First Report of Colletotrichum karstii Causing Glomerella Leaf Spot on Apple in Santa Catarina State, Brazil.

Glomerella leaf spot (GLS) is an emerging disease of apple (Malus domestica Borkh.) that has been reported in regions with a humid subtropical climate, such as southern Brazil, the southeastern United States, and more recently eastern China. GLS is favored by high humidity and temperatures between 23 and 28°C and can result in extensive defoliation when the severity is high. The disease was first reported 1988 in Brazil on cvs. Gala and Golden Delicious in orchards in Paraná State (3), but now is widespread in the country's producing areas. Two Colletotrichum species of different complexes have been associated with GLS, C. gloeosporioides (Penz.) Penz. & Sacc. and its sexual stage Glomerella cingulata (Stoneman) Spaulding & Scherenk, and C. acutatum J. H. Simmonds, although GLS is more commonly associated with the former. In the summer of 2012, necrotic spots were observed on apple leaves (cv. Gala) in Santa Catarina state, Brazil. The first symptoms were reddish-brown spots, evolving to small necrotic lesions 1 to 10 mm long at 7 to 10 days after symptoms were first noted. Pure cultures were obtained by monosporic isolation and grown on PDA at 25°C and with a 12-h photoperiod under fluorescent light. The color of the upper surface of the colony varied from white to gray and the reverse was pink. The conidia length and width ranged from 9.1 to 17.1 µm ( = 12.8) and from 2.9 to 6.8 µm ( = 4.9), respectively, and were cylindrical, hyaline, and straight. After germination, conidia formed oval or circular appressoria measuring between 4.0 and 10.0 ( = 6.3) × 3.0 and 9.0 ( = 5.7). To confirm pathogenicity, susceptible apple seedlings (cv. Gala) were inoculated with a suspension of 1 × 106 conidia.mL-1. Seedlings sprayed with sterile distilled water served as controls. Seedlings were incubated in a moist chamber at 25°C and 100% RH for 48 h. First symptoms appeared 4 days after inoculation and were similar to those observed in the field. The control treatment remained symptomless. The pathogen was reisolated from lesions, confirming Koch's postulates. Fungus was molecularly characterized by sequencing the internal transcribed spacer (ITS) rDNA and glyceraldehyde-3-phosphate dehydrogenase (GAPDH), and the nucleotide sequence was deposited in the GenBank database (KC876638 and KC875408). C. karstii, considered as part of the C. boninense species complex (1), was identified with 100% sequence homology. This species was previously reported in China (4), Thailand, and the United States, affecting Orchidaceae plants (2), and in Brazil it has been reported affecting Carica papaya, Eugenia uniflora, and Bombax aquaticum (1). To our knowledge, this is the first report of C. karstii causing GLS on apple in Brazil. The development of pre-harvest management practices may be warranted to manage this disease. References: (1) U. Damm et al. Stud. Mycol. 73:1, 2012. (2) I. Jadrane. Plant Dis. 96:1227, 2012. (3) T. B. Sutton. Plant Dis. 82:267, 1998. (4) Y. Yang. Cryptogamie Mycologie 32:229, 2011.

First Report of Colletotrichum nymphaeae Causing Apple Bitter Rot in Southern Brazil.

Bitter rot (BR) is a common disease on apples (Malus domestica Borkh.) worldwide. Fruit infection often occurs in warmer weather (25 to 32°C) where high temperatures and humidity strongly favor disease development. Three species causing BR have been reported: Colletotrichum gloeosporioides (Penz.) Penz. & Sacc. and its sexual stage Glomerella cingulata (Stoneman) Spaulding & Schrenk, C. acutatum J. H. Simmonds, and recently C. fragariae in Uruguay (1). In February 2012, typical BR symptoms were observed on apple fruits (cv. Gala) in two orchards located in Santa Catarina, Brazil. Initial symptoms were light brown, slightly sunken lesions that enlarged over time, becoming dark brown and forming sunken or saucer-shaped depressions that extended toward the fruit core in a V-shaped pattern. Acervuli were sparse and the conidia masses varied from dark gray to pale orange in color. To attempt to identify the causal organism, isolations were made from lesions on fully ripened fruits. Pure cultures were obtained following monosporic isolation and grown on PDA at 25°C with a 12-h photoperiod under fluorescent light for 7 to 15 days. The color of the upper surface of the colony varied from white to gray and was orange on the underside. One hundred conidia for each isolate were measured and each was one-celled, hyaline, fusiform, and with a length and width ranging between 8.0 and 25.9 µm (avg. 14.7), and between 2.4 and 9.9 µm (avg. 5.6), respectively. After germination, conidia formed oval appressoria between 5.1 and 9.9 µm (avg. 7.3) × 3.4 and 7.8 µm (avg. 5.2). These morphological characters are consistent with the description of C. nymphaeae (Pass.) Aa (2). To examine pathogenicity, eight 'Pink Lady' apples were inoculated with isolates MANE 25, 137, 143, and 144. A isolate treatments were arranged in a completely randomized design with three replications. Fruits were inoculated with two drops of 10 µl (1 × 106 conidia per ml) using wounded and non-wounded sterile fruit surfaces. Sterile distilled water drops served as controls. Fruit were incubated in a moist chamber at 25°C with a 12-h photoperiod for 10 days. Symptoms were observed at the inoculation site 3 to 4 days after inoculation (DAI) on wounded and 5 to 6 DAI on non-wounded fruits. All of the isolates produced symptoms identical to those observed in Santa Catarina, and each isolate was re-isolated from the apple lesions, confirming Koch's postulates. Fungal isolates were also characterized by sequencing of the internal transcribed spacer (ITS) rDNA using ITS1/IT4 primers and glyceraldehyde-3-phosphate dehydrogenase (GAPDH) using GDF/GDR primers. Isolate sequences had 100% homology to C. nymphaeae. The nucleotide sequences were deposited in GenBank (KC840351, KC840352, KC840353, KC840354, KC875404, KC875405, KC875406, and KC875407). Species identified as C. nymphaeae are considered as part of the C. acutatum species complex and have been reported to occur on water lilies (Nymphaea alba) (3) and causing bitter rot on apples in Korea (2,4). To our knowledge, this is the first report of C. nymphaeae causing bitter rot of apples in southern Brazil. References: (1) S. Alaniz et al. Plant Dis 96:458, 2012 (2) U. Damm et al. Stud. Mycol. 73:37, 2012. (3) D. A. Johnson et al. Mycol. Res. 101:641, 1997. (4) D. H. Lee et al. Plant Pathol. J. 23:37, 2007.

First Report of Colletotrichum acutatum and C. fragariae Causing Bitter Rot of Apple in Uruguay.

Almost 50% of deciduous fruit produced in Uruguay are apples and bitter rot is ubiquitous in the apple-production regions in Uruguay. In rainy and hot seasons (25 to 32°C by day), severe outbreaks of bitter rot occur. In summer 2010, when apple rot incidence reached 70% in some orchards, fruit with typical symptoms of bitter rot were collected from orchards in the south-central region, the main apple-production area. Symptoms included 0.5 to 6.0 cm in diameter, circular, sunken, light brown-to-brown lesions on the fruit surface that contained black, pinhead-sized fruiting structures that produced orange-to-brown conidial masses under high relative humidity. Each lesion progressed to the core of the fruit in a V-shaped pattern. Single-conidial isolates from lesions were examined morphologically (3), and based on sequences of the internal transcribed spacer (ITS) rDNA determined using ITS1/ITS4 primers (4), three species were identified: Colletotrichum acutatum with white-to-pale orange colonies and one-celled, hyaline, fusiform to cylindrical conidia that averaged 14.5 (9.3 to 17.8) × 5.0 (6.9 to 4.0) µm (isolates C11 and C18, GenBank Nos. JN413081 and JN413082, respectively); C. fragariae with white-to-pale gray and/or dark gray colonies and one-celled, hyaline, cylindrical to fusiform conidia that averaged 20.5 (14.3 to 22.9) × 6.0 (4.6 to 7.6) µm (isolate C15 and C37, GenBank Nos. JN413083 and JN413084, respectively); and C. gloeosporioides with white-to-pale gray or gray colonies and one-celled, hyaline, cylindrical to fusiform conidia that averaged 16.5 (13.1 to 20.3) × 6.5 (3.7 to 7.6) µm (isolates C5 and C29, GenBank Nos. JN413079 and JN413080) when grown on potato dextrose agar (PDA) at 25°C. To confirm pathogenicity, two isolates of each Colletotrichum spp. were inoculated onto mature, asymptomatic fruit of cv. Pink Lady (eight fruit per isolate). Each fruit was surface disinfested with 70% ethanol, wounded with a sterile needle, and inoculated with 10 µl of a spore suspension (5 × 105 conidia/ml) of the appropriate isolate. Eight control fruit were each inoculated with 10 µl of sterile water. Inoculated fruit and the control fruit were placed in plastic bags (eight fruit per bag) and incubated at 25°C. Symptoms (sunken, brown lesions each with a V-shaped pattern extending to the core) developed on all inoculated fruit 2 to 4 days after inoculation. No lesions were observed on control fruit. When fungi were reisolated from lesions of inoculated fruit onto PDA and incubated at 25°C, colony and conidial morphology were identical to those of the original isolates, confirming Koch's postulates. This study confirms a previous report of C. gloeosporioides causing bitter rot on apple in Uruguay (1). C. acutaum and C. gloeosporioides are known to cause bitter rot on apple. C. fragariae has traditionally been associated with strawberry and recently with other host plants (2), but not with bitter rot of apple. To our knowledge, this is the first report of bitter rot of apple caused by C. fragariae, and the first report of this disease caused by C. acutatum and C. fragariae in Uruguay. References: (1) S. García. Page 49 in: Guía Para el Manejo Integrado de Plagas y Enfermedades en Frutales. INIA Las Brujas, Canelones, 1998. (2) S. J. MacKenzie et al. Plant Dis. 92:1432, 2008. (3) B. C. Sutton. The Coelomycetes. CAB International Publishing, New York, 1980. (4) T. J. White et al. Page 315 in: PCR Protocols: A Guide to Methods and Applications. Academic Press, San Diego, 1990.

First Report of Cylindrocladiella parva and C. peruviana Associated with Black-foot Disease of Grapevine in Spain.

From 2007 to 2009, Cylindrocladiella-like isolates were recovered from grapevine (Vitis vinifera L.) roots with symptoms of black-foot disease in Spain, where the causal agents of this disease have been previously reported as Campylocarpon and Cylindrocarpon species (1,2). Three representative isolates were selected to confirm their identity: CPa1 and CPa2 from Asturias (northern Spain), and CPe523 from Cuenca (central Spain). Isolates were incubated on malt extract agar (MEA) and Spezieller Nährstoffarmer Agar (SNA) with carnation leaves (4) at 25°C for 10 days in darkness. On MEA, colonies developed light brown, cottony mycelium. On SNA, all three isolates produced chlamydospores in chains, and conidia were zero-to one-septate, but CPa1 and CPa2 produced longer conidia (10.4 to 18.9 [15.3] × 1.7 to 3.1 [2.4] µm) than CPe523 (6.4 to 12.3 [9.7] × 1.6 to 3.3 [2.4] µm). A fragment of the beta-tubulin gene from all isolates was sequenced with primers T1 and Bt2b (1) and deposited in GenBank (Accession Nos. JQ693133, JQ693134, and JQ693135). CPa1 and CPa2 showed high similarity (99%) to Cylindrocladiella parva (AY793486) and CPe523 showed high similarity (99%) to C. peruviana (AY793500), which is in agreement with the corresponding morphological features of these species (4). Pathogenicity tests were conducted with inoculum produced on wheat (Triticum aestivum L.) seed soaked for 12 h in 300 ml of distilled water and autoclaved three times. Inoculum was prepared by inoculating two fungal disks (8 mm in diameter) of a 2-week-old culture of each isolate grown on potato dextrose agar to wheat seed and incubation at 25°C for 4 weeks. One-month-old grapevine seedlings were planted individually in 220-cc pots filled with a potting medium of sterilized peat moss and 10 g of inoculum, and grown in the greenhouse at 25°C in a completely randomized design. Controls were inoculated with sterile, noninoculated wheat seed. There were six replicate plants per isolate, with an equal number of controls, and the experiment was repeated once. Symptoms developed in all plants by 20 days post-inoculation and consisted of reduced vigor, necrotic root lesions, and occasionally mortality, all of which resembled the symptoms from grapevines in the field from which the isolates were originally recovered. Mean shoot dry weights of inoculated plants (0.25, 0.16, and 0.28 g for CPa1, Cpa2, and CPa523, respectively) were significantly lower (P < 0.05) than that of the controls (0.74 g). Mean root dry weights of inoculated plants (0.28, 0.16, and 0.29 g for CPa1, Cpa2, and CPa523, respectively) were also significantly lower (P < 0.05) than that of the controls (0.68 g). Isolates recovered from the roots of inoculated plants were identical morphologically and molecularly to C. parva and C. peruviana, thereby satisfying Koch's postulates. No symptoms were observed on the control plants. These Cylindrocladiella spp. have been reported from nurseries or vineyards in South Africa and New Zealand (3). To our knowledge, this is the first report of C. parva and C. peruviana associated with black-foot disease of grapevine in Spain, and in Europe. References: (1) S. Alaniz et al. Plant Dis. 91:1187, 2007. (2) S. Alaniz et al. Plant Dis. 95:1028, 2011. (3) E. E. Jones et al. Plant Dis. 96:144, 2012. (4) L. Lombard et al. Mycol. Progress DOI 10.1007/s11557-011-0799-1, 2012.

First Report of Pestalotiopsis clavispora Causing Dieback on Blueberry in Uruguay.

During the last 10 years, blueberry (Vaccinium corymbosum) production in Uruguay has increased to more than 850 ha. From 2005, symptoms of dieback characterized by the death of twigs and branches have been frequently observed in blueberry plants cv. O'Neal in orchards located in Uruguay. Symptomatic 4-year-old plants (cv. O'Neal) were collected and small pieces of necrotic tissues were surface disinfected and plated onto potato dextrose agar (PDA) with 0.2 g liter-1 of streptomycin sulfate. Plates were incubated at 25°C in the dark. All affected tissues consistently developed colonies with white and cottony mycelium, turning slightly yellow after 7 to 10 days. Black acervuli distributed in concentric circles were observed after 10 days. Conidia were fusiform, straight, and had five cells. Basal and apical cells were colorless while the three median cells were dark brown. Conidia (n = 50) had an average of 22.1 (16.5 to 28.2) × 6.6 (5.6 to 7.7) µm. All conidia had one basal appendage of 6.1 (3.9 to 14.3) µm and two to four (usually three) apical appendages of 22.8 (17.4 to 42.9) µm. According to colony and conidia morphology, the isolates were initially identified as Pestalotiopsis clavispora (G.F. Atk.) Steyaert (1). To identify, the internal transcribed spacers (ITS1, 5.8S, ITS2) region of rDNA of a representative isolate (Ara-1) was amplified with ITS1/ITS4 primers (4), sequenced, and compared with those deposited in GenBank. The isolate Ara-1 (Accession No. JQ008944) had 100% sequence identity with P. clavispora (Accession Nos. FJ517545 and EU342214). To confirm pathogenicity, isolate Ara-1 was inoculated onto asymptomatic 1-year-old blueberry plants (cv. O'Neal). Mycelial plugs (4 mm in diameter) from an actively growing colony on PDA were applied to same-size bark wounds made with a cork borer in the center of the stems previously disinfected with 70% ethanol and covered with Parafilm. Control plants were inoculated with sterile PDA plugs. Inoculated plants (five per treatment) were randomly distributed in a greenhouse and watered as needed. After 2 weeks, all stems inoculated with P. clavispora showed brown necrotic lesions 2 to 3 cm in length and 1 to 2 mm deep. White mycelium was observed over lesions. Control plants remained symptomless. The pathogen was reisolated from all necrotic lesions, thus fulfilling Koch's postulates. P. clavispora has been reported as associated with blueberry in Hawaii (3) and Chile (2). To our knowledge, this is the first report of P. clavispora causing dieback disease on blueberry in Uruguay. References: (1) E. F. Guba, Monograph of Pestalotia and Monocheatia. Harvard University Press, Cambridge, MA, 1961. (2) J. G. Espinoza et al. Plant Dis. 92:1407, 2008. (3) L. M. Keith et al. Plant Dis. 90:16, 2006. (4) T. J. White et al. Page 315 in: PCR Protocols: A Guide to Methods and Applications. Academic Press, San Diego, 1990.

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

In May 2008, symptoms of black foot disease were observed on 8-year-old grapevines (Vitis vinifera L.) cv. Garnacha in Albuñol (Granada Province, southern Spain). Affected plants showed delayed budding with low vigor. Roots showed black discoloration and necrosis of wood tissues. Root fragments were cut, washed under running tap water, surface sterilized for 1 min in a 1.5% sodium hypochlorite solution, and washed twice with sterile distilled water. Small pieces of discolored or necrotic tissues were plated onto potato dextrose agar (PDA) supplemented with 0.5 g liter-1 of streptomycin sulfate. Plates were incubated at 25°C in the dark for 10 days and all colonies were transferred to PDA. A Cylindrocarpon-like fungus was consistently isolated from necrotic root tissues. Single conidial isolates were obtained and grown on PDA and Spezieller Nährstoffarmer Agar (SNA) and incubated at 25°C for 10 days in darkness. On PDA, the isolates developed white, thick, and cottony to felty abundant mycelium. On SNA, all isolates produced slightly to moderately curved one-septate (22.5-) 25.6 (-27.5) × (5-) 5.63 (-6.25) µm, two-septate (30-) 36.1 (-45) × (6.25-) 7.08 (-7.5) µm, three-septate (37.5-) 47.9 (-52.5) × (6.25-) 7.5 (-8.75) µm, four-septate (47.5-) 53.3 (-62.5) × (7.5-) 7.89 (-8.75) µm, and five-septate (52.5-) 61.8 (-67.5) × (7.5-) 8 (-8.75) µm macroconidia. Microconidia were not observed. DNA sequence of the rDNA internal transcribed spacer region (ITS) was obtained for isolate Cf-270 and deposited in GenBank (Accession No. HQ441249). This sequence showed high similarity (99%) to the sequence of Campylocarpon fasciculare Schroers, Halleen & Crous (GenBank Accession No. AY677303), in agreement with morphological features (1). Pathogenicity tests were conducted with inoculum produced on wheat (Triticum aestivum L.) seeds that were soaked for 12 h in flasks filled with distilled water. Each flask contained 300 ml of seeds that were subsequently autoclaved three times after excess water was drained. Two fungal disks of a 2-week-old culture of C. fasciculare (isolate Cf-270) grown on PDA were placed aseptically in each flask. The flasks were incubated at 25°C for 4 weeks and shaken once a week to avoid clustering of inoculum. Plastic pots (220 cm3) were filled with a mixture of sterilized peat moss and 10 g of inoculum per pot. One-month-old grapevine seedlings were planted individually in each pot and placed in a greenhouse at 25 to 30°C in a completely randomized design. Control plants were inoculated with sterile uninoculated seeds. Six replicates (each one in individual pots) were used, with an equal number of control plants. The experiment was repeated. Symptoms developed on all plants 20 days after inoculation and consisted in reduced vigor, interveinal chlorosis and necrosis of the leaves, necrotic root lesions with a reduction in root biomass, and plant death. The fungus was reisolated from the roots of affected seedlings and identified as C. fasciculare, completing Koch's postulates. No symptoms were observed on the control plants. Black foot disease of grapevines can be caused by different species of Cylindrocarpon and Campylocarpon. C. fasciculare was first reported in South Africa in 2004 (1). To our knowledge, this is the first report of C. fasciculare causing black foot disease of grapevine in Spain as well as other countries in Europe. Reference: (1) F. Halleen et al. Stud. Mycol. 50:431, 2004.

Outbreak of a New Phytophthora sp. Associated with Severe Decline of Almond Trees in Eastern Spain.

Since 2007, a decline of young almond trees (Prunus dulcis) has been observed in different field-grown nurseries in Valencia (east-central Spain). Early symptoms in affected trees included chlorosis, wilting, cankers, and profuse stem gumming. A Phytophthora sp. was consistently isolated from cankers, roots, and soil of affected trees. It was a heterothallic species with amphigynous and/or paragynous antheridia, and its morphological features did not conform to any of the described Phytophthora species. Pathogenicity was proved by artificial inoculation, completing Koch's postulates. All isolates were sensitive to the phenylamide fungicides metalaxyl and mefenoxam. Amplification and sequencing of the internal transcribed spacer (ITS) region, translation elongation factor 1 alpha gene (EF-1α), the ß-tubulin (ß-tub) gene, and the region containing the mitochondrial cytochrome c oxidase subunit 1 (cox1) gene fragment identified the species as Phytophthora taxon "niederhauserii", and phylogenetic analyses placed it in Phytophthora Clade 7b.

First Report of Phaeoacremonium scolyti Causing Petri Disease of Grapevine in Spain.

In May 2007, a survey was conducted to evaluate the phytosanitary status of grapevine propagating materials in a commercial nursery located in Valencia Province (eastern Spain). Fungal isolation was performed on 25 grafted plants (1-year-old grapevines cv. Tempranillo grafted onto 110 R rootstock) because they showed reduced root biomass and black discoloration of the xylem vessels. Sections (10 cm long) were cut from the basal end of the rootstocks, washed under running tap water, surface sterilized for 1 min in a 1.5% sodium hypochlorite solution, and washed twice with sterile distilled water. The sections were split longitudinally and small pieces of discolored tissues were placed onto malt extract agar (MEA) supplemented with streptomycin sulfate (0.5 g L-1). Plates were incubated at 25°C in the dark for 14 to 21 days after which all colonies were transferred to potato dextrose agar (PDA). Togninia minima (Tul. & C. Tul.) Berl. (anamorph Phaeoacremonium aleophilum W. Gams, Crous, M.J. Wingf. & Mugnai) and another Phaeoacremonium sp. were consistently isolated from necrotic tissues. Single conidial isolates of this Phaeoacremonium sp. were grown on PDA and MEA in the dark at 25°C for 2 to 3 weeks until colonies produced spores (3). Colonies were grayish brown on PDA and pinkish white on MEA. Conidiophores were mostly short and unbranched, 15 to 30 (mean 20.8) µm long, often consisting of an elongate-ampuliform phialide. Conidia were hyaline, oblong-ellipsoidal occasionally reniform or allantoid, 2.5 to 5.6 (mean 3.8) µm long, and 1 to 2.1 (mean 1.4) µm wide. On the basis of these characteristics, these isolates were identified as Phaeoacremonium scolyti L. Mostert, Summerb. & Crous (2,3). Identity of isolate Psc-1 was confirmed by PCR-restriction fragment length polymorphism of the internal transcribed spacer region using Phaeoacremonium-specific primers Pm1-Pm2 and restriction enzymes BssKI, EcoO109I, and HhaI (1). Additionally, the ß-tubulin gene fragment (primers T1 and Bt2b) of this isolate was sequenced (GenBank Accession No. EU260415). The sequence showed high similarity (98%) with the sequence of P. scolyti (GenBank Accession No. AY579292). Pathogenicity tests were conducted on 2-month-old grapevine seedlings (cv. Tempranillo) using the isolate Psc-1. Ten seedlings were inoculated when two to three leaves had emerged by watering the roots with 25 mL of a conidial suspension (106 conidia mL-1) harvested from 21-day-old cultures grown on PDA. Ten controls plants were inoculated with sterile distilled water. Seedlings were maintained in a greenhouse at 23 to 25°C. Within 2 months of inoculation, symptoms developed on all of the inoculated plants as crown necrosis, chlorotic leaves, severe defoliation, and wilting. Control plants did not show any symptoms. The fungus was reisolated from internal tissues of the crown area and the stems of all inoculated seedlings, completing Koch's postulates. To our knowledge, this is the first report of P. scolyti causing Petri disease in Spain. References: (1) A. Aroca and R. Raposo. Appl. Environ. Microbiol. 73:2911, 2007. (2) L. Mostert et al. J. Clin. Microbiol. 43:1752, 2005. (3) L. Mostert et al. Stud. Mycol. 54:1, 2006.

Characterization of Cylindrocarpon Species Associated with Black Foot Disease of Grapevine in Spain.

In this work, 82 Cylindrocarpon isolates associated with black foot disease of grapevines in Spain were studied by means of phenotypical characterization, DNA analyses, and pathogenicity tests. Partial sequences of the beta-tubulin (BT) gene, BT1, were amplified using primers BT1a and BT1b. A unique and conserved 52-bp insertion in the BT1 sequence, which is a specific marker for C. macrodidymum, was found in 56 of the isolates. The rest of the isolates (26) were identified as C. liriodendri. The BT phylogeny grouped all the isolates of each species into two well-supported clades. Phenotypical data were subjected to multivariate factorial analysis. According to this study, the isolates were clearly separated into two groups which were in agreement with BT species identification. C. macrodidymum isolates were differentiated from C. liriodendri by producing fewer conidia, presenting longer and wider macroconidia, and lower growth rate at 5 and 10°C. Selected isolates of each species inoculated onto rooted cuttings of grapevine rootstock cv. 110 R caused typical black foot disease symptoms. This is the first report of C. liriodendri and C. macrodidymum causing black foot disease of grapevines in Spain.

First Report of Phaeoacremonium mortoniae Causing Petri Disease of Grapevine in Spain.

In May 2006, symptoms of grapevine decline were observed on 4-year-old grapevines (cv. Cabernet Sauvignon) grafted onto 110 R rootstock in Daimiel (Ciudad Real Province, central Spain). Affected vines had low vigor, reduced foliage, and chlorotic leaves. Cross or longitudinal sections of the rootstock trunk showed black spots and dark streaking of the xylem vessels. Five symptomatic plants were collected and analyzed for fungal isolation. Sections (10 cm long) were cut from the basal end of the rootstocks, washed under running tap water, surface sterilized for 1 min in a 1.5% sodium hypochlorite solution, and washed twice with sterile distilled water. The sections were split longitudinally and small pieces of discolored tissues were plated onto malt extract agar (MEA) supplemented with 0.5 g L-1 of streptomycin sulfate. Plates were incubated at 25 to 26°C in the dark for 14 to 21 days and all colonies were transferred to potato dextrose agar (PDA). A Phaeoacremonium sp. was consistently isolated from necrotic tissues. Single conidial isolates were obtained and grown on PDA and MEA in the dark at 25°C for 2 to 3 weeks until colonies produced spores (3). Colonies were yellowish white on PDA and white-to-pale gray on MEA. Conidiophores were short and unbranched, 12.5 to 37.5 (20.5) µm long, and often consisting of a single subcylindrical phialide. Conidia were hyaline, oblong to ellipsoidal or reniform, 2.5 to 7.5 (4.6) µm long, and 1.2 to 1.9 (1.6) µm wide. On the basis of these characteristics, the isolates were identified as Phaeoacremonium mortoniae (2,3). Identity of isolate Pmo-1 was confirmed by PCR-restriction fragment length polymorphism of the internal transcribed spacer region (Phaeoacremonium-specific primers Pm1-Pm2) with the restriction enzymes BssKI, EcoO109I, and HhaI (1). Additionally, the ß-tubulin gene fragment (primers T1 and Bt2b) of this isolate was sequenced (GenBank Accession No. EF517921). The sequence was identical to the sequence of P. mortoniae (GenBank Accession No. DQ173109). Pathogenicity tests were conducted on 2-month-old grapevine seedlings (cv. Tempranillo) using two isolates, Pmo-1 and a reference isolate of P. mortoniae (CBS-101585) obtained from the Centraalbureau voor Schimmelcultures (Utrecht, the Netherlands). Seedlings were inoculated when two to three leaves had emerged by watering the roots with 25 mL of a conidial suspension (106 conidia mL-1) harvested from 21-day-old cultures grown on PDA. Controls were inoculated with sterile distilled water. There were 20 replicates for each isolate with an equal number of uninoculated plants. Seedlings were maintained in a greenhouse at 23 to 25°C. Within 2 months after inoculation, symptoms developed as reduced growth, chlorotic leaves, severe defoliation, and finally wilting. Control plants did not show any of these symptoms. The fungus was reisolated from internal tissues of the crown area and the stems of all inoculated seedlings, completing Koch's postulates. To our knowledge, this is the first report of P. mortoniae causing young grapevine decline in Spain. References: (1) A. Aroca and R. Raposo. Appl. Environ. Microbiol. 73:2911, 2007. (2) M. Groenewald et al. Mycol. Res. 105:651, 2001. (3) L. Mostert et al. Stud. Mycol. 54:1, 2006.