Neofusicoccum parvum Associated with Grapevine Trunk Diseases in Chile.

During 2009 and 2010, a survey (n = 520) of diseased grapevines (Vitis vinifera L.) was done in vineyards located in Maipo and Colchagua valleys (33°43' to 34°36'S) in Chile. Symptoms of trunk diseases (TD) were observed on >10-year-old grapevines and consisted of short internodes, dead spurs and arms, and dieback. In cross sections, diseased arms and trunks exhibited brown, V-shaped cankers of hard consistency. Collected canker samples from cvs. Cabernet Sauvignon, Carménère, Red Globe, Syrah, and Thompson Seedless were surface sterilized in 75% ethanol for 45 s and plated onto potato dextrose agar modified with 0.005% tetracycline, 0.01% streptomycin, and 0.1% Igepal CO-630 (MPDA; Sigma-Aldrich, St. Louis, MO) for 7 days at 20°C. White-to-gray colonies with aerial mycelium growth turned dark gray after 3 to 5 days and tentatively identified as Botryosphaeriaceae. Hyphal tips of these colonies were transferred to MPDA and kept at 20°C with continuous light. After 30 days, colonies developed black, globose pycnidia with unicellular, hyaline, ellipsoidal, densely granulate, externally smooth, and thin-walled conidia that measured (16.3) 19.3 ± 2.3 (25.9) × (5.8) 7.4 ± 0.8 (9.2) µm (n = 20). Morphologically, these isolates were identified as Neofusicoccum parvum (Pennycook & Samuels) Crous, Slippers & A.J.L. Phillips (2). Nucleotide BLAST analysis of the region ITS1-5.8S-ITS2 of rDNA of N. parvum isolates HMUC-104 and HMUC-105 (GenBank Accession Nos. JF273631 and JF273632) were amplified with ITS4 and ITS5 primers and revealed >99% similarity with the sequence of reference isolate (EU833984). Pathogenicity tests were conducted using isolates HMUC-104 and HMUC-105 on 30-day-old Carménère grapevines (n = 8) rooted in vitro by placing a 3- to 5-mm mycelial plug on the surface of the propagation medium. Additionally, detached green shoots (GS) (n = 5) and dormant canes (DC) (n = 6) 15-cm long were inoculated by placing a 3- to 5-mm mycelial plug underneath a cut aseptically made in the cortex. The GS and DC were placed in humidity chambers at 20 and 25°C, respectively. For controls, an equal number of rooted vines, in vitro vines, GS, and DC were treated with sterile agar plugs. Leaf number (LN), shoot length (SL), and root length (RL) were assessed on rooted plants in vitro after 30 days at 20°C. The extent of vascular discoloration (VD) of GS and DC were determined 15 and 45 days, respectively. N. parvum significantly (P < 0.05) reduced the LN, SL, and RL relative to the control plants. The length of VD varied from 54.86 to 55.39 mm and 14.8 to 15.48 mm in inoculated GS and DC, respectively. No VD symptoms were observed on the controls. N. parvum was reisolated from 100% of the inoculated in vitro plants, GS, and DC, completing Koch's postulates. N. parvum has been documented as a canker pathogen on V. vinifera and is known to contribute to the decline of grapevines. To our knowledge, this is the first report of N. parvum causing bot canker on grapevines in Chile, but has previously been reported in Australia, Spain, and the United States. Of 520 diseased plants in this study, 10 to 15% prevalence was estimated for TD and almost 2% prevalence was associated to N. parvum. Other Botryosphaeriaceae spp. were isolated with N. parvum from grapevine TD in Chilean vineyards (1,3,4). References: (1) J. Auger et al. Plant Dis. 88:1286, 2004. (2) P. W. Crous et al. Stud. Mycol. 55:235, 2006. (3) B. A. Latorre et al. Phytopathology 76:1112, 1986. (4) A. Morales et al. Phytopathol. Mediterr. 49:112, 2010.

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). References: (1) J. Auger et al. Plant Dis. 88:1285, 2004. (2) J. Auger et al. Plant Dis. 88:1286, 2004. (3) M. T. Martin et al. Plant Dis. 93:545, 2009. (4) F. P. Trouillas et al. Mycologia 102:319, 2010.

Mapping genes for resistance to barley stripe rust (Puccinia striiformis f. sp. hordei).

Two genes conferring resistance to the barley stripe rust found in Mexico and South America, previously identified as race 24, were mapped to the M arms of barley chromosomes 7 and 4 in a doubled haploid population using molecular markers and the quantitative trait loci (QTL) mapping approach. The resistance gene on chromosome 7 had a major effect, accounting for 57% of the variation in disease severity. The resistance gene on chromosome 4 had a minor effect, accounting for 10% of the variation in trait expression. Two pairs of restriction fragment length polymorphism markers are being used to introgress the resistance genes to North American spring barley using molecular marker-assisted backcrossing.