First Report of Campylocarpon fasciculare Causing Black Foot Disease of Grapevine in Turkey.

Soil-borne fungal diseases have become an important problem in grapevine nurseries of the Aegean region (western Turkey) in recent years. Reduced vigor, black vascular streaking in basal ends, blackish-sunken necrotic root lesions, and young vine death were observed in 15 grapevine nurseries of Manisa city in May 2011 and 2012. To determine the causal agents, symptomatic young grapevine (Vitis vinifera cv. Sultana 7) plants (grafted on 1103 Paulsen) were collected from nurseries (8 to 10 plants from each). Symptomatic basal end tissues were surface disinfested with 95% ethanol and flame sterilized. The internal tissues were plated onto potato dextrose agar amended with tetracycline (0.01%). Campylocarpon-like fungi were isolated (with 37.9% isolation frequency) from only one nursery (corresponding to 6.7% of all surveyed nurseries). Fungal colonies were incubated for 21 days in the dark to induce sporulation. Fungal colonies produced cottony aerial mycelium and turned chocolate-brown to dark brown on PDA. Abundant macroconidia were observed at branched conidiophores on long and cylindrical phialides. Microconidia were not observed. Macroconidia were generally 2 to 4 septate, cylindrical and slightly curved, with the following dimensions: 2 septate: 33.5 to 40.7 × 6.1 to 7.6 µm (mean: 35.9 × 6.8 µm), 3 septate: 36.2 to 43.4 × 6.6 to 8.3 µm (mean: 37.3 × 7.6 µm), and 4 septate: 48.9 to 53.5 × 7.6 to 8.3 µm (mean: 50.7 × 8.0 µm). Fifty macroconidia were measured. Morphologically, the isolates resembled the published description of Campylocarpon fasciculare Schroers, Halleen & Crous (2,4). For molecular identification, fungal DNA was extracted from mycelium and ribosomal DNA fragments (ITS1, 5.8S ITS2 rDNA), ß-tubulin, and histone H3 genes, amplified with ITS 4-5, Bt 2a-2b, and H3 1a-1b primers (3,5), and sequenced. Sequences were compared with those deposited in GenBank. The isolate (MBAi45CL) showed 99% similarity with Campylocarpon fasciculare isolates AY677303 (ITS), AY377225 (ß-tubulin), and JF735502 (histone H3). The DNA sequences were deposited into GenBank under accessions KJ573392, KJ573393, and KJ573394 for ITS, ß-tubulin, and Histone H3 genes, respectively. To fulfill Koch's postulates, pathogenicity tests were conducted under greenhouse conditions on own-rooted grapevines (Vitis vinifera) cv. Sultana 7. Plants were removed from the rooting bench and the roots were slightly trimmed and submerged in a 107 ml-1 conidial suspension of the isolate for 60 min (5). After inoculation, the rooted cuttings were planted in 1-liter bags containing a mixture of soil, peat, and sand (2:1:1, v/v/v), and maintained in the greenhouse (24°C. 16/8-h day/night, 75% RH). Ten plants were inoculated with the isolate and five plants were submerged in sterile distilled water (control). After 4 months, young vines were examined for vascular discoloration, reduced root biomass, blackish lesions, and recovery of fungal isolates. The experiment was repeated twice. Blackish-brown discoloration of xylem vessels and necrosis in the basal ends was visible in the inoculated plants but not in the control plants. The pathogen was successfully re-isolated from 69.1% of the inoculated plants. This report is important for the new studies aiming at black foot disease control in Turkey viticulture. References: (1) A. Cabral et al. Phytopathol. Mediterr. 51:340, 2012. (2) P. Chaverri et al. Stud. Mycol. 68:67, 2011. (3) N. L. Glass and G. C. Donaldson. Appl. Environ. Microbiol. 61:1323, 1995. (4) F. Halleen et al. Stud. Mycol. 50:431, 2004. (5) T. J. White et al. PCR Protocols: A Guide to Methods and Applications. Academic Press, San Diego, CA, 1990.

First Report of Diaporthe neoviticola Associated with Wood Cankers of Grapevine in Turkey.

In recent years, delayed bud bursting, cane bleaching, shoot dieback, and cankers in 1-year-old canes and perennial arms were observed in vineyards of the Aegean region (western Turkey). These symptoms were frequently observed on the following major table grape (Vitis vinifera) cultivars: 'Alphonse Lavallée,' 'Cardinal,' 'Sultana Seedless,' and 'Trakya Ilkeren' in 2012. To determine the causal agents, symptomatic woody tissues (0.5 cm2) were sampled from the canes of nine Manisa and four Salihli Cities (13 total) grapevine varieties and were plated onto potato dextrose agar amended with tetracycline (0.01%) (PDA-tet). A considerable amount of phomopsis-like fungi were isolated from the symptomatic tissues and fungal colonies were incubated for 2 to 3 weeks to induce sporulation. After incubation for 14 days at 24°C in the dark, white mycelial growth with undulating colony margins, and abundant pycnidia producing hyaline, ellipsoidal, fusoid α-conidia with invisible nuclei, and ß-conidia, were observed on PDA, and they resembled species in the Diaporthaceae (1,2). The α-conidia dimensions were 9.3 to 10.2 × 1.9 to 2.9 µm (avg. 9.7 × 2.4 µm) and ß-conidia were 19 to 24 × 0.5 to 1 µm (avg. 22 × 0.9 µm). For molecular identification, fungal DNA was extracted from mycelial mats and ribosomal DNA fragments (ITS1, 5.8S ITS2 rDNA, amplified with ITS4 and ITS5 primers) (3) were sequenced and the sequences were compared with those deposited in NCBI GenBank in a BLASTn search. The representative isolate (MBAi43AG) showed 99% homology with Diaporthe neoviticola isolate from New Zealand KC145831.1. The DNA sequence of the identified isolate was submitted to GenBank under accession number KF460427. Pathogenicity tests were conducted under controlled conditions (24°C, 16/8 h day/night, and 70% RH) on 1-cm-diameter, detached green grapevine cv. Cabernet Sauvignon canes (with leaves) using the isolate of D. neoviticola specified above. The shoots were wounded by creating a 5-mm-diameter incision with a sterile scalpel. An agar disc with mycelia and pycniospores was placed into each wound and covered with Parafilm. Sterile PDA plugs were used as mock inoculum for the control plants. There were 10 replicates per treatment and the experiment was repeated twice. After 1 month of incubation, the green shoots were examined for the extent of superficial blackish lesions. The average lesion length on inoculated shoots was 18.2 mm for D. neoviticola. No lesions were observed in the control shoots. The fungal isolate was successfully re-isolated from 96% of inoculated shoots to fulfill Koch's postulates. To our knowledge, this is the first report of D. neoviticola causing wood canker and dieback of shoots on grapevine in Turkey. References: (1) R. R. Gomes. Persoonia 31:1, 2013. (2) D. Udayanga et al. Fungal Diversity 56:157, 2012. (3) J. M. van Niekerk et al. Australas. Plant Pathol. 34:27, 2005.

First Report of Wood Canker Caused by Botryosphaeria dothidea, Diplodia seriata, Neofusicoccum parvum, and Lasiodiplodia theobromae on Grapevine in Turkey.

The Aegean region (western Turkey) is the center of table, raisin, and wine grape cultivation. During the 2012 growing season, wood canker symptoms were observed in vineyards in Manisa city. Symptoms adjacent to pruning wounds, including shoot dieback and wedge-shaped wood discolorations observed in cross section, were among the most prevalent symptoms of the vines. To identify the causal agents, symptomatic woody tissues were surface disinfested with 95% ethanol and flame-sterilized and the discolored outer bark was cut away. The internal tissues (0.5 cm2) were excised from cankers of vines and plated onto potato dextrose agar amended with tetracycline (0.01%) (PDA-tet). The most frequently isolated fungi, based on general growth pattern, speed of growth, and colony color, resembled species in the Botryosphaeriaceae family. According to morphological characteristics, four different groups have been identified based on visual discrimination. After DNA extraction, ribosomal DNA fragments (ITS1-5.8S-ITS2) (2) amplified with ITS4 and ITS5 primers were sequenced and sequences were compared with those deposited in NCBI GenBank database. Four different Botryosphaeriaceae isolates were identified, including Botryosphaeria dothidea (MBAi25AG), Diplodia seriata (MBAi23AG), Lasiodiplodia theobromae (MBAi28AG), and Neofusicoccum parvum (MBAi27AG) (Accession Nos. KF182329, KF182328, KF182331, and KF182330, respectively) with species nomenclature based on Crous et al. (1). Pathogenicity tests were conducted under greenhouse conditions (24°C, 16/8-h day/night, 70% RH) on 1-year-old own rooted grapevine (Vitis vinifera) cv. Sultana Seedless seedlings using one isolate from each of the Botryosphaeriaceae species specified above. Stems of grapevine seedlings were wounded by removing bark with 4-mm cork borer and fresh mycelial plugs were inoculated into the holes and covered with Parafilm. Sterile PDA plugs were placed into the wounds of control seedlings. Five vines were inoculated per isolate. The experiment was repeated twice. After 4 months of incubation, grapevine seedlings were examined for the extent of vascular discoloration and recovery of fungal isolates. Mean lesion lengths on wood tissues were 85.3, 17.2, 13.9, and 13.1 mm for N. parvum, B. dothidea, L. theobromae, and D. seriata, and 6.3 mm for control. Each fungal isolate was successfully re-isolated from inoculated seedlings to fulfill Koch's postulates. To our knowledge, this is the first report of multiple species in the Botryosphaeriaceae causing wood canker and dieback on grapevine in Turkey. These results are significant because Botryosphaeriaceae species are known causal agents of grapevine trunk disease worldwide (3). References: (1) P. W. Crous et al. Stud. Mycol. 55:235, 2006. (2) B. Slippers et al. Mycologia 96:83, 2004. (3) J. R. Urbez-Torres. Phytopathol. Mediterr. 50:S5, 2011.

Effect of Fungicide Application on the Management of Avocado Branch Canker (Formerly Dothiorella Canker) in California.

Members of the Botryosphaeriaceae family have been associated with branch cankers of avocado trees (Persea americana) in California. Canker infections are initiated by spores entering the host plant through fresh wounds such as pruning wounds. With high-density planting becoming more common in the California avocado industry, more intensive pruning may increase the occurrence of branch canker. The objective of this study was to evaluate the preventive ability of some commercial fungicides belonging to different chemical families against fungal pathogens associated with avocado branch canker. Initially, 12 fungicides were tested in vitro for their effect on the inhibition of mycelial growth of three isolates of Dothiorella iberica and isolates (five per species) of Neofusicoccum australe, N. luteum, N. parvum, and Phomopsis sp. Subsequently, azoxystrobin, fludioxonil, metconazole, and pyraclostrobin, selected because of their low effective concentrations that reduce 50% of mycelial growth (EC50 values), and myclobutanil, selected for its high EC50 value, were tested in two field experiments. Azoxystrobin and fludioxonil were used in a premix with propiconazole and cyprodinil, respectively, in field trials. Significant differences (P < 0.05) were observed among fungicides in field trials. Azoxystrobin + propiconazole had the highest percent inhibition at 52 and 62% (internal lesion length) in trial 1 and trial 2, respectively, although this level of inhibition was not significantly different from that of metconazole. A significant correlation (r = 0.51, P < 0.05) was observed between internal lesion length data in the field experiment and EC50 data from in vitro fungicide screening. Application of azoxystrobin + propiconazole and metconazole can play a key role in protecting Californian avocado against fungi causing avocado branch canker.

First Report of Wood Canker Caused by Neoscytalidium dimidiatum on Grapevine in California.

In May 2012 in the Coachella valley, Riverside County, California, the decline of vines in table grape (Vitis vinifera) vineyards was observed. Foliar symptoms consisted of shoot blight with wilting and necrosis of leaves and drying and shriveling of berries. In some cases, the entire vine collapsed in the middle of the growing season (apoplexia). Wood cankers in the spurs, cordons, and trunks of affected vines were also present. The nine isolates recovered from the cankers were identified as Neoscytalidium dimidiatum (Penz.) Crous & Slippers based on morphological characteristics and DNA sequence comparisons. Two isolates were grown on potato dextrose agar (PDA) medium and a total of 50 conidia were measured per isolate. Conidia were ellipsoid to ovoid, with a truncate base and an acutely rounded apex, initially aseptate, becoming brown and two-celled at maturity, 7.2 ± 1.2 µm × 3.8 ± 0.4 µm. The rDNA internal transcribed spacer (ITS), and ß-tubulin (BT) loci were amplified using primer pairs and methods previously described (4). A total of five isolates were sequenced. The DNA sequences of one N. dimidiatum grapevine isolate (UCR-Neo1) were deposited in the GenBank database (ITS, KC937066; BT, KC937067). Pathogenicity tests were performed by inoculating 12 grape cuttings cv. Thompson Seedless with isolate UCR-Neo1 and 12 control cuttings with sterile medium using a technique previously described (1). The experiment was repeated twice. After 20 weeks of incubation period in the greenhouse, the lesions length produced by N. dimidiatum averaged 13.5 mm and was significantly longer (P < 0.05) from the control (average 3 mm). N. dimidiatum was reisolated from all the inoculated plants and identified by colony morphology. The incidence of N. dimidiatum in table grape vineyards of the Coachella valley has been estimated at 15%, with nine vines infected out of 60 vines total. This pathogen has been identified in California in walnut nursery causing the death of trees due to the development of canker at the graft union (2). N. dimidiatum has also been identified as the causal agent of shoot blight, canker, and gummosis on citrus in Italy (3). The crop is also being grown in the Coachella valley and these findings warrant further investigation in order to determine the host range, distribution, and incidence of this pathogen in the area. References: (1) K. Baumgartner et al. Plant Dis. 97:912, 2013. (2) S. F. Chen et al. Plant Dis. 97:993, 2013. (3) G. Polizzi et al. Plant Dis 93:1215, 2009. (4) J. R. Urbez-Torres et al. Plant Dis. 92:519, 2008.