PCR-Based Strategy To Detect and Identify Species of Phaeoacremonium Causing Grapevine Diseases

Species of Phaeoacremonium (especially Phaeoacremonium aleophilum) are associated with two severe diseases in grapevines, Petri disease in young plants and Esca disease in adult plants. Phaeoacremonium species grow slowly on culture medium, and it is difficult to identify these species on the basis of morphological characteristics. Primers Pm1 and Pm2 were designed in the ribosomal DNA internal transcribed spacer (ITS) regions ITS1 and ITS2, respectively. They yielded a single amplicon of 415 bp for nine species of Phaeoacremonium that may occur in grapevines. A nested PCR (using general fungal primers ITS1F/ITS4 in the primary reaction) was developed to detect Phaeoacremonium directly in grapevine wood. Molecular detection was more sensitive than the traditional method of culturing in growth medium was. Identification of Phaeoacremonium species was achieved by digesting the PCR-amplified fragment with the restriction enzymes BssKI, EcoO109I, and HhaI. It was possible to distinguish these species by their restriction fragment length polymorphism patterns, except for Phaeoacremonium viticola and Phaeoacremonium angustius, which had 100% similarity in their ITS region sequences. A species-specific PCR amplification of the partial β-tubulin gene using the primer pair Pbr4_1/T1 and Pbr8/T1 was necessary to differentiate P. angustius from P. viticola, respectively. An easy and fast protocol was developed to detect and identify species of Phaeoacremonium in a few hours. Primers defined here can be used in a plant nursery sanitation program to produce plants free of Phaeoacremonium spp. Use of healthy grapevine plants in new plantations is the most effective measure to manage Petri disease.     

Pathogenicity of Phaeoacremonium Species on Grapevines

Several Phaeoacremonium species have been recently described to include some species involved in disease of decline of woody plants and others associated with human infections. Thirteen species are currently reported on grapevines and they are suspected to be involved in Esca and Petri disease. The pathogenic character of new defined species is still unknown and, therefore, pathogenicity studies were conducted in this work. The pathogenicity of the following species was studied on grapevine seedlings and cuttings: Phaeoacremonium aleophilum , P. angustius , P. inflatipes , P. krajdenii , P. mortoniae , P. parasiticum , P. scolyti , P. venezuelense , P. viticola , and Phaeomoniella chlamydospora included as positive control. Two-month-old grapevine seedlings of Vitis vinifera cv. Malvar and cv. Airen were inoculated by watering 10 individual pots with a spore suspension (10⁷ spores/ml) of each Phaeoacremonium species. All inoculated seedlings showed typical symptoms of a vascular disease 2 months after inoculation. Grapevine cuttings of Vitis vinifera cv. Monastrell were vacuum-inoculated with a spore suspension (10⁸ spores/ml) and individually planted. Plants rated after 5 months showed that all Phaeoacremonium species and P. chlamydospora (used as positive control) caused a significant vascular discoloration, while only Phaeomoniella chlamydospora , Phaoeacremonium mortoniae and P. aleophilum caused a significant root weight reduction compared with a non-inoculated control. Phaoeacremonium parasiticum , P. angustius , P. inflatipes and P. venezuelense caused significant foliar symptoms that included interveinal chlorosis and stunted leaves.     

A biomarker for the identification of four <Emphasis Type="Italic">Phaeoacremonium</Emphasis> species using the β-tubulin gene as the target sequence

A real-time polymerase chain reaction (PCR) was developed for the rapid detection and identification of Phaeoacremonium species, the fungi associated with severe diseases in grapevines. A degenerate primer pair (F2bt–R1bt) with homology to the β-tubulin gene was designed to be used in the amplification of 11 species of Phaeoacremonium. Four species-specific probes labelled with three different fluorescent dyes were designed to be used with the degenerate primers in a real-time PCR for the identification of Phaeoacremonium aleophilum, P. parasiticum, P. viticola and P. mortoniae. Combinations of two probes in a duplex real-time PCR allowed to detect and identify a mixture of Phaeoacremonium species and cross-amplifications were not detected. This method was applied to detect Phaeoacremonium species in eight wood fragments from grapevine plants naturally infected, and results were compared with those obtained with nested PCR and culturing on growth media. Real-time PCR detected Phaeoacremonium in 100% of the analysed fragments, whereas nested PCR did only in the 62% of them and requiring subsequent restriction fragment-length polymorphism analysis to identify the species. This method is a sensitive tool to detect and identify Phaeoacremonium species in infected grapevine wood. Real-time PCR assay defined here can be used in a plant nursery program to identify pathogen-free plants in order to manage Petri disease of grapevines.     

Togninia (Calosphaeriales) is confirmed as teleomorph of Phaeoacremonium by means of morphology, sexual compatibility and DNA phylogeny

Petri disease, or black goo, is a serious disease of vines in most areas where grapevines are cultivated. The predominant associated fungus is Phaeomoniella chlamydospora (Chaetothyriales). Several species of Phaeoacremonium (Pm.) also are associated, of which Pm. aleophilum is the most common. Although no teleomorph is known for Phaeoacremonium, the genus Togninia previously has been linked to phaeoacremonium-like anamorphs. To investigate the possible anamorph-teleomorph connection of Phaeoacremonium to Togninia, anamorphs of Togninia minima, T. fraxinopennsylvanica and T. novae-zealandiae morphologically were compared with Pm. aleophilum and some representative cultures were mated in all combinations. Although no interspecies mating proved fertile, matings between isolates of Pm. aleophilum produced a Togninia teleomorph within 3-4 weeks. Certain field isolates of Pm. aleophilum commonly produced the teleomorph, demonstrating that both mating types can occur in the same vine and thus also explaining the genetic diversity observed for this fungus in some vineyards. To elucidate the phylogenetic relationships among these taxa, isolates were subjected to sequence analysis of the nuclear ribosomal internal transcribed spacers (ITS1, ITS2) and the 5.8S rRNA gene, as well as portions of the translation elongation factor 1 alpha (EF-1α) gene. The generic placement of teleomorphs within Togninia (Calosphaeriales) further was confirmed via phylogenetic analyses of 18S small subunit (SSU) DNA. From these sequences, morphological and mating data, we conclude that T. minima is the teleomorph of Pm. aleophilum, and that it has a biallelic heterothallic mating system. An epitype and mating type tester strains also are designated for T. minima.     

Sensitivity of Petri disease pathogens to hot-water treatments in vitro

Petri disease pathogens ( Phaeoacremonium spp. and Phaeomoniella chlamydospora ) are able to colonize the vessels in the xylem of grapevine propagating material. Hot-water treatment (HWT) protocols at 50°C for 30 min have been applied in grapevine nurseries to control these pathogens with variable results. The effect of HWT in vitro at higher temperatures on Pa.   chlamydospora , Phaeoacremonium aleophilum and Phaeoacremonium parasiticum isolates was determined by placing conidial suspensions and plugs of agar with mycelia in Eppendorf vials and incubated in hot water at 49, 50, 51, 52, 53 or 54°C for 30, 45 or 60 min. Conidial germination and the colony growth rate decreased with increased temperature and time combinations. Pa.   chlamydospora was more sensitive than Phaeoacremonium spp. to the experimental temperatures for all periods of time. Pa.   chlamydospora tolerated temperatures of 53°C, while Pm.   aleophilum and Pm.   parasiticum tolerated temperatures of 54°C, although the treatments above 51–52°C drastically reduced conidial germination and mycelial growth. These results highlight the need to develop HWT using temperatures above 51°C to reduce the incidence of fungal infections and ensure high-quality propagating material for grapevine growers.     

Detection of black-foot and Petri disease pathogens in soils of grapevine nurseries and vineyards using bait plants

Background and aims

Little information is currently available regarding the number of species of black-foot and Petri disease pathogens present in soil and their capacity to infect grapevine roots and reach the xylem vessels.

Methods

Seedlings of grapevine rootstock 41-B, and cvs. Bobal and Palomino were planted both in pots containing soil samples collected from commercial vineyards and in nursery fields. Roots and xylem vessels were later analyzed for fungal isolation.

Results

Black-foot pathogens: Ilyonectria alcacerensis, I. macrodidyma, I. novozelandica and I. torresensis were frequently isolated from roots of seedlings grown in all soils evaluated, whereas Petri disease pathogens: Cadophora luteo-olivacea, Phaeoacremonium aleophilum, Pm. parasiticum and Phaeomoniella chlamydospora were only isolated from xylem vessels of seedlings grown in nursery soils, with a low incidence. Ilyonectria alcacerensis, I. novozelandica and I. torresensis were isolated for the first time from grapevines in Spain, and Pm. parasiticum and Ca. luteo-olivacea were detected for the first time in nursery soils.

Conclusions

Our results confirm nursery and vineyard soils as an important inoculum source for black-foot pathogens and demonstrate the presence of several Petri disease pathogens in nursery soils.     

Development of an isolate-specific marker for tracking Phaeomoniella chlamydospora infection in grapevines

Petri disease causes decline of grapevines worldwide. The grapevine endophyte Phaeomoniella chlamydospora is the most important fungal pathogen associated with this disease. Epidemiological studies of this pathogen have been hampered by its common occurrence in the internal tissue of apparently healthy vines. Development of a molecular marker for a single strain would overcome this limitation and aid experiments designed to answer key questions about the biology of this pathogen. Genetic variation analysis of New Zealand and Italian strains of P. chlamydospora detected a potential molecular marker in New Zealand isolate A21. Characterization of the 1010 bp marker band showed that it had 50% identity to moxY, a gene involved in the aflatoxin biosynthetic pathway of Aspergillus parasiticus. Sequencing of the region flanking the 1010 bp product revealed a single nucleotide polymorphism in the 3' border of the marker band. Primers were designed to amplify a 488 bp fragment encompassing this polymorphic site and cleavage of this product with the restriction enzyme BsrI produced three bands only in isolate A21 and two bands in all other isolates tested. The sensitivity of the PCR-RFLP protocol was increased with a nested PCR approach and the protocol optimized for soil and wood samples. When the nested PCR/RFLP procedure was used to determine the persistence of viable and nonviable spores in soil, the results showed that nonviable spores were undetected after 8 wk whereas viable spores still could be detected at 17 wk.     

The Distribution of Grapevine Yellows Disease Associated with the Buckland Valley Grapevine Yellows Phytoplasma

Three blocks of Chardonnay in one vineyard in the Buckland Valley of Victoria, Australia, were surveyed over 4 years for grapevine yellows disease (GYd). Buckland Valley grapevine yellows phytoplasma (BVGYp) was the only phytoplasma detected by polymerase chain reaction (PCR) and restriction fragment length polymorphism (RFLP) analysis in GYd affected grapevines. GYd affected many grapevines and was characterized by remission of disease, some recurrence and occurrences in previously unaffected grapevines. A regional survey of the Buckland Valley indicated that both GYd and BVGYp occurred in the same restricted grape growing area. Within this area, BVGYp was detected in two vineyards that had been established using planting material from different sources. One could therefore speculate that BVGYp was present in these grapevines as a result of aerial transmission and was not present in the original planting material.     

Characterization of Plasmopara-resistance in grapevine using in vitro plants

Although the exact mechanisms by which grapevine cells operate to reduce disease incidence caused by the downy mildew fungus Plasmopara viticola are not fully elucidated, our cytological results obtained from infected in vitro-plants confirm that enhanced disease resistance is associated with an expression of distinct reactions in a chronological order. An increased production of reactive oxygen species (superoxide radicals, 4-6 hours post infection, hpi) was followed by a hypersensitive response (6-8 hpi), an increased activity of peroxidase in cells flanking the infection area and in the vascular tissue (10-12 hpi) and an increased production, accumulation or conversion of phenolic compounds (12-15 hpi). These mechanisms seem also to be present in susceptible varieties as shown after an inoculation with non-host oomycetic pathogens on the basis of peroxidase activity, but they do not become activated after P. viticola infection. The investigation of the peroxidase activity in leaves at several time points after an infection with P. viticola indicated that there is a strong correlation between the POX activity in leaves of in vitro-plants and the resistance of grapevine plants to P. viticola in the field.     

Identification of fungal pathogens associated with grapevine trunk using fluorescent-labelled ribosomal DNA probe

Detection and identification of fungal pathogens associated with grapevine trunk diseases (GTDs) are often difficult and laborious. The aim of the study is to develop a simple and time-saving protocol for the identification of a broad range of fungal species causing GTDs by fragment length analysis of the internal transcribed spacer (ITS) regions 1 and 2 and the large subunit D2 hypervariable region of the ribosomal DNA (rDNA). A total of 32 fungal isolates from declining vines and six type strains, representing 19 different species was included in this study. The majority of the fungi had unique species-specific PCR products ranging from 212 to 370 bp (ITS1), 332 to 428 bp (ITS2) and 320 to 325 bp (LSU-D2), and the combination of three regions identifies all pathogenic fungi tested. The protocol proposed here provides a highly sensitive, reliable and rapid identification method for a broad range of significant grapevine fungal trunk pathogens.     

Development of a transient expression system in grapevine via agro-infiltration

Transient expression of genes using Agrobacterium is a powerful tool for the analysis of gene function in plants. We have developed this method for the analysis of genes involved in disease resistance in grapevine leaves. Our research showed that the quality of the plant material, the plant genotype used for agro-infiltration and the presence of additional virulence factors (carried on plasmid pCH32) in the Agrobacterium strain are all important factors for success of the procedure. After optimising these factors, we consistently achieve sufficient acceptable levels of expression of the markers beta-glucuronidase (GUS) and green fluorescent protein (GFP) using vacuum infiltration of grapevine leaves from plants grown in vitro. We used this procedure to investigate the proposed role of stilbenes in defense against grapevine downy mildew (Plasmopara viticola) by transiently overexpressing stilbene synthase in grapevine leaves, before infection with P. viticola. We found that agro-infiltration itself induces the synthesis of stilbenes in grapevine leaves, thus preventing us to test the effect of the overexpression of stilbene synthase in defense. However, our results revealed that agro-infiltration before P. viticola inoculation had an effect on the development of the infection. Further research is required to show whether stilbenes or some other factor are the causal agent restricting pathogen development. The method described here provides and excellent tool to exploit at the many grapevine genomic resources now available, and will contribute to a better understanding of many areas of grapevine biology.     

Laminarin elicits defense responses in grapevine and induces protection against Botrytis cinerea and Plasmopara viticola

Grapevine (Vitis vinifera L.) is susceptible to many pathogens, such as Botrytis cinerea, Plasmopara viticola, Uncinula necator, and Eutypa lata. Phytochemicals are used intensively in vineyards to limit pathogen infections, but the appearance of pesticide-resistant pathogen strains and a desire to protect the environment require that alternative strategies be found. In the present study, the beta-1,3-glucan laminarin derived from the brown algae Laminaria digitata was shown both to be an efficient elicitor of defense responses in grapevine cells and plants and to effectively reduce B. cinerea and P. viticola development on infected grapevine plants. Defense reactions elicited by laminarin in grapevine cells include calcium influx, alkalinization of the extracellular medium, an oxidative burst, activation of two mitogen-activated protein kinases, expression of 10 defense-related genes with different kinetics and intensities, increases in chitinase and beta-1,3-glucanase activities, and the production of two phytoalexins (resveratrol and epsilon-viniferin). Several of these effects were checked and confirmed in whole plants. Laminarin did not induce cell death. When applied to grapevine plants, laminarin reduced infection by B. cinerea and P. viticola by approximately 55 and 75%, respectively. Our data describing a large set of defense reactions in grapevine indicate that the activation of defense responses using elicitors could be a valuable strategy to protect plants against pathogens.     

Major changes in grapevine wood microbiota are associated with the onset of esca,a devastating trunk disease

Esca, a major grapevine trunk disease in old grapevines, is associated with the colonization of woody tissues by a broad range of plant pathogenic fungi. To identify which fungal and bacterial species are involved in the onset of this disease, we analysed the microbiota from woody tissues of young (10-year-old) grapevines at an early stage of esca. Using meta-barcoding, 515 fungal and 403 bacterial operational taxonomic units (OTUs) were identified in woody tissues. In situ hybridization showed that these fungi and bacteria co-inhabited in grapevine woody tissues. In non-necrotic woody tissues, fungal and bacterial microbiota varied according to organs and seasons but not diseased plant status. Phaeomoniella chlamydospora, involved in the Grapevine trunk disease, was the most abundant species in non-necrotic tissues from healthy plants, suggesting a possible non-pathogenic endophytic behaviour. Most diseased plants (70%) displayed cordons, with their central white-rot necrosis colonized essentially by two plant pathogenic fungi (Fomitiporia mediterranea: 60%–90% and P. chlamydospora: 5%–15%) and by a few bacterial taxa (Sphingomonas spp. and Mycobacterium spp.). The occurrence of a specific association of fungal and bacterial species in cordons from young grapevines expressing esca-foliar symptoms strongly suggests that that microbiota is involved in the onset of this complex disease.     

Alpha-proteobacteria cultivated from marine sponges display branching rod morphology

PCR amplification of two CHS gene fragments of the obligate biotroph Plasmopara viticola, the causal agent of downy mildew of grapevine, is described. While one fragment shows homology to fungal class IV chitin synthases, the other fragment groups with other oomycete chitin synthases to form a novel class of chitin synthases most closely related to class I-III. RT-PCR experiments indicate that PvCHS1 is constitutively expressed, whereas PvCHS2 is specifically transcribed in sporangiophores and sporangia. Analyses of wheat germ agglutinin labeling patterns by confocal laser scanning microscopy show that chitin is present on the surface of hyphal cell walls during in planta growth, and of sporangiophores and sporangia.     

Localization and subcellular association of Grapevine Pinot Gris Virus in grapevine leaf tissues

Despite the increasing impact of Grapevine Pinot gris disease (GPG-disease) worldwide, etiology about this disorder is still uncertain. The presence of the putative causal agent, the Grapevine Pinot Gris Virus (GPGV), has been reported in symptomatic grapevines (presenting stunting, chlorotic mottling, and leaf deformation) as well as in symptom-free plants. Moreover, information on virus localization in grapevine tissues and virus-plant interactions at the cytological level is missing at all. Ultrastructural and cytochemical investigations were undertaken to detect virus particles and the associated cytopathic effects in field-grown grapevine showing different symptom severity. Asymptomatic greenhouse-grown grapevines, which tested negative for GPGV by real time RT-PCR, were sampled as controls. Multiplex real-time RT-PCR and ELISA tests excluded the presence of viruses included in the Italian certification program both in field-grown and greenhouse-grown grapevines. Conversely, evidence was found for ubiquitous presence of Grapevine Rupestris Stem Pitting-associated Virus (GRSPaV), Hop Stunt Viroid (HSVd), and Grapevine Yellow Speckle Viroid 1 (GYSVd-1) in both plant groups. Moreover, in every field-grown grapevine, GPGV was detected by real-time RT-PCR. Ultrastructural observations and immunogold labelling assays showed filamentous flexuous viruses in the bundle sheath cells, often located inside membrane-bound organelles. No cytological differences were observed among field-grown grapevine samples showing different symptom severity. GPGV localization and associated ultrastructural modifications are reported and discussed, in the perspective of assisting management and control of the disease.     

Uwe Joachim Nebert ‐ 70 Jahre

Esca is a very important and destructive disease of grapevine in many grapevine producing countries. Several groups of fungi have been reported from grapevines with esca disease symptoms; however, Phaeoacremonium species are the main hyphomycete fungi involved in this disease. In recent years, esca disease symptoms with consequent decline disease have frequently been observed in vineyards of north-western Iran. Nevertheless, the involvement of Phaeoacremonium spp. with esca disease symptoms in this region remains unknown. During 2008–2010 growing seasons, wood samples were collected from vines showing typical esca disease symptoms such as interveinal leaf chlorosis with subsequent necrosis and various types of internal wood deterioration in north-western parts of Iran. A total of 44 Phaeoacremonium-like hyphomycetes were recovered from sampled materials. Fungal isolates were subjected to tentative morphological identification and were further characterised by using sequenced data from ITS-rDNA and β-tublin gene. Phylogeny inferred using sequence data from ITS-rDNA region and β-tublin gene revealed that Phaeoacremonium aleophilum and P. mortoniae together with Sarocladium strictum (syn. Acremonium strictum) are associated with esca disease symptoms in this region. The pathogenicity of Phaeoacremonium spp. and Sarocladium strictum were verified by the inoculation of cutting root and stems under greenhouse conditions. Isolates of P. aleophilum and P. mortoniae were the most virulent based on the length of vascular necrosis on woody stems that followed by Sarocladium strictum. This is first study on the pathogenicity of Sarocladium strictum on grapevine worldwide and the first report on the occurrence of P. mortoniae on grapevines in north-western Iran.     

Genetic similarity among Cercospora apii-group species and their detection in host plant tissue by PCR/RFLP analyses of the rDNA internal transcribed spacer (ITS)

The objective of this study was to determine the genetic relatedness among the Cercospora and Pseudocercospora species closely related to Cercospora apii by using a polymerase chain reaction/restriction fragment length polymorphism (PCR/RFLP) analysis of the internal transcribed spacer (ITS) region. A single PCR fragment (about 550 bp) was obtained from all Cercospora species categorized as the C. apii-group, Pseudocercospora purpurea, Pseudocercospora conyzae, and Pseudocercospora cavarae. Cercospora caricis yielded a 680 bp PCR fragment. The similarity in the PCR fragment size and RFLP profiles among the C. apii-group isolates, including Pseudocercospora purpurea, and Pseudocercospora conyzae strongly suggests that these species are conspecific. Synonymy with C. apii (lectotype) at a subspecific rank has been proposed. Amplified ITS regions of genomic DNA extracted from spinach leaves showing 12 and 233% leaf spot disease symptoms caused by Cercospora beticola yielded two PCR fragments (i.e., one from the fungus and one from the host plant) and were resolved by electrophoresis of the PCR product in 3% LMP agarose. Digestion of the total PCR product with HinfI restriction enzyme yielded RFLP profiles similar to those obtained from amplified DNA from the causative agent, C. beticola. The method described in this preliminary study offers rapid detection and diagnosis of fungal infections in plants for disease prediction and management and screening of plant materials for quarantine purposes.     

Seasonal Migration of Apolygus spinolae (Hemiptera: Miridae) between Grapevines and Herbaceous Plants

This study was conducted to verify the seasonal migration of Apolygus spinolae (Meyer-Dür) between grapevines and herbaceous plants. Overwintering eggs were hidden in the hair layer under grapevine bud scales. A. spinolae adults were captured on sticky traps in the grapevine yard from late spring to early summer, dwindled through the summer, and captured again in late fall. However, adults were observed from early summer in herbaceous plant fields. A. spinolae adults were abundant during the summer of July and August in the herbaceous field, and thereafter its density decreased through fall. A few or no A. spinolae was found on mesh-netted grapevines after the installation year of the mesh-net, which indicated that A. spinolae adults migrating to lay overwintering eggs during the autumn could not land at the grapevines because of the mesh-net. Damaged shoots by A. spinolae were concentrated near the edge of grapevine yards bordering the herbaceous plants. This distribution pattern of shoot damage was believed to be related to an oviposition behavior of A spinolae, reflecting that adults migrating from herbaceous plants lay eggs more frequently in grapevines adjacent to the summer host plants. Seasonal occurrence of A. spinolae in grapevine yards was suggested as follows: A. spinolae overwinter as eggs in dormant buds on grapevines and hatch in the spring. Nymphs feed on grapevines then develop to adults (spring population), and migrate to herbaceous plants. A. spimolae spends the summer on the herbaceous hosts (summer population). Then, adults migrate back to grapevines in late autumn and lay overwintering eggs.     

Leishmania donovani: intraspecific polymorphisms of Sudanese isolates revealed by PCR-based analyses and DNA sequencing

Four polymerase chain reaction (PCR)-based approaches were used to analyze diversity within 23 Sudanese isolates of Leishmania donovani. Methods compared were fingerprinting with single nonspecific primers, restriction analysis of the amplified ribosomal internal transcribed spacer (ITS) locus, single-stranded conformation polymorphism (SSCP), and sequencing of the ITS region. When PCR fingerprinting and restriction analysis of ITS were applied, highly similar fragment patterns were observed for all strains of L. donovani studied. The ITS1 locus gave five different SSCP profiles among the 23 Sudanese isolates, whereas the ITS2 locus was highly conserved with the exception of 1 isolate. Strains of L. donovani derived from other geographical areas were found to have different ITS2 patterns. SSCP analysis correlated well with results of DNA sequencing and confirmed that SSCP was able to detect genetic diversity at the level of a single nucleotide. SSCP had advantages over the other methods employed for investigation of sequence variation within the species L. donovani. There was no correlation between the form of clinical manifestation of the disease and the PCR fingerprinting, ITS-RFLP, or ITS-SSCP characteristics.     

A Basic Peroxidase Isoenzyme, Marker of Resistance Against Plasmopara viticola in Grapevines, is Induced by an Elicitor from Trichoderma viride in Susceptible Grapevines

The constitutive expression of basic peroxidase isoenzymes in the Plasmopara viticola -resistant ( Vitis vinifera × Vitis rupestris) × Vitis riparia crossing and in the P. viticola -susceptible V. vinifera parent species was studied. The results illustrate that both leaves and stems of the ( V. vinifera × V. rupestris) × V. riparia crossing showed the differential expression of a basic peroxidase isoenzyme B₃ (pl = 8.9), this being almost completely absent from the P. viticola -susceptible V. vinifera parent species. To test whether the basic peroxidase isoenzyme B₃ may be considered as a molecular marker of disease resistance in Vitis spp., suspension cell cultures derived from the P. viticola -susceptible V. vinifera parent species were treated with an elicitor (cellulase Onoztika R-10) from the soil fungus Trichoderma viride , a specific and well-known elicitor of disease resistance reactions in grapevines. The results showed that treatment with the elicitor induces, simultaneously with the activation of the disease resistance mechanism, the appearance of B₃ in the cell cultures. These results suggest that the basic peroxidase isoenzyme B₃ may be considered as a marker of disease resistance in Vitis species since it is present in the P. viticola -resistant ( V. vinifera × V. rupestris) × V. riparia hybrid and is induced by the elicitor Onozuka R-10 in cell cultures of the P. viticola -susceptible Vitis vinifera parent species. This conclusion is supported by the presence of this isoenzyme in other resistant and its absence in other susceptible Vitis spp.