First Report of Diaporthe novem Causing Postharvest Rot of Kiwifruit During Controlled Atmosphere Storage in Chile.

Chile is considered the third major exporter of kiwifruits (Actinidia deliciosa (A. Chev.) C. F. Liang & A. R. Ferguson) worldwide after Italy and New Zealand (1). The genus Diaporthe Nitschke (anamorph: genus Phomopsis) has been reported as causing postharvest rot in kiwifruit (4). During the current study, 1,400 fruits arbitrarily collected from seven controlled atmosphere (CA) rooms after 90 days of storage conditions (2% O2, 5% CO2) determined that 21.5% of the fruit were affected by decay and 0.86% developed symptoms different than those caused by Botrytis cinerea, the main postharvest pathogen associated to kiwifruit. Symptoms were soft rot with brown skin that started at the stem-end and in severe cases affected the entire fruit. Internally, affected fruit showed browning and watery tissues. Twelve affected fruits were surface disinfested (75% ethanol) and small pieces of internal rotten tissues were placed on acidified potato dextrose agar (APDA) for 7 days at 20°C. Twelve isolates were obtained, and four of them were identified morphologically and molecularly as Diaporthe ambigua, a species that has been previously described causing rot in stored kiwifruits in Chile (2). However, eight other flat, white to grayish colonies with sparse dirty-white aerial mycelium at the edge of the dish were obtained (3). Black pycnidia contained unicellular, hyaline, biguttulate, oval to cylindrical alpha conidia, with obtuse ends of (7.9) 6.7 (5.3) × (2.9) 2.5 (2.1) µm (n = 30). These isolates were tentatively identified as a Diaporthe sp. The species identification was determined by sequencing comparison of the internal transcribed spacer (ITS1-5.8S-ITS2) region of the rDNA (GenBank Accession Nos. KJ210020 to 24, KJ210027, and KJ210033) and a portion of beta-tubulin (BT) (KJ210034 to 38, KJ210041, and KJ210047) using primers ITS4-ITS5 and Bt2a-Bt2b, respectively. BLAST analyses showed 99 to 100% identity with D. novem J.M. Santos, Vrandecic & A.J.L Phillips reference ex-type (KC343156 and KC344124 for ITS and BT, respectively) (3). Eighteen mature kiwifruits cv. Hayward were inoculated using a sterile cork borer on the surface of the fruit and placing 5-mm agar plugs with mycelial of D. novem (DN-1-KF). An equal number of fruits treated with sterile agar plugs were used as negative controls. After 30 days at 0°C under CA, all inoculated fruit showed rot symptoms with lesions 7.8 to 16.4 mm in diameter. The same D. novem isolate was inoculated with 30 µl of a conidial suspension (106 conidia/ml) on the surface of 18 ripe kiwifruits that were previously wounded and non-wounded as described above. An equal number of wounded and non-wounded fruits, treated with 30 µl sterile water, were used as negative controls. All inoculated wounded fruits developed rot symptoms with necrotic lesions of 14.1 to 20.2 mm of diameter after 14 days at 25°C. Inoculated non-wounded and negative control fruits remained symptomless. Koch's postulates were fulfilled by re-isolating D. novem only from the symptomatic fruits. To our knowledge, this is the first report of rot caused by D. novem on kiwifruit during cold storage in Chile and worldwide. Therefore, both Diaporthe species appears to be associated to Diaporthe rot of kiwifruit in Chile. References: (1) Belrose, Inc. World Kiwifruit Review. Belrose, Inc. Publishers, Pullman, WA, 2012. (2) J. Auger et al. Plant Dis. 97:843, 2013. (3) R. Gomes et al. Persoonia 31:1, 2013. (4) L. Luongo et al. J. Plant Pathol. 93:205, 2011.

First Report of Fusarium oxysporum f. sp. lycopersici Race 3 and F. oxysporum f. sp. radicis-lycopersici in Tomatoes in the Azapa Valley of Chile.

Tomato (Solanum lycopersicum L.) is an important crop in the Azapa Valley (18°35' S, 69°30' W) in northern Chile, with approximately 600 ha of fresh tomatoes under greenhouses. Cultivars resistant to Fusarium oxysporum f. sp. lycopersici (FOL) races 1 and 2 are mainly used. However, in 2012 and 2013, Fusarium wilt incidence was 2 to 3%. Symptoms appeared unilaterally and consisted of yellowing, leaf wilting of lower leaves, dark brown vascular discoloration, and plant death. The aim of this study was to determine the causal agent of tomato wilt in seven tomato greenhouses in the Azapa Valley. Stem samples (5 × 5 mm) were obtained 10 cm of the stem base from wilted tomatoes 'Naomi' (BIOAMERICA S.A., Chile) or from Maxifort tomato rootstock (De Ruiter Seed, USA), both FOL resistant to races 1 and 2. Samples were washed with tap water, surface sterilized with 1% NaClO for 3 min, and incubated on sterile moist paper towels in petri plates for 5 days at 22°C. Mycelial fragments from white colonies, emerging from diseased tissues, were transferred to PDA. Six Fusarium isolates were characterized by the presence of hyaline macroconidia, mostly 3 to 5 septate, slightly curved (19.2 to 32.1 × 2.9 to 4.5 µm) and single-celled, oval to elongated microconidia (3.1 to 8.9 × 2.0 to 4.0 µm). Chlamydospores were single or in pairs. These isolates were identified as F. oxysporum (3). The identity of F. oxysporum was confirmed by PCR assays using genomic DNA of each isolated and the universal primers Uni F and Uni R that generate a 672-bp PCR product. The pathogenic form and races were determined by PCR assays using the specific primers uni, sp13, sp23, and sprl that were able to discriminate all the three FOL races as well as F. oxysporum f. sp. radicis-lycopersici (FORL) isolates (2). The sp13 and sp23 primers amplified DNA bands of 445 and 518 bp, confirming the identity of FOL race 3. However, sprl amplified a fragment of 947 bp corresponding to FORL (2). Pathogenicity tests were conducted on 25-day-old seedlings (10 seedlings per isolate) of tomato 'Poncho Negro,' which is susceptible to FOL and FORL. Seedling roots were cut, submerged for 5 min in conidial suspension of 2 × 106 conidia/ml, and transplanted to 250-ml plastic containers with sterile substrate (sand/peat, 1:1). Equally treated non-inoculated seedlings were left as controls. The first symptoms induced by each of the five FOL isolates appeared 8 days after incubation under greenhouse and were characterized by yellowing of older leaves, sometimes affecting one side of the plant, vascular discoloration of the stem, and eventually plant death. In contrast, all seedlings inoculated with a FORL isolate developed a necrotic lesion and vascular discoloration at the base of the stems near the soil line, followed by wilting and plant death. Control plants remained asymptomatic. F. oxysporum was re-isolated only from inoculated plants, completing Koch's postulates. FOL and FORL were reported earlier in other tomato growing areas of Chile (1), located over 1,000 km south of the Azapa Valley. However, this is the first report of FOL race 3 and FORL in the Azapa Valley and FOL race 3 is reported for the first time in Chile. References: (1) S. Acuña. Compendio de Fitopatógenos de Cultivos Agrícolas. Servicio Agrícola y Ganadero. Gobierno de Chile, 2008. (2) Y. Hirano and T. Arie. J. Gen. Plant Pathol. 72:273, 2006. (3) J. F. Leslie and B. A. Summerell. The Fusarium Laboratory Manual. Blackwell Publishing, Ames, IA, 2006.

First Report of High Boscalid Resistance in Botrytis cinerea Associated with the H272L Mutation in Grapevine in Chile.

Gray mold (Botrytis cinerea Pers.) is a major disease of grapevine (Vitis vinifera L.) worldwide. Integrated control strategies, including canopy management and fungicide treatments, are needed to control gray mold. Chemical control relies on the use of single mode of action fungicides. The aim of this research was to study the sensitivity of B. cinerea to boscalid, which is a single mode of action fungicide of the succinate dehydrogenase inhibitor (SDHI) fungicide group. Fifty isolates were obtained in 2012 to 2013 from commercial vineyards in central Chile. Vineyards had received two boscalid applications at least for four consecutive years. Briefly, the percent mycelial growth inhibition (MGI) was determined on minimal medium (MM) (2) plus 50 µg m-1 of boscalid (Cantus 50 WP, BASF Chile). Each isolate was tested in triplicate, obtaining 2% highly resistant (HR, MGI ≤25%), 32% moderately resistant (MR, MGI 26 to 50%), 64% low resistant (LR, MGI 51 to 80%), and 2% sensitive (S, MGI ≥81%) phenotypes. Nine isolates were arbitrary selected and compared for MGI on MM plus 50 µg ml-1 of boscalid (1) and conidial germination inhibition (CGI) on yeast extract-bacto peptone-Na acetate (YBA) plus 5 µg ml-1 of boscalid (2,3). Isolates previously determined to be S and HR had the same phenotype for both MGI and CGI. However, all of the MR and LR isolates, determined based on the MGI tests, were identified as S isolates in the CGI tests. Using primer-introduced restriction analysis (PIRA)-PCR (4), the SdhB mutations were detected only in the HR isolate. The amplifications were performed with H272L-fw/H272-rev and were digested by the enzyme BglII, yielding 35- and 85-bp fragments and confirming a mutation at codon 272 (H272L) in the HR phenotype. The efficacy of the label-rate (0.4 g liter-1) boscalid in controlling gray mold was determined on 'Granny Smith' apples. The apples were surface-disinfested (75% ethanol, 30 s), wounded with a sterile syringe, and inoculated with a mycelium plug (5 mm in diameter) or 20 µl of a conidial suspension (106 conidia/ml) of one HR, MR, and S isolate. The inoculum was placed on the wounded sites after boscalid application. Apples were incubated for 7 days at 21°C. Each test had four replicates and the experiment was conducted three times. Boscalid slightly controlled (<6.7% efficacy) gray mold on the apples that were inoculated with mycelium or conidia of the HR phenotype isolate, while the sensitive isolate was highly controlled (>95% efficacy), and the MR isolate was moderately controlled (27 to 34% efficacy). These results demonstrate that mycelium or conidia assays using MM + 50 µg ml-1 boscalid or YBA+5 µg ml-1 boscalid consistently detected HR isolates. The S isolates detected using MGI were also S according with the CGI tests. The presence of the boscalid HR strains of B. cinerea associated with the H272L mutation in grapevine in Chile is reported for the first time in this study. This finding suggests that resistance to boscalid needs to be considered in the design of gray mold control strategies in commercial grapevine orchards. References: (1) D. Fernandez-Ortuño et al Plant Dis. 96:1198, 2012. (2) M.-J. Hu et al. J. Phytopathol. 159:616, 2011. (3) Y. K. Kim and C. L. Xiao. Plant Dis. 94:604, 2010. (4) T. Veloukas et al. Plant Dis. 95:1302, 2011.

First Report of Blossom Blight Caused by Sclerotinia sclerotiorum on Japanese Plum, Nectarine, and Sweet Cherry Orchards in Chile.

Blossom blight of Japanese plum (Prunus salicina), nectarine (P. persica var. nectarina), and sweet cherry (P. avium) was observed in commercial orchards in central Chile in 2012. Disease prevalence of 8% and 1% were estimated in 2012 and 2013, respectively. Early symptoms appeared as small pale-brown necrotic lesions on the petals that eventually affected the entire flowers. White and cottony fungal colonies were consistently isolated on potato dextrose agar acidified with 0.5 ml/liter of 92% lactic acid (APDA), incubated for 5 days at 20°C. Black spherical to elongated sclerotia of 2.5 to 4.2 × 2.8 to 5.3 mm (n = 60) were formed on APDA. This fungus was tentatively identified as Sclerotinia sclerotiorum (Lib.) de Bary. The identity of the fungus was confirmed by BLAST analysis of the internal transcribed spacer (ITS) region (GenBank Accession Nos. KF148604 to KF148609) of rDNA, amplified with PCR primers ITS1/ITS4 (3), demonstrating a 99 to 100% similarity with the reference S. sclerotiorum strains (EU082466 and JX307092). The pathogenicity was studied in detached flowers of 'Larry Ann' Japanese plum, 'Summer Bright' nectarine, and 'Bing' sweet cherry that were inoculated with a mycelial suspension (106 fragments/ml) of six isolates of S. sclerotiorum and incubated for 5 days at 20°C in humid chambers (>80% relative humidity). Inoculated flowers developed a light brown petal necrosis that eventually comprised the entire flower. The same S. sclerotiorum isolates were inoculated in mature fruits of 'Larry Ann' Japanese plum, 'Summer Bright' nectarine, and 'Staccato' sweet cherry. Surface disinfected (1% NaOCl for 1 min) fruits were inoculated by placing a mycelium plug (4 mm in diameter) into a wound made with a sterile scalpel and incubated for 3 days at 20°C in humid chambers. Symptoms consisted on light brown soft lesions that varied from 8.7 to 46.5 mm in diameter. A superficial white and cottony septated mycelium was also obtained. An equal number of non-inoculated flowers and wounded but non-inoculated fruits remained healthy. S. sclerotiorum was re-isolated from 100% of the artificially inoculated flowers and fruits, completing Koch's postulates. S. sclerotiorum was reported causing shoot blight on apricot (P. armeniaca), lemon tree (Citrus limon), and table grapes (Vitis vinifera) in Chile (1,2), and to our knowledge, this is the first report of S. sclerotiorum associated with blossom blight in Japanese plum, nectarine, and sweet cherry in Chile. References: (1) R. Acuña. Compendio de Bacterias y Hongos de Frutales y Vides en Chile. Servicio Agrícola y Ganadero, Santiago, Chile, 2010. (2) B. A. Latorre and M. J. Guerrero. Plant Dis. 85:1122, 2001. (3) T. J. White et al. Page 315 in: PCR Protocols: A Guide to Methods and Applications. M. A. Innis et al., eds. Academic Press, San Diego, CA, 1990.

First Report of Monilinia fructicola Causing Brown Rot on Stored Japanese Plum Fruit in Chile.

In autumn 2013, fruit of Japanese plum (Prunus salicina) cvs. Angelino and Black Kat developed an unusual brown and soft rot after 2 months in cold storage (0°C) on nearly 1% of the fruit. Fruit showed small, circular, light brown spots that eventually destroyed the entire fruit. Small sporodochia appeared on the fruit surface. Fruit was harvested from orchards located near San Francisco de Mostazal (33°59' S, 70°41' W), Chile. Small pieces of diseased tissue were selected from margins of lesions of surface disinfected (96% ethanol) fruit (n = 7) and placed on acidified potato dextrose agar (PDA) plates for 5 days at 20°C. Light brown colonies with even margins and concentric rings of spores were obtained. The conidia of five isolates were one-celled, hyaline, lemon-shaped, (min. 10.7) 14.9 ± 1.5 (max. 18.6) × (min. 8.1) 9.4 ± 0.8 (max. 10.8) µm (n = 30), and borne in branched monilioid chains. This fungus was identified as Monilinia fructicola (G. Winter) Honey (1). Identification was confirmed by amplifying and sequencing the ribosomal ITS1-5.8S-ITS2 region using ITS1 and ITS4 primers (3). BLAST analysis of Chilean plum isolates (GenBank Accession Nos. KF148610 and KF148611) were 99 to 100% identical to isolates of M. fructicola originating from the United States (DQ314727 and HQ846966, respectively) and 100% identical to the first Chilean isolate (JN001480) found in nectarines originating from California at the supermarkets in Santiago in June 2009. Koch's postulates were fulfilled by reproducing brown rot symptoms on mature wounded Japanese plums cv. Angelino (n = 8) inoculated with 10 µl of a conidial suspension (105 conidia/ml) or with a mycelium plug (5-mm diameter). After 2 days in humid chambers (>80% relative humidity) at 25°C, all inoculated fruit developed brown rot symptoms with necrotic lesion means of 15.8 and 21.5 mm in diameter in fruit inoculated with conidia and mycelium, respectively. Non-inoculated control fruit remained healthy. Re-isolations were performed on PDA and the presence of M. fructicola was morphologically confirmed in 100% of the symptomatic fruits. To our knowledge, this is the first report demonstrating the presence of M. fructicola causing brown rot in stored Japanese plums in Chile after its first interception in 2009 in Chile, suggesting that this pathogen has been established in the field. Currently, M. fructicola is a quarantine organism under official control, restricted to Prunus orchards between Santiago and Nancagua in central Chile (2). References: (1) EPPO. EPPO Bull. 39:337, 2009. (2) Servicio Agrícola y Ganadero, SAG, Ministerio de Agricultura, Gobierno de Chile. www.sag.cl , accessed 15 November 2013. (3) 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.

Infection Risk and Critical Period for the Postharvest Control of Gray Mold (Botrytis cinerea) on Blueberry in Chile.

Gray mold (Botrytis cinerea) is a major disease of blueberries (Vaccinium spp.), which require more than 15 days of shipment at 0°C to reach international markets. The aims of this study were (i) to determine the relative susceptibility of the flowering and fruiting stages, (ii) to determine the critical blueberry growth stages for postharvest gray mold control, and (iii) to determine the infection risks on the basis of weather conditions. The epiphytic colonization of B. cinerea of flowers and fruits was demonstrated in blueberry 'Brigitta' and 'Duke' in Antuco and Virquenco. In inoculated flowers and fruits in humid chambers at 20°C, full bloom and mature fruit stages were the most susceptible stages. Fungicide applications at the mature fruit stage appeared as the most critical period for gray mold control in stored fruits. The algorithm proposed, which was based on >6 h of wetness between 14 and 25°C, allowed the estimation of the periods of B. cinerea infection risk. A significant correlation between the B. cinerea infection risk and gray mold incidence in stored fruits was obtained (r = 0.96, P < 0.0001). Therefore, this algorithm has the potential to optimize fungicide applications under field conditions, but field validation of this algorithm remains to be determined.

A Severe Outbreak of Charcoal Rot in Cantaloupe Melon Caused by Macrophomina phaseolina in Chile.

A severe outbreak of charcoal rot was observed in cantaloupe melon (Cucumis melo L.) in the summer of 2011 to 2012 in Curacaví Valley, Chile. Prior to harvest, of 72 plants per cultivar, charcoal rot prevalence varied from 32% to 82% in cvs. Colima, Charantias, Navigator, Origami, Otero, and Samoa. Symptoms were wilting and leaf browning associated with water-soaked lesions at the base of the crown with amber to dark brown exudates. Lesions dried out progressively, turned tan, and cracked. Affected plants declined and died before harvest. Reddish fruit decay was observed. Symptomatic stem and root samples (n = 97) were collected, surface disinfected (96% ethanol, 30 s), plated on PDA acidified with 0.5 ml/liter of 92% lactic acid (APDA), and incubated at 20 ± 1°C. A white, fast-growing mycelium was obtained that turned gray to black after 7 days due to the presence of spherical to oblong black microsclerotia 136 ± 52 µm (n = 80) in diameter. On the basis of colony morphology and microsclerotia, 57 isolates (59%), obtained from 97 melon samples, were tentatively identified as Macrophomina phaseolina (Tassi) Goid. (2,3). The morphological identification of four isolates M1HB-B, M2CO-B, M3CH-R, and M4OT-B (GenBank Accession Nos. JX203630, JX203631, JX203632, and JX203633) was confirmed by sequencing of the internal transcribed spacer region (ITS1-5.8S-ITS2) of rDNA, using primers ITS4 and ITS5, with >99% similarity with the sequences of M. phaseolina (GenBank Accession No. HQ660592) (4). Pathogenicity tests were conducted with isolates M1HB-B, M2CO-B, M3CH-R, and M4OT-B on melon fruits cvs. Colima, Origami, Charantias, and Diva. Four mature melon fruits per cultivar per isolate were surface disinfected with 0.5% sodium hypochlorite for 2 min before inserting a mycelium plug (19 mm2) in a 6 mm diameter hole made with a sterile cork borer. An equal number of perforated fruits in which a sterile agar plug was inserted were left as non-inoculated controls. After 8 days of incubation at 20°C, inoculated fruits developed a spherical, reddish, soft necrotic lesion of 15 to 20 mm in diameter in the pulp. Non-inoculated fruits remained symptomless. The pathogenicity of the four isolates was also studied in 3-month-old melon plants (n = 4) cvs. Colima and Navigator. Plants were inoculated by inserting a mycelial plug (9 mm2) underneath the epidermis of the crown, 5 cm above the soil level. The inoculation site was immediately wrapped with Parafilm to avoid dehydration. An equal number of non-inoculated, but injured plants, treated with a sterile agar plug, were left as controls. After 21 days of incubation under greenhouse conditions (17 ± 5.5°C), all inoculated plants developed water-soaked to dry necrotic lesions, 20 to 70 mm long, yellow to tan in color. No symptoms were obtained in non-inoculated controls. M. phaseolina was reisolated in 84% and 100% of the inoculated plants and fruits, respectively. To our knowledge, this study is the first report of charcoal rot in cantaloupe melon in Chile, previously found on watermelon and melon group inodorus (1). Charcoal rot appears as an emerging disease that aggressively affects current cantaloupe melon cultivars in central Chile. References: (1) G. Apablaza. Cien. Inv. Agr. 20:101, 1993. (2) B. D. Bruton and E. V. Wann. Charcoal rot. Page 9 in: Compendium of Cucurbit Diseases. T. A. Zitter, D. L. Hopkins, and C. E. Thomas, eds. APS, St. Paul, MN, 1996. (3) S. Kaur et al. Crit. Rev. Microbiol. 38:136, 2012. (4) J. Q. Zhang et al. Plant Dis. 95:872, 2011.

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). References: (1) G. A. Díaz et al. Plant Dis. 95:1032, 2011. (2) J. G. Espinoza et al. Plant Dis. 92:1407, 2008. (3) Slippers et al. Mycologia 96:1030, 2004. (4) J. R. Úrbez-Torres Phytopathol. Mediterr. 50:S5, 2011.

First Report of Seimatosporium botan Associated with Trunk Disease of Grapevine (Vitis vinifera) in Chile.

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.

First Report of Diaporthe australafricana Associated with Stem Canker on Blueberry in Chile.

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.

First Report of Nothofagus macrocarpa Dieback Caused by Phytophthora citrophthora and P. nicotianae in Chile.

The genus Nothofagus, family Nothofagaceae, comprises 36 species of trees that are native to the Southern Hemisphere. N. macrocarpa (DC.) F.M. Vásquez & R.A. Rodríguez (Roble de Santiago) is an important deciduous tree, endemic to central Chile (32 to 35°S), and found above 800 m altitude. There is an increasing interest in N. macrocarpa as an ornamental. However, a general dieback (40 to 50% prevalence) was observed at a commercial nursery in Santiago in 2009, limiting its multiplication. Symptoms are wilting, partial defoliation, reddish brown cankers on the crowns, and root necrosis. The purpose of this work was to study the etiology of the dieback in nurseries. Phytophthora was isolated from the roots and cankers of symptomatic plants (n = 3) and soil samples (using apples and avocados as baits) on amended corn meal agar (3) at 20°C for 5 days in the dark. Morphologically, P. citrophthora (Smith & Smith) Leonian, and P. nicotianae Breda de Haan were identified (2). On V8 juice agar (V8) (1), P. citrophthora formed petaloid colonies, grew between 5 and 30°C (optimum of 25°C), and produced deciduous, mono- or bipapillated sporangia of (28.1) 45.0 to 64.1 × (18.8) 32.0 to 39.2 µm. On V8, P. nicotianae produced cottony colonies, grew between 10 and 30°C (optimum of 25°C), and produced spherical, intercalary chlamydospores (mean diameter of 19.6 µm) and persistent, papillate, spherical to ovoid, ellipsoid, obpyriform sporangia of (33.2) 47.5 to 67.6 × (24.1) 30.0 to 48.9 µm. Isolates of P. citrophthora were sexually sterile, but P. nicotianae formed oogonia with amphigenous antheridia in dual cultures with P. cinnamomi (A2 compatibility type). BLAST analysis of the internal transcribed spacer (ITS) region of rDNA of isolates identified as P. citrophthora (IMI 399056 and IMI 399054, GenBank Accession Nos. JF699756 and JF699755) and P. nicotianae (IMI 399055, Accession No. JF699757), amplified by PCR using ITS universal primers (4), revealed 100% similarity with reference isolates of P. citrophthora (Accession Nos. GU259324.1 and GU259317.1) and P. nicotianae (Accession No. GU983635.1). P. citrophthora (n = 2) and P. nicotianae (n = 1) were pathogenic when wounded detached twigs (n = 5) of N. macrocarpa and N. obliqua were inoculated with 20 µl of a mycelial suspension (106 CFU/ml) of either Phytophthora spp. Twigs were placed in a moist chamber at 20°C for 12 days prior to determine the length of the necrotic lesions that developed. An equal number of noninoculated twigs were left as control. Reisolation of P. citrophthora and P. nicotianae from inoculated material was 100%. The length of the necrotic lesions (13 to 80 mm) from inoculated N. macrocarpa and N. obliqua was significantly greater (P < 0.05) compared with the controls. Regardless of Phytophthora isolates, necrotic lesions (53.9 ± 15.8 mm) in infected N. macrocarpa were significantly longer than in N. obliqua (28.6 ± 13.1 mm) (P < 0.0001). To our knowledge, this is the first report of P. citrophthora and P. nicotianae associated with dieback on N. macrocarpa in Chile. Therefore, there is a potential risk of Phytophthora dieback in N. macrocarpa in nature. References: (1) J. Ampuero et al. Plant Dis. 92:1529, 2008. (2) D. C. Erwin and O. K. Ribeiro. Phytophthora Diseases Worldwide. The American Phytopathological Society, St. Paul, MN, 1996. (3) B. A. Latorre and R. Muñoz. Plant Dis. 77:715, 1993. (4) T. J. White et al. PCR Protocols: A Guide to Methods and Applications. Academic Press, San Diego, 1990.

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 Pestalotiopsis clavispora and Pestalotiopsis spp. Causing Postharvest Stem End Rot of Avocado in Chile.

Avocado (Persea americana) production in Chile has increased to more than 33,500 ha. Chilean avocadoes are sent to markets 15 to 45 days away by overseas transport to the United States, Europe, and Asia. Although apparently healthy avocadoes were harvested in 2009, a 10 to 14% incidence of stem end rot appeared after 15 days of cold storage. Symptoms appeared as small, irregular, brown lesions on the peel at the stem end. Lesions enlarged rapidly, became sunken and soft, eventually comprising the entire fruit as ripening progressed. A white mycelium often developed around the stem cavity. A dark brown necrosis of the pulp was observed that comprised a big part of the pulp as the fruits matured. Isolations were performed from 'Hass' avocadoes that developed stem end rot after fruits were kept in humid chambers for 15 days at 5°C plus 6 days at 20°C (n = 50) to simulate a transport period from Chile to U.S. markets or from diseased fruits (n = 50) kept for 15 days at 20°C. Fruits were surface disinfected for 60 s in 75% ethanol, and small pieces of tissue were excised from the margins of the pulp lesions and then plated onto potato dextrose agar (PDA) plus 1 ml/liter of Igepal CO-630 (Sigma-Aldrich, Atlanta, GA) (MPDA). Fungal colonies that developed on PDA were white and cottony, turning slightly yellow after 15 days. Black acervuli appeared after 15 days at 20°C. Conidia (n = 40) were fusiform, (22.2) 27.0 to 30.4 × (6.3) 7.0 to 9.8 µm with a length/width ratio of 3.4 ± 0.4. All isolates had five-celled conidia. Apical and basal cells were colorless, while the three median cells were dark brown. Conidia had one basal appendage (9.3 ± 3.3 µm) and two to four long apical appendages (34.5 ± 6.9 µm). On the basis of colony and conidia morphology, most of these isolates were initially identified as Pestalotiopsis clavispora (G.F. Atk) Steyaert, but other nonidentified species of Pestalotiopsis were also found (3). Identification was confirmed by amplifying and sequencing the internal transcribed spacer (ITS) region of rDNA using ITS1/ITS4 primers of P. clavispora isolate PALUC-12 (Accession No. HQ659767). A BLAST search of the NCBI database showed that isolate PALUC-12 had 100% homology with P. clavispora (No. EU342214.1). Pathogenicity tests were conducted on surface-disinfected (75% ethanol, 30 s) fruits by placing agar pieces (3 mm in diameter) from 7-day-old cultures and a 20-µl drop of 106 conidia/ml on wounded and unwounded stem cavities and equatorial area of five avocado fruits of 'Hass', per isolate tested, at the commercial maturity stage. Inoculated fruits were placed in moist chambers at 25°C for 10 days. Necrotic lesions resembling symptoms that occurred in storage fruits were observed on wounded fruits. No symptoms were observed on unwounded fruits inoculated in the equatorial zone. However, unwounded fruits inoculated in the stem cavity developed a slight necrosis probably because of undetectable wounds made at harvest. Koch's postulates were confirmed after the reisolation of P. clavispora and Pestalotiopsis spp. from diseased fruits. P. versicolor has been reported in South Africa (1), but to our knowledge, this is the first report of P. clavispora causing stem end rot of avocado. P. clavispora has been reported on blueberry in Chile (2). References: (1) J. M. Darvas and J. M. Kotzé. Phytophylactica 19:83, 1987. (2) J. G. Espinoza et al. Plant Dis. 92:1407, 2008. (3) E. F. Guba. Monograph of Pestalotia and Monochaetia. Harvard University Press, Cambridge, MA, 1961.

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.

Neofusicoccum spp. Associated with Stem Canker and Dieback of Blueberry in Chile.

Blueberry (Vaccinium spp.) plantings have significantly increased in Chile during the last decade and, currently, over 10,700 ha are cultivated throughout the country. Among other diseases, stem canker and dieback has been frequently observed in commercial plantations with incidences between 15 and 45%. The aim of this study was to identify and characterize Neofusicoccum spp. causing stem canker and dieback of blueberry in Chile. Three species, N. arbuti, N. australe, and N. parvum, were identified based on colony and conidia morphology, and nucleotide sequence analysis of the internal transcribed spacer (ITS) region (ITS1-5.8S-ITS2). These Neofusicoccum spp. were found alone or coexisting with Pestalotiopsis spp., Truncatella spp., or Phomopsis spp. Koch's postulates showed all Neofusicoccum spp. isolated from infected plants to be pathogenic when inoculated on blueberry fruit and twigs using both mycelia and conidia suspension. All blueberry cultivars tested, including, Brigitta, Bluecrop, Brightwell, Duke, Elliott, Misty, and O'Neal, were susceptible to Neofusicoccum spp. infection. Pathogenicity tests showed N. parvum to be the most virulent species and Elliott to be the most susceptible cultivar. This report represents the first description of N. arbuti, N. australe, and N. parvum as canker-causing agents on blueberry in Chile.

Identification of Phytophthora cryptogea as the Cause of Rapid Decline of Petunia (Petunia × hybrida) in Chile.

Phytophthora cryptogea was consistently isolated from diseased tissue taken from the crown and necrotic roots of grandiflora type petunia (Petunia × hybrida) that were collected in gardens in five public parks in Santiago, Chile in 2004 and 2005. Symptoms included leaf wilting and foliar chlorosis, followed by partial necrosis, and extensive dark-brown to reddish cankers in the crown. Disease incidence was over 50% and infected plants died within 7 to 10 days after transplanting. This pathogen was identified on the basis of colony morphology, morphological characterization of the sexual and asexual reproductive structures, and temperature range. The identification of Phytophthora cryptogea was further corroborated by the internal transcribed spacer sequence analysis (GenBank accession number EF093534). Isolates of P. cryptogea were pathogenic on 10-week-old white grandiflora petunia plants that were inoculated on the roots or on the crown using mycelium fragments, or via soil inoculation using zoospores. A rapid decline was observed after soil inoculations with zoospores. Root fresh weight decreased significantly and the root rot index and severity of foliage symptoms increased significantly (P ≤ 0.05), relative to noninoculated plants after 14 days of incubation. Two isolates (Ph-1 and Ph-2) were pathogenic on bell pepper and one isolate (Ph-1) was pathogenic on tomato after root inoculation. Two isolates (Ph-2 and Ph-3) were pathogenic on the fruit of avocado, bell pepper, cherry tomato, cucumber, kiwifruit, lemon, pear, pepino, and potato tubers, demonstrating the pathogen's ability to cause postharvest infection of fruit of a wide range of host plants. The efficacy of mefenoxam at 0.1 mg/ml mixed with either chlorothalonil at 1.0 mg/ml or mancozeb at 1.6 mg/ml was demonstrated in this study, whereas chlorothalonil and mancozeb alone did not control disease development. No significant differences were obtained between foliage and soil drench applications. This study demonstrated that P. cryptogea is the cause of the rapid decline found on petunia in Santiago, Chile and, to our knowledge, this is the first report giving a detailed description of a disease caused by P. cryptogea on petunia.

Outbreaks of Cladosporium Rot Associated with Delayed Harvest Wine Grapes in Chile.

Severe outbreaks of Cladosporium rot have occurred on berries in clusters of late harvest wine grapes (Vitis vinifera L.) during the 2003 to 2006 growing seasons. This disease was especially prevalent on Cabernet Sauvignon (CS) vineyards in central Chile where disease incidence commonly comprised 50 to 100% of the clusters at harvest. Symptoms appeared on mature grapes (total soluble solids [TSS] >22%) and were characterized by berry dehydration, a firm decay affecting a small portion of the berry and a superficial olive-green mold. Isolations made on acidified potato dextrose agar (APDA) consistently yielded olive-green colonies after 7 days at 20°C. On the basis of colony morphology and morphological characteristics of conidiophores and conidia, Cladosporium herbarum (Pers.:Fr) Link and C. cladosporioides (Fres.) de Vries were identified (1). These species were primarily separated by the presence of rough and smooth conidial surfaces, respectively. Koch's postulates were completed by inoculating 80 wounded mature (20% TSS) CS berries and incubating them at 10, 20, and 30°C. An equal number of wounded but noninoculated berries were left as controls. Berries were surface sterilized (75% ethanol for 30 s and 0.5% sodium hypochlorite for 60 s), inoculated with 10 µl of a 106 conidial suspension per ml applied to the wounds made with a sterile hypodermic needle. Dark, necrotic lesions, 1 to 6 mm in diameter, and a dark mycelial colony appeared on the surface after 7 days in chambers with relative humidity of >95%. Disease incidence was significantly (P < 0.05) influenced by temperature, with 90, 100, and 49% of inoculated berries becoming infected when incubated at 10, 20, or 30°C, respectively. C. herbarum and C. cladosporioides appeared to be equally pathogenic, producing symptoms similar to naturally infected CS berries, and were reisolated (100%) on APDA. These same isolates were pathogenic when tested on mature (TSS >16%) Thompson Seedless (TS) berries. Fungicide sensitivity tests were performed on detached TS berries challenged by placing 10 µl of a 106 conidial suspension per ml of C. herbarum on injured berries. Boscalid (0.5 mg/ml, Cantus WG; BASF, Santiago, Chile), iprodione (0.5 mg/ml, Rovral WP; Bayer Crop Science, Santiago, Chile), pyraclostrobin (0.085 mg/ml, Comet SC; BASF), and pyraclostrobin (0.009 mg/ml) mixed with boscalid at 0.017 mg/ml (Bellis WG; BASF) provided a significant control (P < 0.05) with efficacy between 84.3 and 95.9%. Azoxystrobin (0.188 mg/ml, Quadris SC; Syngenta Crop Protection, Santiago, Chile), kresoxim methyl (0.067 mg/ml, Stroby SC; BASF), and trifloxystrobin (0.06 mg/ml, Flint WG; Bayer Crop Science) provided partial control with efficacy between 23.1 and 42.1%. Cladosporium spp. have been previously reported (2). However, severe outbreaks of Cladosporium rot occur when berries become partially senescent because of a considerable delay in harvest (3). This appears to favor the development of these pathogens. References: (1) B. U. Heuchert et al. Schlechtendalia 13:1, 2005. (2) W. Hewitt. Compendium of Grape Diseases. R. Pearson and A. Goheen, eds. The American Phytopathological Society, St. Paul, MN, 1988. (3) Ph. Pszczolkowski et al. Cien. Inv. Agr. 28(3):157, 2001.

First Report of Practical Resistance to QoI Fungicides in Venturia inaequalis (Apple Scab) in Chile.

Resistance to the QoI fungicide (kresoxim-methyl) was detected in Venturia inaequalis (Cooke) Winter isolates from apple scab lesions collected from apple orchards in Chile after 5 to 6 years of commercial use of QoI fungicides. To reduce or prevent resistance development, QoI fungicides have been applied in blocks of two or three consecutive applications followed by an application of a fungicide with a different mode of action. However, the efficacy of kresoxim-methyl declined dramatically, providing almost no disease control in several orchards under high scab pressure in southern Chile, and resistance to the QoI fungicides was suspected. Conidia from at least five sporulating leaf scab lesions were collected from each of 10 and 14 orchards in 2003 and 2004, respectively. Fungicide sensitivity was determined by using a conidial germination test on potato dextrose agar acidified with 0.5 ml/liter of 95% lactic acid (APDA) containing kresoxim-methyl (Stroby 50 SC) at 0.0, 0.01, 0.1, and 1.0 µg/ml. The kresoxim-methyl concentration (0.01 µg/ml) has been used as a discriminatory dose in previous resistance studies (2). Germination was assessed for at least 100 conidia at each inhibitor concentration after 18 to 24 h of incubation at 20°C (1,2). Sensitivities were expressed as the percentage of germination relative to that on unamended APDA (1,2). These assays were repeated and all isolates with 50% effective dose (ED50) at >0.1 µg/ml were considered resistant. Isolates from eight of 10 and two of 14 orchards were resistant to kresoxim-methyl in 2003 and 2004, respectively. Relative germination varied from 42.4 to 100% for isolates from resistant orchards on 0.1 µg/ml of kresoxim-methyl, with eight isolates having ED50 at >0.1 µg/mL. Conidia from isolates obtained in 2003 also germinated on APDA amended with 1.0 µg/ml of kresoxim-methyl, with two isolates having ED50 at >1.0 µg/mL. Relative to the mean ED50 of 0.02 µg/ml of kresoxim-methyl determined for relative germination in North America baseline population of V. inaequalis (2), resistance factors for these Chilean isolates were estimated to range from 5 to 50. Conidia of resistant isolates continued to germinate in the presence of 0.1 µg/ml of kresoxim-methyl when 100 µg/ml of salicylhydroxamic acid was added to the amended APDA, suggesting that an alternative oxidase pathway was not responsible for the in vitro results obtained (2). A G143A target mutation has been detected in a V. inaequalis isolate from Chile, although no details concerning this finding were reported (Fungicide Resistance Action Committee, online publication, 2004 [ http://www.frac.info ]). The poor control of apple scab with QoI fungicides in Chilean orchards appears to be due to the development of resistant strains of V. inaequalis that occurred despite the limited use of these materials in blocks of two or three consecutive applications, as initially suggested for resistance management. Farmers have been advised to make only one application of QoI fungicide per year in a tank mixture with another type of fungicide or even to abandon the use of QoI fungicides for apple scab control. To our knowledge, this is the first report of a severe outbreak of apple scab due to the presence of populations of V. inaequalis resistant to QoI fungicides under commercial conditions in Chile. References: (1) R. B. Küng Färber et al. Pest Manage. Sci. 58:261, 2002. (2) G. Olaya and W. Köller. Plant Dis. 83:274, 1999.

First Report of Phytophthora cryptogea Root and Crown Rot on Peach Trees in Chile.

Phytophthora cryptogea Pethybridge and Lafferty was consistently isolated from the Nemaguard rootstock (Prunus persica × P. davidiana) of peach trees in Chile. Symptoms included reddish necrotic tissues at the base of the trunk often extending to the main roots, root rot, gummosis, foliar chlorosis, lack of vigor, and dieback, with severely infected trees dying. Isolations with corn meal agar (Difco, Detroit, MI) amended with antibiotics and fungicides (ACMA) (2) and pure cultures were obtained by hyphal tip transfers to ACMA. Ten isolates from different locations were identified by morphology and growth at cardinal temperatures (1). These isolates produced hyphal swellings and sporangia by using carrot juice broth (2) for 48 h followed by the addition of 1% (w/v) nonsterilized soil extract. Sporangia were nonpapillate, internally proliferating, noncaducous, ovoid to obpyriform, and with mean dimensions of 36 × 24 µm. All isolates were typed as A1 using a P. cinnamomi A2 culture as the test isolate and produced oospores on clarified V8 juice agar amended with thiamine, tryptophan, and ß-sitosterol (2) after 15 to 30 days at 20°C in the dark. Mycelia grew between 5 and 30°C, with optimal growth at 20°C and no growth at 35°C. The ITS sequence analysis performed by IMI (CABI Bioscience, Wallingford, UK) indicated a close match with P. cryptogea, P. drechsleri, and P. erythroseptica, however, peach isolates were differentiated from P. drechsleri and P. erythroseptica by the absence of mycelial growth at 35°C and the heterothallic production of oospores, respectively. Four P. cryptogea isolates were pathogenic when inoculated onto 1-year-old twigs detached from a Nemaguard tree, causing reddish necrotic lesions varying from 7 to 29 mm long after 15 days of incubation at 20°C. Two-year-old P. persica var. nectarina cv. Ruby Diamond budded on Nemaguard and cultivated in 100-liter containers in open field conditions were inoculated with 500-ml of a mycelial suspension (4.5 × 106 propagules per ml) per plant added to 15-cm-deep holes around the trunk. Plants were immediately irrigated and maintained at field capacity during the experiment. An equal number of noninoculated trees treated similarly were left as controls. During the spring, plants developed a general chlorosis and declined within 90 days postinoculation. Tests were repeated with similar results. In other preliminary tests, plants developed cankers characterized by gumming and reddish necrotic lesions underneath the bark following trunk inoculations with mycelium in agar of the same isolates obtained on ACMA. These tests were also repeated. The pathogen was consistently reisolated from symptomatic tissue. To our knowledge, this is the first report of P. cryptogea as a pathogen on peach trees in Chile. References: (1) F. J. Newhook et al. Mycol. Pap. No. 143. CMI, Kew, Surrey, UK, 1978. (2) W. F. Wilcox and B. A. Latorre. Plant Dis. 86:1357, 2002.

Identities and Geographic Distributions of Phytophthora spp. Causing Root Rot of Red Raspberry in Chile.

Five identified and two unidentified Phytophthora spp. were isolated from diseased roots of dead or declining red raspberry (Rubus idaeus L.) plants sampled from 18 plantations along a >1,000-km north-south axis in Chile. The array of Phytophthora spp. isolated was strongly associated with geographical location. P. fragariae var. rubi was recovered from 75 and 60% of the plantations in the southern (40°16' to 40°53' S latitude) and central (34°35' to 37°23' S latitude) production sectors, respectively, but was not recovered from any plantation in the northern sector (32°43' to 33°45' S latitude). Similarly, P. megasperma and P. gonopodyides were recovered from multiple plantations in the southern and central sectors but were not recovered in the northern sector. In contrast, P. cryptogea was recovered from 80% of the plantations in both the northern and central sectors but not from any plantation in the south, whereas P. citricolawas isolated from diseased plants in all sectors. In subsequent pathogenicity trials, P. citricola, P. cryptogea, and an unidentified Phytophthora sp. were equally and highly virulent on 'Heritage' red raspberry in each of three greenhouse experiments. The other species were less virulent in the experiment when soil temperatures were highest (mean weekly maximum = 27.5°C) relative to the other two experiments when temperatures were more moderate (mean weekly maxima of 19.9 and 23.7°C). Isolates identified as P. cryptogea were very similar to P. cryptogea isolates recovered previously from kiwi fruit in Chile and from deciduous fruit trees in California with respect to morphological characters and electrophoretic banding patterns of soluble mycelial proteins. Using the same criteria, isolates identified as P. gonopodyides were very similar to isolates recovered earlier from deciduous fruit crops in New York, which previously were identified as P. cryptogea sensu lato but are hereby reclassified as P. gonopodyides.