J. Auger

Black Dead Arm and Basal Canker of Vitis vinifera cv. Red Globe Caused by Botryosphaeria obtusa in Chile

Several years ago, Vitis vinifera cv. Red Globe vines (over 6 years old), started to show disease symptoms approximately 10 weeks after bud break. Symptoms first appeared on the leaves at the base of the shoots and then spread to other leaves, continuing to appear throughout the growing season. Two forms of the disease (severe and mild) were observed, each case leading to premature leaf fall. The severe form was characterized by dieback of one or more shoots accompanied by leaf drop and shriveling and drying of fruit clusters. The mild form was characterized by wine-red spots on the leaf margins or the leaf blade, which coalesced to form large zones of necrosis between the veins and the margins of the leaf. Fruit clusters may wither. If the bark is scraped off, a brown streak, 1 to 2 cm wide, was often seen in the wood. The streaking began at the base of the affected shoot and extended upward to the ground level, eventually resulting in a basal canker. Botryosphaeria obtusa (Schwein.) Shoemaker (anamorph = Sphaeropsis malorum Berk.) was isolated from 86% of samples from vines that were 6 to 10 years old from 12 locations in IV, V, VI, and metropolitan regions of Chile. Isolations were made from brownstreaked wood. Isolates were identified on the basis of a previous description (1,2) and internal transcribed spacer (ITSI-5.8S-ITS2) rDNA sequences identical to those of B. obtusa (culture KJ9356, GenBank Accession No. AF027759). B. obtusa is established as one of the main fungi associated with black dead arm of grapevine (2,3). Pathogenicity tests were completed by inoculating approximately 20 μl of mycelial suspension via injection into the pith of 16 single-node, rooted cuttings of V. vinifera cv. Red Globe. Sixteen control cuttings were injected with an equal volume of sterile distilled water. Twenty weeks after inoculation, all B. obtusa-inoculated cuttings exhibited brown streaks in the wood extending 50 to 60 mm from the point of inoculation. The wood streaking observed in inoculated plants was identical to symptoms observed in naturally infected black dead arm vines in the vineyard. No symptoms were observed in the controls. B.obtusa was reisolated from the region of brown streaking in all the inoculated cuttings. B. obtusa was not isolated from the water-treated controls. To our knowledge, this is the first report of B. obtusa causing black dead arm and basal canker on Red Globe grapevine in Chile. The fungal isolates have been deposited in the plant pathology laboratory of the Sanidad Vegetal Department of Agronomy Faculty of the University of Chile under the name V. vinifera/B. obtusa from N 1 to 16. References: (1) G. Cristinzio. Inf. Fitopatol. 28:21, 1978. (2) P. Larignon and B. Dubos. Phytoma 538:26, 2001. (3) A. J. L. Phillips. J. Phytopathol. 146:327, 1998.

First report of fenhexamid resistant isolates of Botrytis cinerea on grapevine in Chile.

Botrytis cinerea Pers. (teleomorph Botryotinia fuckeliana (de Bary) Whetzel) is a haploid, filamentous ascomycete that causes gray mold on many economically important crops in temperate regions, especially grapevine. The management of gray mold on table grape in Chile involves cultural and chemical methods. Currently, protection programs are based on several fungicide families (dicarboximides, anilinopyrimidines, mixture of anilinopyrimidines and phenylpyrroles, and hydroxyanilides [fenhexamid]). During the last 25 years, B. cinerea developed resistance to virtually all specific fungicides used to control gray mold. Field resistance to benzimidazoles, phenylcarbamates, and dicarboximides was detected soon after their introduction. Recent studies using PCR-duplex and specific primers for the detection of transposable elements on Chilean B. cinerea isolates recovered from different table grape cultivars corroborated the presence of two sibling cryptic populations, transposa and vacuma (3). Some vacuma isolates have shown natural resistance to fenhexamide (HydR1) and it has been separated into two groups on a molecular basis using a marker gene (Bc-hch): Group I, fenhexamid-resistant vacuma isolates; Group II, vacuma and transposa isolates sensitive to this fungicide (HydS) (2). Group I and II isolates can not interbred (1,2). Other B. cinerea resistant phenotypes, HydR2 and HydR3, have been reported as belonging to Group II (1,4). Single-spore isolates of B. cinerea (472) were collected from different table grape cultivars from 13 locations in the Chilean Central Valley. The isolation was done during harvest time from rotting berries. Fenhexamid (Teldor; Bayer CropScience, Monheim, Germany) was diluted to 10 μg a.i./ml and added to the solid medium (10 g of glucose, 1.5 g of K2HPO4, 2 g of KH2PO4, 1 g of (NH4)2SO4, 0.5 g of MgSO4·H2O, 2 g of yeast extract, and 12.5 g of agar in 1 liter) to reach concentrations of 0, 0.025, 0.05, and 0.1 μg a.i./ml. A 5-mm mycelial plug from each isolate of B. cinerea was cut from the edge of 4-day-old colonies placed in the center of petri dishes with the described fungicide-amended medium and incubated at 20°C for 5 days. Two measurements, octogonal diameters, were taken from each of three replicates per treatment. Means were calculated and the diameter of the inoculated plug was subtracted from each mean. For each isolate, a linear regression of the percent inhibition of mycelial growth versus the Log10 transformation for each of the four concentrations of fenhexamid was obtained. The 50% effective concentration of fenhexamid (EC50) was calculated with the regression equation for each isolate. So, 95.3% of B. cinerea isolates were sensitive (EC50 under 0.083 μg/ml), 1.9% were less sensitive (EC50 between 0.084 and 0.1 μg/ml), and 2.8% (13 isolates) were resistant EC50 values ranging from 0.1 to 8.4 μg/ml. Through PCR-restriction fragment length polymorphism, according to the Bc-hch gene restriction pattern, all resistant isolates analyzed belong to Group II of B. cinerea (Bc-hch2) (2). To our knowledge, this is the first report of fenhexamid resistant isolates of B. cinerea on grapevine in Chile and South America. It would be necessary to study the population dynamics of these isolates, although failure of botrytis control in the field with this compound has not been reported. References: (1) C. Albertini et al. Mycol. Res. 106:1171, 2002. (2) E. Fournier et al. Mycologia 97:1251, 2005. (3) T. Giraud et al. Mol. Biol. Evol. 14:1177, 1997. (4) P. Leroux et al. Phytoma 599:31, 2006.

First Report of Verticillium Wilt of Gold Kiwifruit, Actinidia chinensis Cv. Hort 16A, Caused by Verticillium albo-atrum in Chile

Gold kiwifruit, Actinidia chinensis Planch cv. Hort 16A, was first planted in Chile in 2003 and vines started dying within 2 years. By the end of the 2007-2008 growing season, as much as 80% of the plants in several orchards had died. The disease was characterized by a conspicuous reddish brown discoloration of the xylem and the sudden wilting and dieback of plants any time during the growing season. In the spring, entire plants or parts of plants failed to break buds. In others, the buds broke, but juvenile leaf clusters then wilted and died. On severely affected plants, scion watershoots wilted and died. The disease was often accompanied by shallow cracking of the bark and slight sponginess of the underlying cortex. The disease was apparently most severe in sites that had been planted to Gold kiwifruit immediately after removal of apple, pear, citrus, or grape. Orchards planted following long-term maize, wheat, or grass culture were almost disease free. A fungus was consistently isolated from symptomatic vascular tissue disinfected in 1% sodium hypochlorite and plated on potato dextrose agar. Conidiogenous cells were arranged in verticels; conidia were hyaline, elliptical, single celled, and measured 3.5 to 8.5 × 1.8 to 4.3 μm (average 5.5 × 2.5 μm). Dark, resting mycelium developed after 1 to 2 weeks of incubation. On the basis of these morphological characteristics, the fungus was identified as Verticillium albo-atrum Reinke & Berthier. Identification was confirmed by sequencing part of the internal transcribed spacer (ITS) region with primers ITS1 and ITS4. The sequence of a representative isolate showed high homology (98% identity over a length of 494 bp) with a DNA fragment (NCBI Accession No. 108476) of V. albo-atrum from alfalfa. To complete pathogenicity tests, 20 healthy, 1-year-old Hort 16A kiwi vines grafted on Hayward kiwifruit (A. deliciosa Chevalier) seedlings were inoculated by injection of 20 μl of 106 conidia/ml into stems of the scion. Twenty control plants were injected with an equal volume of sterile distilled water. Plants were held in a controlled environment facility at 24°C with 16 h of light per day. Eight weeks after inoculation, typical wilting and dieback symptoms developed on 90% of the plants. Control plants injected with water remained healthy. Verticillium wilt has never been reported on kiwifruit (A. deliciosa) in Chile. V. albo-atrum has a rather narrow host range and is mainly reported as a pathogen on alfalfa, hop, soybean, tomato, and potato (1). To our knowledge, this is the first report of V. albo-atrum causing wilt and dieback on Gold kiwifruit (A. chinensis) cv. Hort 16A. The fungal isolates have been deposited in the Plant Pathology Laboratory of the Sanidad Vegetal Department of Agricultural Sciences Faculty of University of Chile under the name Actinidia chinensis/V. albo-atrum No. 1 to 8. Reference: (1) E. K. Ligoxigakis et al. Phytoparasitica 30:511, 2002.

Effects of Phaeomoniella chlamydospora ana Phaeoacremonium aleophilum on grapevine rootstocks

G. A. Diaz, M. Esterio, and J. Auger. 2009. Effects of Phaeomoniella chlamydospora and Phaeoacremonium aleophilum on grapevine rootstocks. Cien. Inv. Agr. 36(3):381-390. Cuttings of fi ve grapevine (Vitis vinifera) rootstocks were wounded and immediately inoculated with suspensions (approximately 5x10 3 conidia·mL -1 ) of either Phaeomoniella chlamydospora, Phaeoacremonium aleophilum or a mixture of both species. The presence of these endophyte fungi affected the quality of each of the fi ve rootstocks. Among the roostocks investigated in this study, 1103P and 101-14 MG were less susceptible to the infection caused by Pa. chlamydospora and Pm. aleophillum.

First Report of Phaeomoniella chlamydospora on Vitis vinifera and French American Hybrids in Chile

Phaeomoniella chlamydospora (W. Gams, Crous. M.J. Wingfield & L. Mugnai) Crous & Gams (= Phaeoacremonium chlamydosporum) was isolated during the growing seasons of 2003-2004 from roots, trunks, and cordons of grapevines, including cvs. Cabernet Sauvignon, Merlot, Pinot noir, Thompson seedless, Ruby seedless and root stock 3309C, and Kober 5BB, from 10 locations in V, VI, VII, and metropolitan regions of Chile. P. chlamydospora was isolated from 82% of samples from vines 2 to 18 years old that showed decline symptoms in the field. Isolates were identified on the basis of a previous description (1) and internal transcribed spacer (ITS1-5.8S-ITS2) rDNA sequences identical to those of P. chlamydospora isolated from Vitis vinifera (culture CBS 22995, GenBank Accession No. AF 197973). P. chlamydospora is established as a member of the petri and esca disease complex and as a pathogen of grapevines (2,3). Pathogenicity tests were completed by injecting into the pith of 50 single-node, rooted cuttings of Pinot noir and 3309C, approximately 20 μl of a 106 conidia per ml suspension, obtained from four isolates from Chile and one from California. Ten control cuttings of Pinot noir and 3309C were injected with an equal volume of sterile distilled water. Twenty-four weeks after inoculations, all P. chlamydospora-inoculated cuttings exhibited dark streaking of the vascular tissue extending 40 to 45 mm from the point of inoculation. The vascular streaking observed in inoculated plants was identical to symptoms observed in declining vines in the vineyard. No symptoms were observed in the controls. P. chlamydospora was isolated from the region of vascular streaking in 85% of inoculated cuttings. P. chlamydospora was not isolated from the water-treated controls. The reisolated P. chlamydospora was verified with means of morphological characters and polymerase chain reaction amplification with the species-specific primers (3). P. chlamydospora is widespread and readily isolated from declining grapevines in Chile and other grape growing regions of the world. To our knowledge, this is the first report of P. chlamydospora from the cultivars cited above in Chile. References: (1) M. Groenewald et al. Mycol. Res. 105:651, 2001. (2) L. sparapano et al. Phytopathol. Mediterr. (Suppl.)40:376, 2001. (3) S. Tegli et al. Phytopathol. Mediterr. 39:134, 2000.