ABSTRACT
Grapevines are planted on 180,000 ha in Chile. In 2010 and 2011, necrotic lesions and hard texture were observed on woody tissue on 10-year-old vines of cvs. Cabernet Sauvignon, Carménère, Moscatel de Alejandría, and Pedro Jimenez in Ovalle (lat. 30°58' S) and Cauquenes (lat. 35°58' S). Symptoms were on 10 to 25% of the arm cross sections, resembling symptoms caused by Botryosphaeriaceae (4). Prevalence of 5% was estimated visually in Ovalle (n = 920 grapevines) and Cauquenes (n = 350 grapevines). Small pieces (3 mm) of necrotic tissues from the margins of lesions in cordons (n = 32) were surface sterilized (96% ethanol, 15 s), and plated on acidified PDA plus 0.5 ml/liter of 92% lactic acid, 0.005% tetracycline, 0.01% streptomycin, and 0.1% Igepal CO-630 (Sigma-Aldrich, St. Louis, MO) (APDA). The plates were incubated at 20°C for 14 days. Isolates (n = 12) were obtained from the yellow to dark green slimy colonies with white irregular margins, staining brown the underside of APDA plates. Black acervuli and ellipsoid to fusiform conidia were obtained. Conidia were triple septated, with hyaline upper and bottom cells and brown middle cells (n = 30) of 17.7 ± 1.2 × 5.8 ± 0.8 µm. A basal conidial appendage (6.2 ± 1.0 µm) was always obtained, but conidia having appendages at both ends also were observed. Morphologically, these isolates were identified as Seimatosporium botan Sat. Hatak. & Y. Harada (2). The identification of isolates sei-302 and sei-316 was confirmed by amplifying and sequencing the region ITS1-5.8S-ITS2 of rDNA using ITS4 and ITS5 primers (GenBank Accession Nos. JN088482 and JN088483). BLAST analyses showed 100% similarity with S. botan (Accession No. HM067840) (2). Pathogenicity tests were conducted with isolates sei-302 and sei-316 on detached green shoots (GS) and on rooted 2-year-old vines 'Carménère.' Rooted vines were inoculated at the base of canes and trunks. Inoculations were performed by placing a mycelial agar plug taken from APDA on a wound aseptically made with a cork borer. Wounds were sealed with Parafilm to avoid a rapid dehydration. The inoculated GS were incubated for 2 weeks in a moisture chamber (relative humidity >80%) at 20°C. Inoculated 2-year-old vines were placed in a lath-house for 7 and 15 months for canes and trunk inoculation, respectively. An equal number of GS and vines were inoculated with sterile agar plugs and left as controls. Necrotic lesions with mean of 23.7 ± 2.5 mm on GS, 50.5 ± 3.4 mm on canes, and 41.9 ± 2.3 mm on trunks developed. No significant difference (P < 0.05) was obtained in lesion length between S. botan isolates. After 7 months, 40% of inoculated canes had died. No symptoms were observed in GS controls and rooted control vines treated with sterile agar plugs. S. botan was reisolated from 93 to 100% of the inoculated samples. Previously, S. botan was reported as pathogenic in Paeonia suffruticosa (1), and Seimatosporium sp. was isolated from V. vinifera in California, but their pathogenicity was not demonstrated (3). To our knowledge, this is the first report of pathogenic isolates of S. botan associated with trunk disease of grapevines. These results contribute to the knowledge of the trunk disease of grapevines worldwide. References: (1) Y. Duan et al. Plant Dis. 95:226, 2011. (2) S. Hatakeyama et al. Mycoscience 45:106, 2004. (3) Z. Morales et al. Phytopathol. Mediterr. 49:109, 2010. (4) J. R. Úrbez-Torres. Phytopathol. Mediterr. 50:S5, 2011.
ABSTRACT
Stem cankers of blueberry (Vaccinium corymbosum L.) have been observed on as much as 15% of the plants in plantations in central and southern Chile since 2006. Symptoms consisted of apical necrosis of the shoots and brown-to-reddish necrotic lesions on the stems. Internally, a brown-to-reddish discoloration of the vascular tissue can be observed. Twenty, single-plant samples were collected in 12 blueberry plantings (approximately 33°27' to 40°53'S). Isolations from the margins of the necrotic lesions on the stems were made by plating small pieces (5 mm) on potato dextrose agar acidified with 0.5 µl/ml of 92% lactic acid (APDA). The plates were incubated at 20°C for 5 to 7 days, and hyphal tips of white colonies with septate and hyaline mycelium were transferred to APDA. Colonies were then transferred to autoclaved Pinus radiata needles on 2% water agar and incubated for 20 days at 20°C. Twelve isolates producing black pycnidia and alpha conidia were tentatively identified as a Phomopsis sp. (teleomoph Diaporthe Nitschke). Other fungi, including Botryosphaeriaceae spp. and Pestalotiopsis spp., were also isolated. Alpha conidia were smooth, unicellular, hyaline, fusoid, biguttulate, and 6.4 to 7.9 × 2.3 to 3.3 µm (n = 20). Beta conidia were not observed. The internal transcribed spacer (ITS) region of the rDNA was amplified using primers ITS1 and ITS2 (4) and sequenced. BLASTn analysis of the 473-bp fragment (GenBank Accession No. JQ045712) showed 100% identity to Diaporthe australafricana Crous & J.M. van Niekerk from Vitis vinifera (3). The pathogenicity of D. australafricana was studied on blueberry cv. O'Neal using detached stems (n = 4) in the laboratory, on 2-year-old potted plants (n = 4) in a greenhouse, and on attached stems of mature plants (n = 4) established in the ground. Inoculations were done by placing mycelial plugs taken from 7-day-old APDA cultures in a 7-mm long incision made on the stems. Inoculations with sterile mycelium plugs served as negative controls. Inoculation sites were wrapped with Parafilm to avoid rapid dehydration. Dark brown, necrotic lesions on the internal tissues were obtained on all inoculated stems 15 days after inoculation. Mean lesion lengths were 18.0 ± 7.4 mm on detached stems, 7.8 ± 6.9 mm on stems of 2-year-old plants, and 7.3 ± 2.5 mm on mature plants in the field. No symptoms developed on control stems. Reisolations were successful in 100% of the inoculated stems and D. australafricana was confirmed by the presence of pycnidia and alpha conidia. To our knowledge, this is the first report of D. australafricana causing stem canker in V. corymbosum. Previously, this pathogen has been reported to be affecting Vitis vinifera in Australia and South Africa (3). These results do not exclude that other plant-pathogenic fungi may be involved in this syndrome (1,2). References: (1) J. G. Espinoza et al. Plant Dis 92:1407, 2008. (2) J. G. Espinoza et al. Plant Dis. 93:1187, 2009. (3) J. M. van Niekerk et al. Australas. Plant Pathol. 34:27, 2005. (4) T. J. White et al. Page 315 in: PCR Protocols: A Guide to Methods and Applications. M. A. Innis et al., eds. Academic Press, NY, 1990.