Grapevine Cankers Research Articles

Risk assessment of Xanthomonas citri pv. viticola for the EU.

Following a request from the European Commission, the EFSA Panelon Plant Health performed a risk assessment of Xanthomonas citri pv. viticola (Xcv). This pest causes bacterial canker of grapevine and is reported from Brazil and India. Two scenarios were considered: scenario A0 (current practice) and A2 (additional control measures). For the fresh grape import pathway, scenario A0 results in an order of magnitude of about one entry per 10 years (median; 90% uncertainty interval between ca. one entry per 18,000 years and ca. five entries per year). For the Vitis spp. plants for planting for research/breeding purposes import pathway, the risk of entry is several orders of magnitude smaller than the risk due to fresh grape import. This outcome is also obtained under scenario A2. The key entry uncertainties include import volume and transfer (for plants for planting), transfer and the disaggregation factor (for fresh grapes) and the limited availability of epidemiological data. The extent of the area favourable for Xcv establishment in the EU is uncertain, illustrating the limitations of climate suitability assessments when based on few data points and little epidemiological information. Nevertheless, the risk of Xcv establishment is only slightly lower than the risk of Xcv entry, i.e. no major establishment constraints are expected for most entries. Similarly, the risk of Xcv establishment is assessed as only slightly lower under current climate compared to the climate of 2041-2060. For grapevine growing areas in the EU with average yearly temperature above 17°C, the lag phase between establishment and spread is expected to be about 3 years (median; 90% range between ca. 6months and ca. 6 years). Under the same scenario, the rate of spread by natural means is assessed to be ca. 300m/year (median; 90% range between ca. 35 and ca. 800m/year). The spread rate would be considerably higher considering movements of plants and cutting tools or machinery. The percentage of grapevine plants infected by Xcv in production sites as yearly average over a 30-year production cycle is estimated to be ca. 17% (median; 90% range between ca. 1.5% and ca. 46%) in table grapes and ca. 12% (median; 90% range between ca. 0.7% and ca. 37%) in wine grapes. Impacts have been reported to be severe in Brazil and India, but the estimates provided here show that there is considerable uncertainty about expected impacts in the EU.

Photodynamic inactivation of Lasiodiplodia theobromae: lighting the way towards an environmentally friendly phytosanitary treatment.

The fungus Lasiodiplodia theobromae is one of the main causal agents of trunk canker and dieback of grapevine. The objective of this work was to evaluate the efficiency of photodynamic inactivation (PDI) of L. theobromae with synthetic and natural photosensitizers and irradiation with either sunlight or artificial photosynthetically active radiation. Although the growth of the mycelium could not be completely prevented with natural sunlight irradiation, phenothiazine dyes (methylene blue, MB; toluidine blue O, TBO), riboflavin and a cationic porphyrin (Tetra-Py+-Me) caused complete inhibition under continuous irradiation with artificial light. Free radicals were the main cytotoxic agents in the PDI with MB, indicating the predominance of the type I mechanism. PDI with MB or Tetra-Py+-Me may represent a promising approach for the sanitation of vine material in greenhouse nurseries, in order to reduce the risk of infection upon grafting.

First Report of Diaporthe cynaroidis and D. australafricana Associated with Walnut Branch Canker in Chile

In 2017, with more than 40,000 planted hectares, Chile became the second exporter of English walnut (Juglans regia) worldwide with a production of about 100,000 tons1. In 2017, low incidence of branch canker from old pruning wounds was observed in young walnut orchards cv. ‘Chandler’ in the Maule region of Chile. Fifty symptomatic wood samples were collected from ten trees in two orchards. Isolates were recovered from the wood necrotic lesions displaying a light to dark brown discoloration in the cortical and vascular tissue as previously described2. The isolates were identified as Diaporthe cynaroidis and Diaporthe australafricana based on morphological characteristics and DNA sequencing. Both species were grown on Potato Dextrose Agar medium and sterile grape leaves to induce sporulation but only D. cynaroidis produced alpha conidiospores. Alpha conidia were fusiform to ellipsoid, with a truncate base and an acutely rounded apex, initially aseptate, becoming brown at maturity, with size of 6.1 µm ± 0.5 × 2.5 µm ± 0.1. Beta conidia were extremely rare. Fungal DNA was extracted from fungal colonies with the DNeasy Plant Mini Kit (Qiagen, Hilden, Germany). The internal transcribed spacer (ITS), elongation factor (EF) and s-tubulin (sT) loci were amplified using primer pairs (ITS1/ITS4, EF728F/EF987R, and Bt2a/Bt2b) and methods previously described3. One isolate from each species was sequenced and D. cynaroidis (isolate #2248) and D. australafricana (isolate #2249) showed 99.13% and 99.65% sequence homology with NCBI voucher specimens MB#506209 and MB#344439, respectively. Sequences were deposited in the GenBank database; D. australafricana accession numbers ITS, KC343038; EF, KC343764; BT, KC344006; and D. cynaroidis accession numbers: ITS, KC343058; EF, KC343784; BT, KC344026. Pathogenicity tests were performed by drilling a hole in stems of two-year old walnut trees cv. ‘Chandler’ (n=20) and inoculating with D. cynaroidis and D. australafricana isolates using 25 µl of inoculum at 1×104 spores/µl. Ten control plants were treated with 25 µl sterile water. The experiment was repeated two times on separate sets of trees. After 16 weeks of incubation in a shaded open house, the lesions were light to dark brown in both the cortical and vascular tissues. The lesion length produced by D. australafricana averaged 31.9 mm ± 8.8 mm, D. cynaroides averaged 26.5 mm ± 7.1 mm and both were significantly longer (P <0.0082 and P <0.0061, respectively) than the control, which averaged 1.3 mm ± 0.6 mm. Both Diaporthe species were re-isolated from all the inoculated plants and identified by colony morphology in order to fulfill Koch’s postulate. Previously, only D. neotheicola and D. rushicola were reported to be pathogenic to walnut in California2 and Spain4. D. australafricana was only reported as the causal agent of stem canker of grapevine (Vitis vinifera L.) in Australia and South Africa5 and of blueberry (Vaccinium corymbosum L.) in Chile6. Additionally, D. cynaroidis has been identified as a pathogen of king protea (Protea cynaroides L.) in South Africa6. Blueberry, grape, and king protea are all grown in Chile and these findings warrant further investigation in order to determine the host range, distribution and incidence of these pathogens in the area.

Xanthomonas citri pv. viticola Affecting Grapevine in Brazil: Emergence of a Successful Monomorphic Pathogen.

The pathovar viticola of Xanthomonas citri causes bacterial canker of grapevine. This disease was first recorded in India in 1972, and later in Brazil in 1998, where its distribution is currently restricted to the northeastern region. A multilocus sequence analysis (MLSA) based on seven housekeeping genes and a multilocus variable number of tandem repeat analysis (MLVA) with eight loci were performed in order to assess the genetic relatedness among strains from India and Brazil. Strains isolated in India from three related pathovars affecting Vitaceae species and pathogenic strains isolated from Amaranthus sp. found in bacterial canker-infected vineyards in Brazil were also included. MLSA revealed lack of diversity in all seven genes and grouped grapevine and Amaranthus strains in a monophyletic group in X. citri. The VNTR (variable number of tandem repeat) typing scheme conducted on 107 strains detected 101 haplotypes. The total number of alleles per locus ranged from 5 to 12. A minimum spanning tree (MST) showed that Brazilian strains were clearly separated from Indian strains, which showed unique alleles at three loci. The two strains isolated from symptomatic Amaranthus sp. presented unique alleles at two loci. STRUCTURE analyses revealed three groups congruent with MST and a fourth group with strains from India and Brazil. Admixture among populations were observed in all groups. MST, STRUCTURE and e-BURST analyses showed that the strains collected in 1998 belong to two distinct groups, with predicted founder genotypes from two different vineyards in the same region. This suggest that one introduction of grape planting materials contaminated with genetically distinct strains took place, which was followed by pathogen adaptation. Genome sequencing of one Brazilian strain confirmed typical attributes of pathogenic xanthomonads and allowed the design of a complementary VNTR typing scheme dedicated to X. citri pv. viticola that will allow further epidemiological survey of this genetically monomorphic pathovar.

Infection by Xanthomonas campestris pv.viticola under temperature increase and carbon dioxide concentrations

The experiments were carried out under controlled conditions to evaluate the impact of increased temperature and concentration of carbon dioxide on infection of Xanthomonas campestris pv viticola, the causal agent of bacterial canker in Vine seedlings. It proceeded the evaluation of the following epidemiological components: incubation period (PI), severity (SEV) and using that data were calculated the area under the disease progress curve (AUDPC). It used grape seedlings (Italia, Crimson Seedless, Sugraone and Selection 8) inoculated with bacterial suspension (108 CFU mL-1). The experimental design was completely randomized, factorial arrangement 4x4 (cultivar x temperature) and 4x2 (cultivar x carbon dioxide concentration) which was carried out twice. The data were subjected to variance analysis. Increasing temperature reduced bacterium’s incubation period with significant differences between genotypes. For Selection 8 and Crimson temperature increase caused enhancement on severity and AUDPC. For Seleção 8 the incubation period (PI) was extended from 7.93 to 30.18 days when the concentration changed from 390 to 770 µmol/mol. The increased CO2 concentration reduced AACPSD and SEV for Sugraone and Selection 8. The results show that the temperature and carbon dioxide (CO2) concentration of the air may have different effects on bacterial canker of grapevine.

Biofilm formation by Xanthomonas campestris pv. viticola affected by abiotic surfaces and culture media

Biofilms are dense surface-associated communities formed by microorganisms. Formation of these structures by the plant pathogenic bacterium Xanthomonas campestris pv. viticola (bacterial canker of grapevine) had not previously been studied. The ability of seven strains of this bacterium to adhere to abiotic surfaces and to form biofilms in vitro was evaluated under different conditions. The surfaces tested were polystyrene and glass using microtiter plates and tubes, respectively. Four liquid culture media were used, nutrient-dextrose-yeast extract (NYD), yeast extract-dextrose-calcium carbonate (YDC), Kado 523 (KADO) and Luria-Bertani (LB). The biofilm architecture was examined by scanning electron microscopy (SEM) and confocal laser scanning microscopy (CLSM). Seven strains adhered to polystyrene in the microtiter plates and formed biofilms in all culture media at weak, moderate, and strong levels. In glass tubes, only strains Xcv229 and Xcv158 formed biofilms. SEM of Xcv229 and Xcv158 revealed typical biofilm architectures. CLSM showed that only Xcv229 formed an initial matrix structure characteristic of biofilms. The X. campestris pv. viticola strains exhibited different levels of biofilm formation in different culture media, of which LB and KADO were the best. Therefore, bacterial growth in polystyrene microtiter plates using LB and KADO media is a good qualitative method for the detection of biofilms of this pathogen.

Identification, Distribution, and Pathogenicity of Diatrypaceae and Botryosphaeriaceae Associated with Citrus Branch Canker in the Southern California Desert.

Several members of the families Botryosphaeriaceae and Diatrypaceae are known as canker and dieback pathogens of a number of woody hosts. Because desert citrus production in California can occur in proximity to table grape production, it was suspected that fungi associated with grapevine cankers might also be associated with citrus branch canker and dieback decline. To determine the fungi associated with branch canker and dieback disease of citrus in the southern California desert regions, surveys were conducted from 2011 to 2013 in the major citrus-growing regions of Riverside, Imperial, and San Diego Counties. Cankered tissues were collected from branches showing symptoms typical of branch canker and dieback. Various fungal species were recovered from necrotic tissues and species were identified morphologically and by phylogenetic comparison of partial sequences of the internal transcribed spacer (ITS) region (ITS1-5.8S-ITS2), β-tubulin gene, and elongation factor 1-α genes with those of other species in GenBank. Four fungi, including Neoscytalidium hyalinum, Eutypella citricola, E. microtheca, and an unnamed Eutypella sp., were associated with branch canker. N. hyalinum was the most frequently recovered fungus from symptomatic tissues (31%) followed by E. citricola (10%), E. microtheca (4%), and the Eutypella sp. (2%). In pathogenicity tests, all fungi caused lesions when inoculated on 'Lisbon' lemon (citrus) branches. Lesions caused by the Eutypella sp. were significantly longer than those of the other Eutypella spp.; however, they did not differ significantly from those produced by N. hyalinum. The most-parsimonious unrooted trees based on the combined data of ITS and partial β-tubulin gene region sequences showed three distinct clades of Eutypella spp. (E. citricola, E. microtheca, and an unidentified Eutypella sp.). Similarly, ITS and partial translation elongation factor 1-α gene region sequences differentiated two species of Neoscytalidium, N. hyalinum and N. novaehollandiae. Identifying the diversity, distribution, and occurrence of these fungal pathogens is useful for the management of citrus branch canker and dieback disease in the desert citrus-growing regions of California.

Occurrence of Eutypella microtheca in grapevine cankers in Mexico

Trunk diseases cause slow decline and loss in productivity of grapevine. In surveys carried out from 2010 to 2012 in vineyards in Baja California (Mexico), fungal isolates were obtained from Cabernet Sauvignon grapevines with cankers. Morphological studies and phylogenetic analyses, using the internal transcribed spacer (ITS) region of the rDNA and a partial sequence of the β-tubulin gene, identified two isolates as Eutypella microtheca . Pathogenicity tests, using grapevine green tissue, fulfilled Koch´s postulates. To our knowledge, this is the first report of E. microtheca occurring on grapevine in Mexico and highlights the need of considering this fungus as another potential threat to vineyards in this region.

Phomopsis Dieback: A Grapevine Trunk Disease Caused by Phomopsis viticola in California.

Field surveys recently conducted in California and in other grape-growing regions in the United States showed Phomopsis viticola to be one of the most prevalent fungi isolated from grapevine perennial cankers in declining vines. The current study has not only confirmed the presence of P. viticola from grapevine cankers in California but also has for the first time revealed the occurrence of Diaporthe ambigua, D. eres, and D. neotheicola in symptomatic grapevine wood in California by means of morphological studies and multi-gene sequence analysis. Pathogenicity trials conducted on mature cordons of Vitis vinifera 'Syrah' and 'Red Globe', as well as on lignified Syrah dormant canes, showed P. viticola isolates from California to be capable of causing perennial cankers. Lengths of vascular discoloration caused by P. viticola were similar to those caused by Eutypa lata and several Botryosphaeriaceae spp., which are well-known grapevine trunk disease pathogens. Additionally, a lack of spring growth was commonly observed in dormant canes inoculated with P. viticola spore suspensions in two pathogenicity trials. As part of this study, V. vinifera 'Cabernet Sauvignon' and 'Zinfandel' wood was shown to be more susceptible to infection by P. viticola than 'Barbera', 'Chardonnay', 'Merlot', and 'Thompson Seedless' wood. After more than 40 years overlooking P. viticola as a grapevine wood pathogen, this study provides strong evidence of the role of P. viticola as a canker-causing organism, and suggests its addition to the fungi involved in the grapevine trunk disease complex. Results from this study suggest D. ambigua and D. neotheicola to be saprophytes or weak pathogens on grapevine wood.

Draft Genome Sequence of Neofusicoccum parvum Isolate UCR-NP2, a Fungal Vascular Pathogen Associated with Grapevine Cankers.

Neofusicoccum parvum, a member of the Botryosphaeriaceae family, is a vascular pathogen that causes severe decline and dieback symptoms in grapevines worldwide. The draft genome of the grapevine isolate N. parvum UCR-NP2 provides a first glimpse into the complex set of putative virulence factors that this pathogen may use to rapidly colonize plants.

Dothiorella vidmadera, a novel species from grapevines in Australia and notes on Spencermartinsia

Strains of a coelomycete isolated from grapevine cankers insoutheasternAustraliaand identifiedasDothiorella iberica in previous studies are redescribed in this study as a novel species based on morphological characters and phylo- genetic analyses of DNA sequences of the internal transcribed spacer region (ITS1-5.8S-ITS2), and partial sequences of the translation elongation factor 1-α and β-tubulin genes. Dothiorella vidmadera sp. nov. is most closely related to D. iberica, D. americana and D. sarmentorum, but differs in morphological characters and DNA sequences. All four spe- cies are known to be associated with Botryosphaeria dieback of grapevines. Additionally, an unidentified ascomycete iso- lated from dead wood of grapevines in Western Australia is described. Phylogenetically, strains were most closely related to Spencermartinsia viticola, and bore conidia with morpho- logical characters and dimensions consistent with published descriptions. However, ascospores were shorter and narrower than previously reported and lacked the terminal apiculi that typify the genus.

Pathogenicity and epidemiology of Botryosphaeriaceae species isolated from grapevines in Australia

Botryosphaeriaceae species are among the most common fungi isolated from grapevine (Vitis vinifera) cankers in Australia. Thirty-eight isolates comprising eight Botryosphaeriaceae species isolated from grapevine cankers throughout New South Wales and South Australia were used in a pathogenicity study on mature wood of 15-year-old Chardonnay grapevines. Experiments showed that all eight species were able to infect grapevines under field conditions causing vascular discoloration and/or staining of the wood (lesions). However, differences in pathogenicity were evident among strains and species (P < 0.001). Neofusicoccum parvum and Lasiodiplodia theobromae were the most pathogenic based on lesion length, followed by Neofusicoccum australe, Botryosphaeria dothidea and Diplodia mutila and thereafter by Dothiorella viticola, Dothiorella iberica and Diplodia seriata, which were the least pathogenic. Growth rates also differed significantly among species depending on temperature (P < 0.001). Lasiodiplodia theobromae and B. dothidea grew optimally between 29 and 30 °C, Diplodia and Neofusicoccum spp. between 25 and 27 °C, and Dothiorella spp. at temperatures between 22 and 24 °C. Temperature-growth relationships correlated well with previous data on the prevalence and distribution of Botryosphaeriaceae species in Australian vineyards. Due to their pathogenicity, prevalence, distribution and tolerance to a wide range of environmental conditions, the Botryosphaeriaceae pose a significant threat to the Australian wine industry.

Evaluation of DMI fungicides against species of Diplodia and Neofusicoccum associated with Botryosphaeria canker of grapevine

C. Torres, B.A. Latorre, P. Undurraga, and X. Besoain. 2013. Evaluation of DMI fungicides against species of Diplodia and Neofusicoccum associated with Botryosphaeria canker of grapevine. Cien. Inv. Agr. 40(1):131-138. The Botryosphaeriaceae species have been widely studied and are considered a serious problem to the production of grapes worldwide. To aid in the development of control strategies for these fungi, in vitro experiments were conducted to evaluate the effectiveness of demethylation inhibiting (DMI) fungicides against Diplodia seriata, D. mutila, Neofusicoccum australe and N. parvum isolated from grapevine (Vitis vinifera). Fungicidal effectiveness was evaluated over a range of increasing concentrations using measurements of both the inhibition of conidial germination and the effects on mycelial growth. The effective concentrations (EC) necessary to inhibit conidial germination and mycelial growth by 50% (EC 50 ) and 90% (EC 90 ) were estimated by linear regression analysis between x = log 10 of the fungicidal concentration and y = the Probit value of the inhibition percentage. The results demonstrate that tebuconazole, myclobutanil, prochloraz and prochloraz plus epoxiconazole exhibit considerable in vitro activity against D. mutila, D. seriata, N. australe and N. parvum. These DMI compounds were able to arrest conidial germination and inhibit mycelial growth. The EC 50 values obtained for conidial germination were generally higher than the EC 50 values obtained for mycelial growth, suggesting that mycelia are more sensitive to the DMI compounds than are conidia. However, the effectiveness of these DMI compounds in the vineyard must be studied before any final conclusions can be reached.

First Report of Neofusicoccum australe Associated with Botryosphaeria Canker of Grapevine in Chile.

A survey of trunk diseases was conducted in 2010 in vineyards (n = 14) in central Chile (latitude 33°51' to 36°30'), specifically of Vitis vinifera 'Cabernet Sauvignon,' which is the main wine-grape cultivar (38,806 ha) in Chile. The following symptoms of trunk disease were observed in 5- to 19-year-old grapevines: short internodes, dead spurs, dead cordons (arms), and shoot dieback. Upon cutting into cordons and trunks of symptomatic vines, brown, V-shaped cankers of hard consistency were observed. A total of 56 wood cankers were collected, and small pieces of symptomatic wood (approximately 4 mm in diameter) taken from the canker margin were surface disinfected (75% ethanol, 30 s) and placed on acidified PDA (0.5 ml of 96% lactic acid per liter; APDA), which was incubated for 4 to 7 days at 24°C. Colonies, tentatively identified as a species within the Botryosphaeriaceae based on the presence of whitish-to-gray aerial mycelium and exhibiting rapid growth (4 to 5 cm colony diameter in 48 h), were hyphal-tip purified to APDA for identification. Colonies produced globose, black pycnidia with unicellular, hyaline, ellipsoidal, densely granulate, externally smooth, and thin-walled conidia of 17.0 ± 0.7 ± 6.7 ± 0.4 μm (n = 20). A yellow pigmentation was observed at the center of 48-h colonies on APDA. Morphologically, these isolates were identified as Neofusicoccum australe (Slippers, Crous & M.J. Wingfield) Crous, Slippers & A.J.L. Phillips (2,3). BLASTn searches of the ITS rDNA region, amplified with PCR primers ITS4/ITS5 (532 bp), and a 400-bp section of the beta-tubulin subunit 2 gene amplified with primers Bt2a and Bt2b of N. australe (GenBank Accession No. JX290091 and JX679868, respectively) revealed 99% similarity with the ITS and beta-tubulin sequences of N. australe reference strains EF638778 and HQ392761, respectively. Pathogenicity tests were conducted using N. australe isolate Vid1559 on 2-year-old Cabernet Sauvignon plants (n = 4), which were inoculated by wounding the woody stem with a scalpel approximately 1 cm below the most basal bud, placing an 8-mm mycelial plug taken from a 7-day culture into the wound, and then sealing the wound with Parafilm. Non-inoculated controls (n = 4) were 'mock' inoculated with sterile agar plugs. After 3 months under field conditions, during spring and summer, the woody stems were examined for vascular discoloration (VD), characteristic of a wood canker. Inoculated plants had stems with light-brown, necrotic VD with a mean length of 15.2 cm, measured from the inoculation point. No VD was observed on the controls. N. australe was reisolated from 100% of the inoculated plants, completing Koch's postulates. Of 14 vineyards surveyed, 8% were infected with N. australe. N. australe is known as a trunk pathogen of grape (4), and other species of Botryosphaeriaceae have been associated with grapevine trunk disease in Chile (1). To our knowledge, this is the first report of N. australe causing Botryosphaeria canker of grape in Chile, where the pathogen is previously reported on blueberry (2).

First Report of Cryptovalsa ampelina and Eutypella leprosa Associated with Grapevine Trunk Diseases in Chile.

Trunk diseases (TD) of grapevines (Vitis vinifera L.) have increased considerably in Chile with an incidence of more than 25% found in ≥7-year-old vineyards. Only species of Botryosphaeriaceae, Phaeoacremonium, and Phaeomoniella were associated with TD in Chile (1,2). From 2009 to 2010, isolations were made from the grapevines 'Cabernet Sauvignon', 'Carmenere', 'Flame Seedless', and 'Pinot Noir' collected in central Chile (33°27' to 34°39'S, 71°17' to 71°33'W). These grapevines showed cankers and vascular necrosis of trunks, arms, and spurs along with a general decline and dieback. Isolations were performed in potato dextrose agar (PDA) plus 0.005% tetracycline, 0.01% streptomycin, and 0.1% Igepal CO-630 (Sigma-Aldrich, St. Louis, MO), for 14 days at 20°C. On the basis of colony morphology and conidia production, two Libertella-like species were obtained in 26 (7.8%) of 335 trunk samples. On the basis of the internal transcribed spacer region (ITS4 and ITS5) of rDNA, Cryptovalsa ampelina (Nitschke) Fuckel (GenBank Accession Nos. HQ694976 and HQ694977), and Eutypella leprosa (Persoon) Berlese (HQ694974 and HQ694975) were identified, showing 98 to 100% similarity with the sequences of C. ampelina (GQ293913) and E. leprosa (AJ302463.1). C. ampelina produced white-to-creamy, smooth colonies with a creamy underside. Colonies of E. leprosa were white-to-gray with a white underside. Orange conidial masses were exuded after 30 days at 20°C. Conidia on PDA (n = 20) were unicellular, hyaline, filiform, slightly curved, and (19.8) 23.4 ± 2.6 (28.3) × (1.1) 1.4 ± 0.2 (1.8) μm and (19.2) 23.9 ± 3.0 (27.6) × (1.0) 1.2 ± 0.1 (1.5) μm for E. leprosa and C. ampelina, respectively. Stromatic perithecia of C. ampelina, embedded in the bark, were observed in dead pruning residues of infected vines (4). Pathogenicity tests were conducted with two isolates of each species, on 30-day-old 'Carmenere', rooted in vitro (n = 12), that were inoculated by placing a 5-mm agar plug on the surface of the propagation medium. Additionally, 15 cm long pieces (n = 5) of 1-year-old canes from 'Carmenere', 'Chardonnay', and 'Red Globe' were inoculated by placing a 5-mm agar plug underneath a cut aseptically made in each cane. An equal number of noninoculated plants and canes, but treated with sterile agar plugs, were used as controls. Leaf number (LN), shoot length (SL), and root length (RL) were assessed on plants in vitro after 28 days at 20°C. The extent of vascular discoloration (VD) obtained in canes was determined after 45 days in humid chambers at 20°C. One-way analysis of variance was performed and mean differences were studied by Tukey's test. C. ampelina and E. leprosa significantly (P < 0.05) reduced the LN, SL, and RL relative to the control plants. They also caused a VD of 10.1, 11.6, and 9.8 mm and 11.2, 13.4, and 10.0 mm in 'Carmenere', 'Chardonnay', and 'Red Globe', respectively. No symptoms were observed on the control canes. C. ampelina (100%) and E. leprosa (75%) were reisolated from inoculated plants and canes. To our knowledge, this is the first report of C. ampelina and E. leprosa on grapevines in Chile. However, their relative importance as causal agent of trunk disease remains to be determined. C. ampelina and E. leprosa have been associated with grapevine cankers in the United States and Spain (3,4).

Taxonomy and DNA phylogeny of Diatrypaceae associated with Vitis vinifera and other woody plants in Australia

The Diatrypaceae occur worldwide and comprise a number of pathogens of woody crops, forest and ornamental tree species. Despite the taxonomic difficulties within this family, interest in the Diatrypaceae has increased recently, mainly due to the recent detection of these fungi in the premium grape growing regions of California. In the present study, we investigated the diversity and host range of diatrypaceous fungi from prominent wine grape growing regions in South Australia, New South Wales and Western Australia. Approximately 100 isolates were collected from grapevine and other woody plants and compared with reference collections from the United States and Europe. Phylogenetic analyses of the complete sequence of the internal transcribed spacer (ITS) region of the ribosomal DNA and partial sequence of the β-tubulin gene, combined with morphological analyses separated 12 species. These included the previously described species Cryptovalsa ampelina, C. rabenhorstii, Diatrype brunneospora, Eutypa lata, E. leptoplaca, Eutypella australiensis, E. citricola, a Cryptosphaeria sp. and a Diatrype sp., whereas Diatrypella vulgaris, Eutypella cryptovalsoidea and E. microtheca are described as new. Seven species were isolated from grapevine but the prevalence of Diatrypaceae in grapevine cankers varied among the regions surveyed. In many instances in WA and NSW, these newly reported fungi were more widespread and abundant than E. lata. This study provides new information to assist with diagnosis of the causal agents of dieback and canker diseases in Australia and development of management strategies. Further studies to characterize the pathogenicity of diatrypaceous species to grapevines and to elucidate the biology of these fungi are underway.

Grapevine bacterial canker in the State of São Paulo, Brazil: detection and eradication

Symptoms of bacterial canker of grapevine in the variety Red Globe were observed in August 2009 in an orchard at Tupi Paulista, Sao Paulo State, Brazil, and the causal agent Xanthomonas campestris pv. viticola was identified by pathological and molecular tests. Eradication procedure was adopted and approximately 4,700 plants were destroyed. A survey was conducted on grape-producing regions in the state of Sao Paulo, which found no other contaminated orchard, and this bacterial species is considered absent in the state.

Botryosphaeria dothidea causing canker of grapevine newly reported in China

In the summer of 2009 in Zhejiang, Jiangsu, Guangxi, Shanxi, Beijing, Tianjin and Hebei Provinces, a decline of grapevines (Vitis vinifera) was identified with symptoms of dieback on one or more shoots, accompanied by elongated oval-shaped cankers in the wood, shrivelling and rot of fruit clusters. A total of 121 diseased samples were collected from affected areas and from these samples more than 1000 infected tissue pieces were used for pathogen isolation. Tissue from the lesion margins was surface-sterilized by placing in 75% ethanol for one min, then rinsing with sterilized water (three times) before placing on potato dextrose agar plates incubated at 28°C for 5 days (Niekerk et al., 2004). Hyphae from the colony margins were transferred to fresh agar plates to obtain pure isolates. Botryosphaeria dothidea was isolated from 44 of the diseased samples. Colonies of B. dothidea were white, becoming grey or dark brown with age. Pycnidia started to develop after 10 days. Conidia measured 17–32 × 4–9 μm. Some isolates were used for ITS sequence analysis to confirm the morphological identification. The primers ITS1 and ITS4 were used to amplify part of the nuclear rDNA operon by PCR (White et al., 1990). Ten ITS sequences were obtained (GenBank Accession Nos. GU226844 to GU226850, GU226852, GU226854 and GU226855). All sequences showed 99–100% homology with B. dothidea (Urbez-Torres et al., 2006). This is the first report of grapevine canker associated with B. dothidea in China. The research was supported by the Earmarked Fund for Modern Agro-industry Technology Research System (Ref. No. nycytx-30).

First Report of Diplodia corticola Causing Grapevine (Vitis vinifera) Cankers and Trunk Cankers and Dieback of Canyon Live Oak (Quercus chrysolepis) in California.

The botryosphaeriaceous fungus Diplodia corticola A. J. L. Phillips, Alves & Luque was shown to be the most prevalent canker and dieback pathogen in cork oaks (Quercus suber L.) in the Iberian Peninsula causing a general decline of the trees as a consequence of canker formation in the trunks (1). In addition, D. corticola has been recently reported as a grapevine pathogen causing cankers in the vascular tissue of 1-year-old canes, spurs, and cordons in Texas (3). In 1998, Jacobs and Rehner reported one isolate of D. corticola from oak in California, but no information regarding the oak species from which the isolate was obtained and its virulence were available in the study (2). In 2009, D. corticola was isolated on potato dextrose agar (PDA) amended with 0.01% tetracycline hydrochloride from symptomatic grapevine cordons and on acidified PDA from the trunk of a canyon live oak tree from Sonoma and Plumas counties, respectively. Two grapevine isolates (UCD1260So and UCD1275So) and one oak isolate (CDFA519) were examined and morphologically compared with previously identified D. corticola isolates CBS678.88 and UCD2397TX from cork oak from Spain and grapevine in Texas, respectively. D. corticola colonies from California were characterized by moderately fast-growing, dark olivaceous, and dense aerial mycelium on PDA. Conidia were obtained from pycnidia formed on pine needles placed on 2% water agar. Conidia were hyaline, contents granular, aseptate, thick walled, ellipsoidal, sometimes becoming dark brown and septate with age. Nucleotide sequences of three genes (ITS1-5.8S-ITS2, a partial sequence of the beta-tubulin gene BT2, and part of the translation elongation factor EF1-α) from D. corticola isolates UCD1260So, UCD1275So, and CDFA519 from California were amplified. All DNA sequences from grapevine and oak tree isolates from California showed 99 to 100% homology with D. corticola isolates previously identified and deposited into GenBank. All DNA sequences obtained from Californian isolates were also deposited into GenBank. Pathogenicity tests were conducted by inoculating detached Vitis vinifera cv. Red Globe dormant canes and canyon live oak branches with agar plugs of isolates UCD1260So, UCD1275So, and CDFA519 (10 inoculations per isolate per host) as described by Úrbez-Torres et al. (3). The same number of grapevine canes and oak branches were inoculated with noncolonized agar plugs as controls. Six weeks after inoculation, the extent of vascular discoloration that developed from the point of inoculation was measured. D. corticola isolates UCD1260So, UCD1275So, and CDFA519 caused an average vascular lesion length of 30.4, 29.6, and 24 mm and 15, 13.2, and 8.6 mm in grapevine dormant canes and oak branches, respectively. Furthermore, D. corticola isolates from grapevine were pathogenic in oak branches and vice versa. Reisolation of D. corticola from discolored vascular tissue of inoculated material was 100%. The extent of vascular discoloration from inoculated grapevine canes and oak branches was significantly greater (P < 0.05) compared with the controls (1.8 and 2 mm, respectively). No fungi were reisolated from the slightly discolored tissue of the controls. To our knowledge, this is the first report of D. corticola causing grapevine cankers and oak trunk cankers in California.

First Report of Grapevine Cankers Caused by Lasiodiplodia crassispora and Neofusicoccum mediterraneum in California.

Several species in the Botryosphaeriaceae family cause perennial cankers in the vascular tissue of grapevines and are responsible for the disease known as bot canker in California (3). Tissue from grapevine vascular cankers from samples submitted to our laboratory in the summer of 2009 were plated onto potato dextrose agar (PDA) amended with 0.01% tetracycline hydrochloride. Lasiodiplodia crassispora (Burgess & Barber) and Neofusicoccum mediterraneum (Crous, M.J. Wingf. & A.J.L. Phillips) were identified based on morphological and cultural characters as well as analyses of nucleotide sequences. L. crassispora isolates were characterized by a fast-growing, white mycelium that turned dark olivaceous with age on PDA. Conidia from pycnidia formed in cultures were thick walled and pigmented with one septum and vertical striations when mature. Conidia measured (25.8-) 27.5 to 30.5 (-33.4) × (12.1) 14.3 to 16.8 (-18.2) μm (n = 60). Pycnidia contained septate paraphyses. N. mediterraneum was characterized as having moderately fast-growing, light green mycelia on PDA. Pycnidia formation was induced with pine needles placed on 2% water agar. Conidia from pycnidia were hyaline, ellipsoidal, thin walled, unicellular, and measured (18.2-) 20.5 to 27.8 (-29) × (5.1) 5.9 to 6.5 (-7.2) μm (n = 60). DNA sequences of the internal transcribed spacer region (ITS1-5.8S-ITS2), part of the β-tubulin gene (BT2), and part of the translation elongation factor 1-α gene (EF1-α) from L. crassispora (UCD23Co, UCD24Co, and UCD27Co) and N. mediterraneum (UCD695SJ, UCD719SJ, UCD720SJ, UCD749St, and UCD796St) grapevine isolates from California were amplified and sequenced. Consensus sequences from L. crassispora and N. mediterraneum from California showed 99 to 100% homology with L. crassispora and N. mediterraneum isolates previously identified and deposited in GenBank (1,2). Sequences from the examined DNA regions of all isolates were deposited at GenBank (GU799450 to GU799457 and GU799473 to GU799488). Pathogenicity tests using three isolates per species were conducted on detached dormant canes of cv. Red Globe. Ten canes per isolate were inoculated by placing a 7-day-old 5-mm-diameter agar plug from each fungal culture into a wound made with a drill on the internode (4). Twenty shoots were inoculated with noncolonized PDA plugs for negative controls. Six weeks after inoculations, necrosis was measured from the point of inoculation in both directions. One-way analysis of variance was performed to assess differences in the extent of vascular discoloration and means were compared using Tukey's test. L. crassispora isolates caused an average necrotic length of 21.1 mm, which was significantly lower (P < 0.05) than the average necrotic length of 35.6 mm caused by the N. mediterraneum isolates. Reisolation of L. crassispora and N. mediterraneum from necrotic tissue was 100% for each species. The extent of vascular discoloration in infected canes was significantly greater (P < 0.05) than in control inoculations (8 mm) from which no fungi were reisolated from the slightly discolored tissue. To our knowledge, this is the first report of L. crassispora and N. mediterraneum as pathogens of Vitis vinifera and as a cause of grapevine cankers in California.