Chemical and biological protection of grapevine propagation material from trunk disease pathogens

Wound protection during all stages of grapevine propagation is of utmost importance to prevent infection of propagation material by decline and dieback pathogens. In semi-commercial nursery trials, grapevine rootstock and scion cuttings were soaked in water (control), chemical or biological sanitation products prior to cold storage, prior to grafting (machine- or hand-grafting) and prior to planting in field nurseries. Natural infection levels in basal ends and graft unions of uprooted nursery grapevines were evaluated 8 months after planting. Total pathogen incidences in the water-treated control plants ranged from 30% in basal ends to 13.5% in graft unions. Phaeomoniella chlamydospora was the most commonly isolated pathogen, followed by Phaeoacremonium, Cylindrocarpon + Campylocarpon, Botryosphaeria and Phomopsis spp. Machine-grafted unions generally had lower pathogen incidences compared with hand-grafted graft unions. In general, repeated soak-treatments of propagation material in the tested products resulted in reduced pathogen incidences in nursery grapevines. However, products containing T. harzianum (Trichoflow-T), hydrogen peroxide (Bio-sterilizer) and 8-hydroxyquinoline sulphate (Chinosol) gave inconsistent results, whereas Bronocide (a blend of halogenated alcohols and water) proved to be a good sterilising agent, but reduced certifiable plant yield significantly. Benomyl (at 100 g/100 l), Sporekill (a patented didecyldimethylammonium chloride formulation at 150 ml/100 l) and captan (at 1000 ml/100 l) were consistently the best treatments as growth parameters were not negatively influenced and pathogen incidences in basal ends and graft unions of uprooted plants were reduced.     

Co‐infection by Botryosphaeriaceae and Ilyonectria spp. fungi during propagation causes decline of young grafted grapevines

Decline of newly planted, grafted grapevines is a serious viticultural problem worldwide. In the Riverina (New South Wales, Australia), characteristic symptoms include low fruit yields, very short shoots and severely stunted roots with black, sunken, necrotic lesions. To determine the cause, roots and wood tissue from affected plants in 20 vineyards (Vitis vinifera cv. Chardonnay grafted to V. champini cv. Ramsey rootstock) were assayed for microbial pathogens. Ilyonectria spp. (I. macrodidyma or I. liriodendra, producers of phytotoxin brefeldin A, BFA, and cause of black foot disease of grapevines) and Botryosphaeriaceae spp. (predominantly Diplodia seriata) were isolated from rootstocks of 100 and 95% of the plants, respectively. Togninia minima and Phaeomoniella chlamydospora (cause of grapevine Petri disease) were isolated from 13 and 7% of affected plants, respectively. All Ramsey rootstock stems of grafted plants sampled from a supplier nursery were infected with Ilyonectria spp. and D. seriata. Diplodia seriata, but not Ilyonectria spp., was also isolated from 25% of canes sampled from the rootstock source block. Root inoculation of potted, disease‐free Chardonnay plants with Ilyonectria isolates from diseased vineyards caused typical disease symptoms, while co‐inoculation with Botryosphaeriaceae spp. increased disease severity. This is the first study to show that a major cause of young grapevine decline can be sequential infection by Botryosphaeriaceae from rootstock cuttings and Ilyonectria spp. from nursery soil. Although the Petri disease fungi were less common in young declining grafted grapevines in the Riverina, they are likely to contribute to the decline of surviving plants as they mature.     

Occurrence of <Emphasis Type="Italic">Phaeoacremonium</Emphasis> spp. and <Emphasis Type="Italic">Phaeomoniella chlamydospora</Emphasis> in grape propagation materials and young grapevines

During a 6-year study, grapevine propagation materials and young grapevines were analysed to evaluate the presence of internal wood discolouration and the occurrence of fungal species involved in Petri disease. The intensity of wood discolouration increased with the ageing of the plants. The maximum incidence of dark streaks was observed in the rootstock while necrosis originating from buds or nodes were notably present in the trunk and cordon of older vines. In contrast, the highest levels of brown-red halo symptoms, defined as discoloured areas around the pith, were recorded in the early growth stages. Phaeoacremonium spp. and Phaeomoniella chlamydospora were usually isolated from the rooted-grafts and the 3-year old plants, respectively. The number of infected grapevines increased with age. Most of the P. chlamydospora strains were isolated from dark streaks or dots, while Phaeoacremonium spp. were detected in brown-red halo symptoms and other symptomatic or asymptomatic wood. The greatest incidence of the two fungal taxa was recorded in the lower parts of the grapevine, including the roots and rootstock.     

Evaluation of the grapevine nursery propagation process as a source of <Emphasis Type="Italic">Phaeoacremonium</Emphasis> spp. and <Emphasis Type="Italic">Phaeomoniella chlamydospora</Emphasis> and occurrence of trunk disease pathogens in rootstock mother vines in Spain

Five commercial nurseries were sampled in 2007 to evaluate the grapevine nursery propagation process as a source of Petri disease pathogens (Phaeoacremonium spp. and Phaeomoniella chlamydospora). Samples were taken at four stages of the propagation process: pre-grafting hydration tanks, scissors used for cutting buds, grafting machines and peat used to promote root development. All samples were analysed using two different techniques: nested PCR using specific primers for Phaeoacremonium spp. (Pm1/Pm2) and Pa. chlamydospora (Pch1/Pch2); and fungal isolation by culturing on semi-selective medium. Either Phaeoacremonium spp. or Pa. chlamydospora were detected at any of these stages, and more importantly they were viable since they were detected by isolating on culturing medium. Additionally, the importance of grapevine rootstock mother fields as sources of inoculum in the nurseries was studied. Fourteen grapevine rootstock mother fields were surveyed in 2006 and 2007 for the occurrence of fungal trunk pathogens. A total of 16.4% and 30% of the plants sampled in 2006 and 2007, respectively were infected. Petri disease pathogens (Pa. chlamydospora, Phaeoacremonium aleophilum, Pm. parasiticum) and several Botryosphaeriaceae species (Neofusicoccum parvum, Botryosphaeria dothidea, Lasiodiplodia theobromae, N. australe, N. mediterraneum and N. vitifusiforme) and Phomopsis viticola were isolated. This is the first time N. mediterraneum has been isolated from grapevines and the first report of N. australe, N. mediterraneum and N. vitifusiforme in Spain. This work shows that grapevine rootstock mother plants and the propagation process of grapevine plants should be considered as important sources of inoculum for fungal trunk pathogens, and especially of Petri disease pathogens.     

Detection of Botryosphaeriaceae species within grapevine woody tissues by nested PCR, with particular emphasis on the Neofusicoccum parvum/N. ribis complex

Several species of Botryosphaeriaceae and Phaeomoniella chlamydospora are common agents of grapevine decline worldwide. Currently, the use of culture independent PCR based techniques for detection of Botryosphaeriaceae within grapevine tissues has been limited to Botryosphaeria dothidea. In the present study, two Botryosphaeriaceae specific nested PCR assays were developed. One with a narrow target range, to detect Neofusicoccum parvum and the closely related species complex (Neofusicoccum parvum/N. ribis sensu Pavlic et al. Molecular Phylogenetics and Evolution 51:259–268, 2009) and another, with a wider range, to detect all 17 species of Botryosphaeriaceae which have been reported as potential wood pathogens of grapevine. The effectiveness of these assays was validated in vivo on naturally infected wood samples collected from standing vines and dormant grafted rooted cuttings commercialized in Italy by different nurseries in different years. All samples were also screened by means of a previously published nested PCR assay specific for Phaeomoniella chlamydospora. It was found that: 1) propagation material may play an important role as source of primary inoculum, not only of Phaeomoniella chlamydospora, as previously reported, but also for members of the Botryosphaeriaceae, among which Neofusicoccum parvum, Botryosphaeria dothidea and Diplodia seriata are the most common, and 2) multiple infections by different species belonging to Botryosphaeriaceae and/or Phaeomoniella chlamydospora occur frequently both in standing vines and propagation material. This last finding supports the hypothesis that at least some of the non-specific symptoms of grapevine decline may be due to the presence of different pathogens within host tissues.     

A Survey of Trunk Disease Pathogens within Rootstocks of Grapevines in Spain

Grapevine trunk disease pathogens, and specifically Petri disease pathogens, can be spread by planting infected plants. Due to the increasing incidence of Petri disease and other young grapevine declines reported lately in Spain, a sampling of plants used before for new vineyards were carried out in 2002 and 2004. A total number of 208 plants (grafted and non grafted) were collected, of which 94 plants (45.2%) were infected with at least one of the following pathogens: Phaeomoniella chlamydospora, and species of Phaeoacremonium, Botryosphaeria, Cylindrocarpon, and Phomopsis. Species of the genera Phaeoacremonium and Botryosphaeria isolated in 2004 were identified using morphological and molecular characters. Species of Phaeoacremonium identified were P. aleophilum and P. parasiticum; and those of Botryosphaeria were B. obtusa, B. dothidea and B. parva. This is the first report of P. parasiticum and B. parva occurring on grapevines in Spain. Distribution of pathogens within the plants was studied in 2004. Phaeomoniella chlamydospora was not detected in the graft union of any plant; however, species of Botryosphaeria and Phomopsis were detected along the plant, but mainly in the graft union; Phaeoacremonium aleophilum was detected along the grafted plants, but not in rooted rootstocks. The results suggest that infected plants used for new plantings in Spain are an important source of primary inoculum of the pathogens associated with grapevine trunk diseases in the field.     

Detection of black-foot disease pathogens in the grapevine nursery propagation process in Spain

Two commercial nurseries located in Comunidad Valenciana region (central-eastern Spain) were sampled in 2010 to evaluate whether the grapevine nursery propagation process could be a source of black-foot disease pathogens. Samples were taken from four sources of the propagation process: pre-grafting hydration tanks, scissors used for cutting buds, omega-cut grafting machines, and peat used for callusing. DNA from these samples was extracted and multiplex nested-PCR using primers specific for “Cylindrocarpon” pauciseptatum, Ilyonectria liriodendri and I. macrodidyma-complex (composed of I. alcacerensis, I. estremocensis, I. macrodidyma, I. novozelandica, I. torresensis, and two undescribed species) was used to identify the species present. Ilyonectria liriodendri and I. macrodidyma-complex were detected in hydration tanks, scissors, grafting machines and peat, I. macrodidyma-complex being the most frequent. Additionally, ten grafted cuttings each from five grapevine scion/rootstock combinations were collected from each nursery immediately after callusing, and again after one growing season in a nursery field. Roots of these grafted cuttings and plants were sampled to isolate the fungal pathogens. Only I. torresensis was isolated after callusing, while I. liriodendri, I. novozelandica and I. torresensis were isolated after one growing season, showing the highest incidence at this latter sampling time. Moreover, DNA was extracted from roots and analyzed as described before. Ilyonectria liriodendri and I. macrodidyma-complex were also detected at both sampling times. The use of the multiplex nested-PCR technique improved the detection of I. liriodendri and I. macrodidyma-complex from grafted cuttings and plants in both nurseries. This work shows that the grapevine nursery propagation process should be considered as a potential infection source for black-foot disease pathogens, and confirms that infections caused by Ilyonectria spp. in grapevine planting material increase markedly after one growing season in nursery fields.     

AFLP markers reveal two genetic groups in the French population of the grapevine fungal pathogen <Emphasis Type="Italic">Phaeomoniella chlamydospora</Emphasis>

Phaeomoniella chlamydospora, (Chaetothyriales, Herpotrichiellaceae) is one of the main causal agents of Petri disease and esca on grapevines. We have used AFLP markers to study the population genetic structure of 74 isolates collected at different spatial scales: 56 isolates originated from vines with esca disease sampled from four French vineyards (Poitou-Charentes, Aquitaine, Languedoc-Roussillon, Alsace); 18 isolates were collected from a single plot (Aquitaine vineyard). Significant linkage disequilibrium indicated that P. chlamydospora populations are not panmictic, whereas the level of haplotypic diversity observed, 72 single multilocus haplotypes identified in total among the 74 isolates analysed, suggest that reproduction in this species may not be strictly clonal. Clustering analyses suggests the presence of two genetically differentiated but sympatric clusters of isolates. The level of differentiation between the two clusters is high (F _ST = 0.23) and significant at 13 out of the 21 loci analyzed. The most plausible explanation for this pattern of admixture is the coexistence in P. chlamydospora French populations of two predominant clonal lineages. Finally, the low level of spatial genetic differentiation in this study is consistent with the spread of this fungus through the transport of infected plant material by human activities.     

Immunological detection of Phaeoacremonium aleophilum,a fungal pathogen found in esca disease

Phaeoacremonium aleophilum is a fungal pathogen implicated in esca, a devastating disease affecting grapevines around the world. We have previously reported that the fungus secreted in its culture medium a variety of polypeptides, which allowed us to develop a serological method of detection. In this method, rabbit antibodies raised against the polypeptide fraction recognized secreted fungal proteins with high sensitivity (commonly 1 ng). These antibodies were specific since they cross-reacted with polypeptides secreted by various strains of Pm. aleophilum, but not with the proteins secreted by many other fungal pathogens implicated in other grapevine infections. Importantly, as shown by ELISA tests and immunolocalization, they did not cross-react with the secreted polypeptides of Eutypa lata (agent of Eutypa dieback), Neofusicoccum parvum (found in Black Dead Arm), and Phaeomoniella chlamydospora (also found in the fungal complex inducing esca). This serological approach permitted the development of a reliable dot-blot method to detect the presence in woody members of Pm. aleophilum in selectively infected trunk and canes. This method of diagnosis is rapid, easy to perform and non destructive for grapevines. Advantages and drawbacks of the method are discussed.     

Evaluation of Greek grapevine cultivars for resistance to <Emphasis Type="Italic">Phaeomoniella chlamydospora</Emphasis>

In the present study, four Greek (Agiorgitiko, Asyrtiko, Roditis and Xinomavro) and one international (Soultanina) grapevine cultivars (Vitis vinifera L.) were screened for their resistance to Phaeomoniella chlamydospora. Artificial inoculation was carried out by drilling a hole into the trunk and injecting a concentrated conidial suspension into the vessels. Disease reactions were evaluated in an 87-day assessment period, on the basis of external symptoms (disease incidence, disease severity and mortality) and by calculating the relative areas under disease progress curves (relative AUDPC). The extension of vascular browning as well as the isolation ratio along the inoculated vine trunks were also taken into account as additional parameters for evaluating resistance. The results indicated that the resistance of grapevine cultivars to P. chlamydospora varied significantly. ‘Agiorgitiko’ and ‘Soultanina’ were susceptible, whereas ‘Asyrtiko’ and ‘Xinomavro’ were resistant; ‘Roditis’ showed an intermediate level of resistance. Cultivars’ resistance was mostly distinguished in terms of the extension of vascular browning and pathogen isolation ratio. On the contrary, the disease incidence, final disease severity, mortality and relative AUDPC provided less distinctive efficiency in resistance evaluation. The robust methodology presented here could be useful in rapid evaluation experiments for future screening programs to search and recognize natural resistant sources within grapevine genotypes against P. chlamydospora.     

Temporal spore dispersal patterns of grapevine trunk pathogens in South Africa

Trunk disease pathogens of grapevines, viz. Phaeomoniella chlamydospora, Eutypa lata and several species in Botryosphaeriaceae, Phaeoacremonium and Phomopsis are known to infect fresh pruning wounds by means of air-borne inoculum released after rainfall or prolonged periods of high relative humidity. Recent surveys have demonstrated that most or all of these pathogens are present in climatically diverse grape growing regions of South Africa. However, the factors controlling spore dispersal of these pathogens in vineyards were largely unknown. To address this question, spore trapping was done in a Chenin Blanc vineyard in the Stellenbosch area, South Africa, for 14 weeks during the grapevine pruning period from June to mid-September of 2004 and 2005. Hourly recordings of weather data were done by a weather station in the row adjacent to the spore trap. Spores of E. lata and Phomopsis and species in Botryosphaeriaceae were trapped throughout the trapping periods of 2004 and 2005, with higher levels of trapped spores recorded in 2005. The spores of all three pathogens were trapped during or after periods of rainfall and/or high relative humidity. In neither of the 2 years were spores of Pa. chlamydospora or Phaeoacremonium spp. trapped. Results indicated that spore event incidence, as well as the amount of spores released during a spore event of above-mentioned pathogens, were governed by rainfall, relative humidity, temperature and wind speed prior to and during the spore events.     

Genetic diversity of Grapevine rupestris stem pitting-associated virus isolates from Tunisian grapevine germplasm

Grapevine rupestris stem pitting-associated virus (GRSPaV) is one of the most widespread grapevine viruses and is transmitted mainly by grafting. GRSPaV presence was tested in 487 samples representative of the Tunisian grapevine germplasm (including autochthonous, table, wine, wild grape, and rootstock varieties) from different Tunisian regions. GRSPaV infection was detected in 51.3% of samples from different Tunisian regions, among which the table grapevine cultivars were the most commonly infected (68.7%). Genetic variability of GRSPaV isolates from wild and cultivated grapevines was assessed by sequencing the partial capsid protein (CP) gene of 19 Tunisian isolates and 1 Italian GRSPaV isolate from Sicily, and the partial RNA-dependent RNA polymerase (RdRp) gene of 13 Tunisian GRSPaV isolates. According to phylogenetic analysis of CP nucleotide sequences obtained in this study and sequences retrieved from GenBank, Tunisian isolates fell into four phylogenetic groups already described (I, II, III, and IV) and two new phylogenetic groups (VI and VIII). Phylogenetic analysis of the partial RdRp gene revealed that Tunisian isolates of GRSPaV are distributed into four phylogroups. This study highlights the importance of regular monitoring of GRSPaV infections in Tunisia, with special regard to those grapevine accessions employed in conservation and selection programmes. In particular, the presence of new GRSPaV genetic variants and infection of wild grapevines must be taken into account in order to choose a correct control strategy.     

Genetic structure of the grapevine fungal pathogen Phaeomoniella chlamydospora in southeastern Australia and southern France

In this study, 18 microsatellite loci were used to examine the genetic structure and mode of reproduction of Phaeomoniella chlamydospora, the fungus that causes Petri disease of grapevine and is involved in another grapevine disease, esca. A total of 60 southeastern Australian isolates were tested and compared with 64 isolates from southern France. The French population possessed relatively high genotypic diversity (G = 29·2) whilst the Australian population showed low genotypic diversity (G = 11·1), consistent with a limited founder population. Haplotypes from four different grapevine cultivars were not significantly differentiated (Φpt values effectively zero). Likewise, genetic differentiation of haplotypes from different regions within each country was not significant, although small but significant genetic differentiation (9%) was identified between Australia and France. Based on bootstrapped cluster analysis, there did not appear to be any genetic groups within the overall sample of isolates. Significant linkage disequilibrium identified in both countries, together with overrepresented, widespread identical haplotypes, indicated limited genetic recombination and a largely clonal structure consistent with the absence of an observed sexual cycle in this species.     

Characterisation and detection of Pythium and Phytophthora species associated with grapevines in South Africa

Replant and decline diseases of grapevines not only cause quantitative and qualitative yield losses, but also result in extra costs when vineyards have to be replanted. This study investigated the role of Pythium and Phytophthora in the decline syndrome in South Africa by determining (1) the species associated with nursery and established vines, and (2) pathogenicity of Ph. sp. niederhauserii and P. vexans relative to known grapevine pathogens. Quantitative real-time PCR (qPCR) assays were also developed for detection of the most prevalent oomycete groups. In total, 26 Pythium and three Phytophthora species were identified from grapevine nurseries and established vineyards. The most common infections in sampled nursery vines were caused by P. vexans (16.7%), followed by P. ultimum var. ultimum (15.0%) and P. irregulare (11.7%). In established vineyards, P. irregulare (18.0%) and P. vexans (6.2%) were also among the three most prevalent species, along with P. heterothallicum (7.3%). Three Phytophthora species were also identified from the sampled established vines, of which Ph. cinnamomi (5.1%) was predominant, followed by Ph. sp. niederhauserii (1.1%). In established vineyards a higher incidence and more diverse species composition was observed in spring and winter, than in summer. Pathogenicity studies showed that some Ph. sp. niederhauserii and P. vexans isolates were as aggressive as the known grapevine pathogens Ph. cinnamomi and P. irregulare. Sensitive qPCR assays were developed for the detection of P. ultimum var. ultimum, P. irregulare, P. vexans and the genus Phytophthora. These assays will be invaluable in limiting pathogen dispersal through screening of nursery material. This is especially important since pathogenic species were also isolated from healthy looking vines in nurseries.     

Isolation,production and in vitro effects of the major secondary metabolite produced by Trichoderma species used for the control of grapevine trunk diseases

Antibiosis has been shown to be an important mode of action by Trichoderma species used in the protection of grapevine pruning wounds from infection by trunk pathogens. The major active compound from Trichoderma isolates known to protect grapevine pruning wounds from trunk pathogen infection was isolated and identified. The compound, a 6‐pentyl‐α‐pyrone (6PP), was found to be the major secondary metabolite, by quantity, which accumulated in the culture filtrate of T. harzianum isolate T77 and the two T. atroviride isolates UST1 and UST2. Benzimidazole resistant mutants generated from these isolates also produced 6PP as their main secondary metabolite, except for a mutant of T77 that had lost its ability to produce 6PP. The isolates UST1 and UST2 were co‐cultured with the grapevine trunk pathogens Eutypa lata and Neofusicoccum parvum in a minimal defined medium and a grapevine cane‐based medium (GCBM). Co‐culturing UST1 with N. parvum induced 6PP production in the minimal defined medium and the GCBM. The production of 6PP by UST2 was induced in the GCBM, while co‐culturing with the two trunk pathogens either reduced or had no effect on 6PP production. Mycelial growth and ascospore/conidia germination of E. lata, N. australe, N. parvum and Phaeomoniella chlamydospora were inhibited by 6PP in a concentration‐dependent manner. The results show that the presence of N. parvum and grapevine wood elicits the production of 6PP, suggesting that this metabolite is involved in Trichoderma–pathogen interactions on grapevine pruning wounds.     

Effects of three esca-associated fungi on Vitis vinifera L.: IV. Diffusion through the xylem of metabolites produced by two tracheiphilous fungi in the woody tissue of grapevine leads to esca-like symptoms on leaves and berries

Two naphthalenone pentaketides (scytalone and isosclerone) and α-glucans (pullulan) are produced in vitro and in planta by Phaeomoniella chlamydospora (Pch) and Togninia minima (Tmi), two tracheiphilous fungi associated with the ‘esca’ disease of grapevines. The possible role of such fungal metabolites in inducing symptoms on leaves and berries was studied in a vineyard of Vitis vinifera cv. Italia located in southern Italy. During early spring, pruning off two to four branches per vine allowed samples of xylem sap to be collected. Vine bleeding, assessed as ml day⁻¹ vine⁻¹, reached its maximum at bud burst and stopped within 28 days. The total amount of sap collected from healthy vines was about a quarter of that from esca-affected vines. During the same period, the leaf water potential of diseased vines increased progressively (i.e. showed less negative values), indicating a dysfunction in water and nutrient supply to the new growth. Both fungi were isolated from the xylem sap and from the woody tissue of branches and the trunk of diseased vines. Conidia isolated from the sap showed a high germination rate (>90%). Bioactive concentrations of the two pentaketides were detected in xylem sap, leaves and berries at various stages of seasonal growth. Exopolysaccharides, including pullulan, were found in the xylem sap. Absorption of culture filtrates of Pch and Tmi, as well as weak solutions of purified preparations of scytalone, isosclerone or pullulan, on detached leaves and berries caused symptoms similar to those shown by the esca-affected vines in the field.     

Inoculum sources of Botryosphaeriaceae species in New Zealand grapevine nurseries

This study investigated the sources of inoculum of Botyosphaeriaceae species in three commercial grapevine nurseries in New Zealand. Botryosphaeriaceae propagules were detected on the surfaces of 33 to 100?% of canes and 33?% of dead grapevine materials using microscopy, plating assays and PCR with Botryosphaeriaceae multi-species primers (Bot100F/Bot472R). Isolations also showed that 15 to 68?% of the canes were internally infected by Botryosphaeriaceae species, with Neofusicoccum luteum and N. parvum being the most predominant species. Incidence varied between nurseries, with Nursery 5 having highest N. luteum incidence (53?%) and Nursery 3 having highest N. parvum incidence (88?%). Botryosphaeriaceae DNA was detected in rain-water run-off from the mother-vine canopy, but not in soil samples collected around infected mother-vines. However, samples from the nursery propagation stages had no visible or viable Botryosphaeriaceae propagules although PCR using multi-species primers detected Botryosphaeriaceae DNA from wash and hydration tanks, on grafting tools and in callusing media from the different nurseries. The single-stranded conformational polymorphism (SSCP) was able to identify N. parvum, N. luteum and Diplodia mutila as the most common species present in the nursery system. Overall results indicated that the canes grown in mother-vine blocks were the most likely source of inoculum for infection of young grafted plants by Botryosphaeriaceae species.     

Detection and quantification of Ilyonectria spp. associated with black‐foot disease of grapevine in nursery soils using multiplex nested PCR and quantitative PCR

Three nursery fields and three rootstock mother fields from commercial nurseries located in Comunidad Valenciana region (central‐eastern Spain) were surveyed in July 2011 to detect the presence and to quantify Ilyonectria spp. in the soil. In each field, ten soil samples were taken randomly with a soil probe at a depth of 10–30 cm, and 10–20 cm from the base of the plant. Three replicate subsamples (10 g each) were taken from each soil sample. DNA was extracted and a multiplex nested PCR with species‐specific primer pairs (Mac1/MaPa2, Lir1/Lir2 and Pau1/MaPa2) was used to identify the species present. Among the 180 soil DNA samples analysed, Ilyonectria spp. were detected in 172 of them. Ilyonectria macrodidyma complex was the most frequently detected, being identified in 141 samples from all the fields evaluated. However, I. liriodendri was detected in only 16 samples, but was present in all open‐root field nurseries and in two rootstock mother fields. In addition, quantitative PCR (qPCR) assays were done to assess the levels of I. liriodendri and I. macrodidyma‐complex DNA in the soil samples. Detection of Ilyonectria spp. DNA using qPCR correlated with the fields found positive with the nested multiplex PCR. DNA concentrations of Ilyonectria spp. ranged from 0·004 to 1904·8 pg μL^?1. In general, samples from rootstock mother fields showed the highest DNA concentrations. The ability to detect and quantify Ilyonectria spp. genomic DNA in soil samples from nursery fields and rootstock mother fields confirms soils from both field types as important inoculum sources for black‐foot pathogens.     

Control of black foot disease in grapevine nurseries

Black foot disease of grapevines is a decline and dieback disease caused by a soilborne pathogen complex including Cylindrocarpon liriodendri , C. macrodidymum , Campylocarpon fasciculare and Campyl. pseudofasciculare . These pathogens cause primary infections of roots and basal ends of grafted cuttings in nursery soils. Thirteen fungicides were screened in vitro for mycelial inhibition of these pathogens. Prochloraz manganese chloride, benomyl, flusilazole and imazalil were the most effective fungicides tested, and were subsequently included in semi-commercial field trials. Basal ends of grafted cuttings were dipped in various chemical and biological treatments prior to planting in open-rooted nurseries. Black foot pathogens were not isolated from grafted cuttings prior to planting. Additional treatments involved soil amendments with Trichoderma formulations and hot water treatment of dormant nursery grapevines. Field trials were evaluated after eight months. Isolations from uprooted plants revealed low levels of black foot pathogens in the roots of untreated control plants, and significantly higher levels in basal ends of rootstocks. The incidence of black foot pathogens, as well as that of Petri disease pathogens, was not significantly and/or consistently reduced by the majority of chemical or biological treatments. However, these pathogens were not isolated from uprooted plants that were subjected to hot water treatment. It is therefore recommended that hot water treatment of dormant nursery plants be included in an integrated strategy for the proactive management of these diseases in grapevine nurseries.     

Role of basidiomycete fungi in the grapevine trunk disease esca

Phaeomoniella chlamydospora and wood-rotting basidiomycete fungi, namely Fomitiporia spp., are known esca pathogens. However, the effect of their mixed infections and the sequence of infection on disease development is unclear. To determine the effects of single and co-inoculations on symptoms, potted Vitis vinifera ‘Crimson Seedless’ was inoculated with P. chlamydospora either alone or in combination with one of four basidiomycetes: Coprinellus radians, Fomitiporia langloisii, F. polymorpha and the novel species Tropicoporus texanus. Basidiomycetes were isolated from vines with foliar symptoms of esca in California and Texas. In sequential co-inoculations, the effects of different sequences of infection (P. chlamydospora first, basidiomycete 6 months later; and vice versa) were tested, compared to simultaneous co-inoculations. Plants inoculated with P. chlamydospora either alone or in combination with a basidiomycete (in any sequence) did not differ significantly in the length of black-streaking lesions. In plants inoculated only with a basidiomycete, the appearance of large brown lesions, coupled with the absence of this wood symptom from control plants, suggests that C. radians, F. langloisii and T. texanus are pathogenic. Foliar symptoms resembling those of esca in the field (marginal and/or interveinal scorching, combined with red and/or yellow discoloration) were statistically more frequent among plants inoculated with F. polymorpha or T. texanus, either simultaneously or following P. chlamydospora, compared to single inoculations. Sequential co-inoculations of a basidiomycete before or after P. chlamydospora were associated with similar lesion lengths, suggesting that basidiomycetes may not require infection by P. chlamydospora in order to extensively colonize the wood.