iranian vineyards, which have been detected only in grapevines.

Spatial pattern of Bois noir: case study of a delicate balance between disease progression and recovery.

Bois noir (BN) is the most important phytoplasma disease of Euro-Mediterranean area and induces severe loss of production and even the death of vines. Understanding the delicate balance between disease progression and recovery of BN infected plants over space and time is crucial to set up management tools. The data collected and analysed allowed to provide insights into dispersal pattern of the disease, caused by'Candidatus Phytoplasma solani'. Point pattern analysis (PPA) was applied to assess the spatial arrangement of symptomatic plants and the spatial correlation of disease levels in four vineyards. For spatio-temporal patterns of BN, a mark-correlation function was applied. Space-time PPA over multiple years (2011-2015) provided graphical visualisation of grapevines more severely affected by BN along the borders of the vineyards, mainly in 2011 when disease incidence was high. PPA across the symptomatic plants in the four vineyards confirmed this visual trend: an overall aggregated pattern at small (<10 m) spatial scales (2013) that were more evident later at all spatial scales (0-15 m). Application of this innovative spatial approach based on point and surface pattern analyses allowed the spread and severity of BN to be monitored, to define the dispersal routes of the pathogen. Such data will contribute to better understand the distribution of symptomatic plants over space and time and to define a model for preventive strategies to reduce future infections.

Morphological and Molecular Identification of Seedborne Fungi in Squash (Cucurbita maxima, Cucurbita moschata).

Squash is one of the most important crops of tropical and temperate regions, and it can be affected by several fungal pathogens. Most of these pathogens infect the seeds, which become an efficient vehicle to disperse seedborne pathogens over long distances, with consequent severe crop losses. The main objective of this study was the identification of the principal seedborne fungi in seeds extracted from 66 samples of asymptomatic and symptomatic squash fruit (Cucurbita maxima, Cucurbita moschata) collected in two countries, Tunisia and Italy. The symptoms of fruit decay were identified and classified according to lesion size. Following the blotter test, 14 fungal species were detected from the seeds. Seedborne fungi were identified in all fruit samples tested, including asymptomatic fruit. The most frequent fungi from Tunisian seeds were Alternaria alternata (25.1%), followed by Stagonosporopsis cucurbitacearum (24.6%), Fusarium solani (16.6%), Rhizopus stolonifer (13.3%), F. fujikuroi (7.8%), Albifimbria verrucaria (3.3%), and Stemphylium vesicarium (2.3%). For the fruits from Italy, the most frequently identified fungal species in seed samples were Alternaria alternata (40.0%), followed by F. fujikuroi (20.8%), Stemphylium vesicarium (3.0%), and Curvularia spicifera (2.1%). Morphological identification was confirmed by molecular diagnosis using the available species-specific primers. Furthermore, specific primers were designed to identify Albifimbria verrucaria, Paramyrothecium roridum, and Stemphylium vesicarium. Application of seed-health testing methods, including such conventional and molecular diagnostic tools, will help to improve seed quality and crop yields.

Molecular Typing of 'Candidatus Phytoplasma solani' in Iranian Vineyards.

Grapevine (Vitis vinifera L.) is one of the most important horticultural crops in Iran, with >200,000 ha of cultivated area. Recently, outbreaks of the grapevine yellows Bois noir that is associated with phytoplasma strains related to 'Candidatus Phytoplasma solani' were recorded in several Iranian regions. This has resulted in severe economic losses. We carried out a survey in 2015, followed by collection of leaf samples from symptomatic grapevines and weeds. Because no information is available on the molecular epidemiology of 'Ca. P. solani' in Iran, multiple gene analyses were carried out here according to molecular characterization of the tuf and vmp1 genes. From the molecular characterization, all of the samples (i.e., grapevines, weeds) were infected with tuf b type. Detailed molecular characterization of the vmp1 gene (i.e., PCR-restriction fragment length polymorphism, sequence analysis) defined five molecular types: V1, V4, V10, V15, and V20. The abundance of Convolvulus arvensis in vineyards and detection of the same 'Ca. P. solani' molecular types in grapevines and weeds suggest that C. arvensis has a major role in Bois noir epidemiology of Iranian vineyards. Therefore, control strategies should be developed to manage these host plants to reduce inoculum sources of the phytoplasma in vineyards.

Detection of 'Candidatus Phytoplasma solani' in roots from Bois noir symptomatic and recovered grapevines.

'Candidatus Phytoplasma solani' is the causal agent of Bois noir (BN) in grapevine (Vitis vinifera). It is usually detected in leaves, where typical disease symptoms are seen. However, little information is available on the presence of this phytoplasma in grapevine roots. Here, we investigated 'Ca. P. solani' in roots collected from 28 symptomatic, 27 recovered and eight asymptomatic grapevine plants. Protocols based on high-resolution melting (HRM) combined with real-time quantitative PCR (qPCR-HRM) and nested-qPCR-HRM were developed to identify 'Ca. P. solani' tuf-type variants with single nucleotide polymorphisms. In all, 21.4% of roots from symptomatic plants were positive to 'Ca. P. solani' using qPCR-HRM, and 60.7% with nested-qPCR HRM. Also, 7.4% of roots from recovered plants were positive using qPCR-HRM, which reached 44.4% using nested-qPCR HRM. These analyses identified tuf-type b1 on 88.2% of the positive samples from symptomatic grapevines, and 66.6% from recovered grapevines, with all other samples identified as tuf-type a. This study reports the presence of 'Ca. P. solani' in the roots of both symptomatic and recovered grapevines. These qPCR-HRM and nested-qPCR-HRM protocols can be applied to increase the sensitivity of detection of, and to simplify and speed up the screening for, 'Ca. P. solani' tuf-types.

Phenotypic and Molecular Investigations on Hypovirulent Cryphonectria parasitica in Italy.

Chestnut blight is caused by the fungus Cryphonectria parasitica. As one of the most ecologically important diseases of Castanea spp., C. parasitica can rapidly kill trees. In Europe, mitigation of disease severity took place spontaneously through colonization of C. parasitica by mycoviruses, which reduced the virulence of the fungus. In the framework of a survey, 138 C. parasitica isolates were identified, and virulent/hypovirulent phenotypes were determined through morphological properties and pathogenicity tests. For a pool of four hypovirulent isolates, dsRNA was extracted, cDNA synthesized, and a library subjected to next-generation sequencing. The bioinformatics analysis allowed detecting and reconstructing the complete genome of Cryphonectria hypovirus 1 (CHV-1), denoted as CHV-1 Marche. When compared with the available genomes of other hypoviruses that affected the virulence of C. parasitica, available in databases, CHV-1 Marche showed some nucleotide diversity. The approach used in this study was effective to explore the virome inside a pool of hypovirulent C. parasitica isolates. Next-generation sequencing allowed us to exclude the presence of any other ssRNA and dsRNA viruses infecting the fungus and determine CHV-1 as the only responsible of hypovirulence of C. parasitica in the analyzed samples.

Genetic Variability of Stolbur Phytoplasma in Hyalesthes obsoletus (Hemiptera: Cixiidae) and its Main Host Plants in Vineyard Agroecosystems.

Bois noir is an economically important grapevine yellows that is induced by 'Candidatus Phytoplasma solani' and principally vectored by the planthopper Hyalesthes obsoletus Signoret (Hemiptera: Cixiidae). This study explores the 'Ca. P. solani' genetic variability associated to the nettle-H. obsoletus and bindweed-H. obsoletus systems in vineyard agroecosystems of the central-eastern Italy. Molecular characterization of 'Ca. P. solani' isolates was carried out using polymerase chain reaction/restriction fragment length polymorphism to investigate the nonribosomal vmp1 gene. Seven phytoplasma vmp-types were detected among the host plants- and insect-associated field-collected samples. The vmp1 gene showed the highest polymorphism in the bindweed-H. obsoletus system, according to restriction fragment length polymorphism analysis, which is in agreement with nucleotide sequence analysis. Five vmp-types were associated with H. obsoletus from bindweed, of which one was solely restricted to planthoppers, with one genotype also in planthoppers from nettle. Type V12 was the most prevalent in both planthoppers and bindweed. H. obsoletus from nettle harbored three vmp-types, of which V3 was predominant. V3 was the only type detected for nettle. Our data demonstrate that planthoppers might have acquired some 'Ca. P. solani' profiles from other plant hosts before landing on nettle or bindweed. Overall, the different vmp1 gene rearrangements observed in these two plant hosts-H. obsoletus systems might represent different adaptations of the pathogen to the two host plants. Molecular information about the complex of vmp-types provides useful data for better understanding of Bois noir epidemiology in vineyard agroecosystem.

In-vineyard population structure of 'Candidatus Phytoplasma solani' using multilocus sequence typing analysis.

'Candidatus Phytoplasma solani' is a phytoplasma of the stolbur group (16SrXII subgroup A) that is associated with 'Bois noir' and causes heavy damage to the quality and quantity of grapevine yields in several European countries, and particularly in the Mediterranean area. Analysis of 'Ca. P. solani' genetic diversity was carried out for strains infecting a cv. 'Chardonnay' vineyard, through multilocus sequence typing analysis for the vmp1, stamp and secY genes. Several types per gene were detected: seven out of 20 types for vmp1, six out of 17 for stamp, and four out of 16 for secY. High correlations were seen among the vmp1, stamp and secY typing with the tuf typing. However, no correlations were seen among the tuf and vmp1 types and the Bois noir severity in the surveyed grapevines. Grouping the 'Ca. P. solani' sequences on the basis of their origins (i.e., study vineyard, Italian regions, Euro-Mediterranean countries), dN/dS ratio analysis revealed overall positive selection for stamp (3.99, P=0.019) and vmp1 (2.28, P=0.001). For secY, the dN/dS ratio was 1.02 (P=0.841), showing neutral selection across this gene. Using analysis of the nucleotide sequencing by a Bayesian approach, we determined the population structure of 'Ca. P. solani', which appears to be structured in 3, 5 and 6 subpopulations, according to the secY, stamp and vmp1 genes, respectively. The high genetic diversity of 'Ca. P. solani' from a single vineyard reflects the population structure across wider geographical scales. This information is useful to trace inoculum source and movement of pathogen strains at the local level and over long distances.

Effects of an innovative strategy to contain grapevine Bois noir: field treatment with resistance inducers.

Grapevine Bois noir (BN) is a phytoplasma disease that is widespread in most viticultural regions of the world, and it can result in heavy reductions to yields and grape juice quality. At present, there is no effective strategy to reduce the incidence of BN-infected grapevines. However, phytoplasma-infected plants can recover through spontaneous or induced symptom remission. Five elicitors (chitosan, two glutathione-plus-oligosaccharine formulations, benzothiadiazole, and phosetyl-Al) were applied weekly to the canopy of BN-infected 'Chardonnay' grapevines from early May to late July. The best and most constant recovery inductions were obtained with benzothiadiazole and the two glutathione-plus-oligosaccharine formulations. The plants that recovered naturally or following the elicitors showed qualitative and quantitative parameters of production no different from healthy plants. In another vineyard, diseased plants showed reduced shoot length and production compared with healthy plants, and there were no negative effects on these parameters for grapevines sprayed with a glutathione-plus-oligosaccharine formulation. The application of resistance inducers promoted the recovery of BN-infected grapevines with no adverse effects on the plants. Therefore, grapevine can be used as a model species to test this innovative strategy to contain phytoplasma diseases.

Effects of Bois noir on carbon assimilation, transpiration, stomatal conductance of leaves and yield of grapevine (Vitis vinifera) cv. Chardonnay.

Bois noir (BN) is one of the main phytoplasma diseases of grapevine (Vitis vinifera). It is widespread, and can cause severe losses in European vineyards. The infective agent colonizes phloem elements and induces visible symptoms of leaf yellowing or reddening after a relatively long incubation period. As the most sensitive cultivars to BN, Chardonnay plants were grouped as healthy or symptomatic in spring, based on the records from the previous year. Leaf gas exchange and chlorophyll a fluorescence were measured weekly from July to September in healthy plants, and in symptomatic and asymptomatic leaves from symptomatic plants. The midday relative water content (mRWC) was measured once per month. The detection of phytoplasma DNA by nested-polymerase chain reaction revealed BN infection in symptomatic leaf samples at the end of September. A significant decrease in pigment content and maximum quantum efficiency of photosystem II (Fv/Fm) of these symptomatic leaves was detected from July to September, although in the asymptomatic leaves of the symptomatic plants the net photosynthesis (Pn) decrease was not significant. In the leaves from the healthy plants, Pn and transpiration were relatively stable. Of note, in July, an initially healthy plant showed a strong Pn reduction that was followed by visible leaf yellowing symptoms only in August. The phytoplasma infection also stimulated significant reductions in mRWC of the symptomatic leaves, with a final large decrease in yield.

Colonization of Vitis spp. wood by sGFP-transformed Phaeomoniella chlamydospora, a tracheomycotic fungus involved in Esca disease.

To evaluate wood colonization and interactions with Vitis spp. of Phaeomoniella chlamydospora, a fungal agent involved in Esca disease, isolate CBS 229.95 was transformed using a pCT74 construct which contained the genetic markers for synthetic green fluorescent protein (sGFP) and hygromycin B phosphotransferase. Nine stable P. chlamydospora fungal transformants (Pch-sGFP lines) were obtained using polyethylene-glycol-mediated transformation of protoplasts. These were characterized for sgfp and hygromycin B phosphotransferase (hph) genome insertions and for sGFP fluorescence emission, using quantitative polymerase chain reaction and fluorimetric systems, respectively. No correlation was observed between sgfp copy number genome insertion and sGFP fluorescence expression. Cuttings of Vitis vinifera 'Montepulciano', 'Verdicchio', 'Sangiovese', 'Biancame', and 'Cabernet Sauvignon'; and the grapevine rootstocks 'Kober 5BB', 'SO4', '420A', '1103P', and V. rupestris were inoculated by immersion in a conidial suspension of the selected fungal Pch-sGFP71 line and incubated at 4 ± 1 and 25 ± 1°C. Wood colonization was estimated through epifluorescence microscopy and was affected by incubation temperature. After 6 months at 4 ± 1°C, the fungal growth was completely inhibited. At 25 ± 1°C, the highest extent of wood colonization was recorded in Montepulciano and Verdicchio, with the lowest in the rootstocks SO4 and V. rupestris. The expression of the Pch-sGFP71 transformed line was localized in the xylem area, primarily around the vessels. The use of sGFP-transformed P. chlamydospora helped to clarify different aspects associated with the location of this pathogen in grapevine tissue, before disease symptom expression.