First Report of Fusarium temperatum Causing Seedling Blight and Stalk Rot on Maize in Spain.

In Europe, several diseases of maize (Zea mays L.) including seedling blight and stalk rot are caused by different Fusarium species, mainly Fusarium graminearum, F. verticillioides, F. subglutinans, and F. proliferatum (3). In recent years, these Fusarium spp. have received significant attention not only because of their impact on yield and grain quality, but also for their association with mycotoxin contamination of maize kernels (1,4). From October 2011 to October 2012, surveys were conducted in a maize plantation located in Galicia (northwest Spain). In each sampling, 100 kernels and 10 maize stalks were collected from plants exhibiting symptoms of ear and stalk rot. Dried kernels and small stalk pieces (1 to 2 cm near the nodes) were placed onto potato dextrose agar medium and incubated in the dark for 7 days. Fungal colonies displaying morphological characteristics of Fusarium spp. (2) were subcultured as single conidia onto SNA (Spezieller Nahrstoffarmer agar) (2) and identified by morphological characteristics, as well as by DNA sequence analysis. A large number of Fusarium species (F. verticillioides, F. subglutinans, F. graminearum, and F. avenaceum) (1,2) were identified. These Fusarium species often cause ear and stalk rot on maize. In addition, a new species, F. temperatum, recently described in Belgium (3), was also identified. F. temperatum is within the Gibberella fujikuroi species complex and is morphologically and phylogenetically closely related to F. subglutinans (2,3). Similar to previous studies (3), our isolates were characterized based on the presence of white cottony mycelium, becoming pinkish white. Conidiophores were erect, branched, and terminating in 1 to 3 phialides. Microconidia were abundant, hyaline, 0 to 2 septa; ellipsoidal to oval, produced singly or in false heads, and on monophialides, intercalary phialides, and polyphialides. Microconidia were not produced in chains. No chlamydospores were observed (3). Macroconidia in carnation leaf agar medium (2) were hyaline, 3 to 6 septate, mostly 4, falcate, with a distinct foot-like basal cell (2,3). DNA was amplified with primers ITS1/ITS4 and EF1/EF2 (3). Partial sequences of gene EF-1α showed 100% homology with F. temperatum (3) (GenBank Accession Nos. HM067687 and HM067688). DNA sequences of EF-1α gene and ITS region obtained were deposited in GenBank (KC179824, KC179825, KC179826, and KC179827). Pathogenicity of one representative isolate was confirmed using a soil inoculation method adapted from Scauflaire et al., 2012 (4). F. temperatum isolate was cultured on sterile wheat grains. Colonized wheat grains (10 g) were mixed with sterilized sand in 10 cm diameter pots. Ten kernels per pot were surface disinfected in 2% sodium hypochlorite for 10 min, rinsed with sterilized water, drained (4), placed on the soil surface, and covered with a 2 cm layer of sterilized sand. Five pots were inoculated and five uninoculated controls were included. Pots were maintained at 22 to 24°C and 80% humidity for 30 days. Seedling malformations, chlorosis, shoot reduction, and stalk rot were observed on maize growing in inoculated soil and not from controls. F. temperatum was reisolated from the inoculated seedlings but not from the controls. References: (1) B. J. Bush et al. Phytopathology 94:88, 2003. (2) J. F. Leslie et al. The Fusarium Laboratory Manual, page 388. Blackwell Publishing, 2006. (3) J. Scauflaire et al. Mycologia 103:586, 2011. (4) J. Scauflaire et al. Eur. J. Plant Pathol. 133:911, 2012.

First Report of Phytophthora alni subsp. uniformis on Black Alder in Spain.

Phytophthora alni is the causal organism responsible for devastating losses occurring on riparian alders stands in Europe. This emergent hybrid pathogen has multiple variants that have been placed in three subspecies (1). P. alni subsp. uniformis and P. alni subsp. multiformis are reported to be less aggressive than P. alni subsp. alni, though all are considered pathogenic. In Spain, P. alni subsp. alni was detected for the first time in 2009 in Galicia (northwestern Spain) causing root and collar rot on riparian alder populations (3,4), but other subspecies had not been identified. In April 2011, a survey along the Deza River in Galicia was carried out to clarify the Phytophthora sp. associated with the alder decline. Thirty riparian Alnus glutinosa stands, from both sides of the river, were surveyed. Samples of bark and roots of 18 alder stands that showed symptoms of Phytophthora rot and soil from all 30 stands were collected. Roots and tissue from fresh, active, inner bark lesions from 54 trees were transferred to selective medium V8-PARPH agar and incubated for 7 days at 22°C in the dark. P. alni subsp. alni (1) was isolated from roots, bark, or soil in five alder stands. Another Phytophthora sp. was isolated from the bark of one symptomatic tree located in Silleda (Pontevedra), transferred to carrot agar (CA), and incubated in the dark. On CA, the isolate produced irregular and appressed colonies with an optimum growth temperature of 22 to 23°C. The isolate was homothallic with smooth-walled oogonia with a diameter ranging from 36 to 50 µm and two-celled, amphigynous antheridia (1). In soil extract, noncaducous, nonpapillate, ellipsoid-to-ovoid sporangia were produced. Average sporangium were 43.4 × 30.1 µm with a length/breadth ratio of 1.43. Internal proliferation occurred. Amplification of DNA was accomplished by sequence characterized amplified region (SCAR)-PCR primers (2). The amplicon sizes obtained were identical to P. alni subsp. uniformis. Internal transcribed spacer (ITS) (DC6-ITS6/ITS4) and nadh1 (NADHF1/NADHR1) mitochondrial gene regions were also amplified and deposited in GenBank (Nos. JN880411 and JN880410). Comparison of the sequences showed 100% homology with P. alni subsp. uniformis (GenBank Nos. GU259293 and DQ202489). Pathogenicity was tested on 10 3-year-old black alder plants grown in pots. A shallow wound was made with a scalpel at the root collar level of each plant. A 5-mm-diameter mycelia plug, taken from the margin of a 7-day-old culture grown on CA, was inserted in every wound and sealed with Parafilm. Five black alder control plants received only sterile CA agar plugs. Plants were kept at 24°C and 80% humidity. After 3 months, wilting of shoots, dead leaves, and dark stained necroses of the bark tissue varying in length from 0.8 to 5 cm were observed on inoculated plants. Control plants remained healthy. P. alni subsp. uniformis was recovered from inoculated plants, but not from controls. To our knowledge, this is the first time that P. alni subsp. uniformis has been reported in Spain. The presence of a new subspecies in a new region can result in hybridization between individuals of different species or subspecies. This process may allow the rapid evolution and adaptation of these species to new hosts or environmental conditions. References: (1) C. M. Brasier et al. Mycol. Res. 108:1172, 2004. (2) R. Ioos et al. Eur. J. Plant Pathol. 112:323, 2005. (3) C. Pintos et al. Plant Dis. 94:273, 2010. (4) A. Solla et al. Plant Pathol.59:78, 2010.

First Report of Cankers and Dieback Caused by Neofusicoccum mediterraneum and Diplodia corticola on Grapevine in Spain.

In November 2010, four grapevine plants of cv. Crimson from a vineyard located in Sevilla (south Spain) revealed trunk cankers. Several pathogens were isolated, including Cylindrocarpon liriodendri (2), Phaeoacremonium aleophilum (2), Pleurostomophora richardsiae, Neofusicoccum parvum, and Botryosphaeria dothidea (2). Among Botryosphaeriaceae fungi isolated on potato dextrose agar (PDA) were two types that did not fit the above mentioned species. Isolates of type 1 produced an abundant, gray mycelium with a diurnal zonation that gradually became dark olivaceous. Mycelium growth occurred from 5 to 37°C with an optimum at 28°C. Conidia were hyaline, fusiform, aseptate, thin walled, but gradually became obscured and septate with age, and measured (18.4-) 21.4 (-24.3) × (4.2-) 5.5 (-7.2) µm with a length/width (L/W) ratio of 4.0 ± 0.5 (n = 100). Isolates of type 1 were identified as N. mediterraneum (3). Single-spore cultures of type 2 developed a whitish, dense, aerial mycelium and remained white up to 10 days on PDA and darkened to gray thereafter. Mycelium growth occurred from 3 to 37°C with an optimum at 29 to 30°C. Conidia were hyaline, aseptate, thick walled, oblong to cylindrical, sometimes becoming light brown and one or two septate after discharge, and measured (24.6-) 30.2 (-42.8) × (10.9-) 14.3 (-18.6) µm with a L/W ratio of 2.1 ± 0.2 (n =100). Isolates of type 2 were identified as Diplodia corticola (1). Nucleotide sequences of the ribosomal internal transcribed spacer (ITS) region and the -tubulin genes were used to confirm the identifications through BLAST searches in GenBank. Comparison of the sequences of types 1 and 2 showed 99 to 100% homology with N. mediterraneum (HM443604 (4) and GU251836) and D. corticola (AY268421 (1) and EU673117), respectively. Representative sequences of N. mediterraneum (JF949757 and JF949756) and D. corticola (JF949758 and JF949759) were deposited in GenBank. The pathogenicity of one representative isolate of each of N. mediterraneum and D. corticola was confirmed by inoculating 10 detached grapevine canes (averaging 12 mm in diameter and 30 cm long) per isolate. A shallow wound was made with a scalpel on the internodes. A colonized 6-mm agar plug, from the margin of an actively growing colony, was inserted in every wound and sealed with Parafilm. Ten grapevine canes controls received only sterile PDA agar plugs. Canes were maintained at 25°C and 70% humidity. After 5 weeks, all inoculated canes developed cankers and pycnidia around the inoculation site. Vascular necroses that developed on the inoculated canes were an average of 28.6 mm for N. mediterraneum and 27.7 mm for D. corticola. One-way analysis of variance and Tukey's test confirmed significant differences in the extent of vascular necroses. The average necroses length in the inoculated canes was significantly greater (P < 0.05) than the average length of discoloration induced by the simulated inoculation process in the control. Both pathogens were reisolated from all inoculated plants but not from controls. To our knowledge, this is the first report of N. mediterraneum and D. corticola as pathogens on grapevine in Spain. References: (1) A. Alves et al. Mycologia 96:603, 2004. (2) A. Aroca and D. Gramaje et al. Eur. J. Plant. Pathol. 126:165, 2010. (3) P. W. Crous et al. Fungal Planet. No. 19, 2007. (4) F. P. Trouillas et al. Plant. Dis. 94:1267, 2010.

First Report of Cylindrocladium buxicola on Buxus sempervirens in Spain.

Cylindrocladium buxicola Henricot, included in the EPPO alert list until November 2008, causes a dangerous foliar disease on Buxus spp. that has been recorded in several European countries and New Zealand (3,4). Buxus sempervirens L. (common boxwood) is one of the oldest ornamental garden plants in Europe. In September 2008, we received 10 2- to 3-year-old potted plants of B. sempervirens cv. Suffruticosa from a nursery in Galicia (northwest Spain) where ≈60% of the plants were affected and had finally defoliated. Diseased plants showed dark brown-to-black spots on the leaves and black streaks on the stems (3,4). To induce sporulation, diseased leaves and stem pieces were incubated in damp chambers at 22°C. A Cylindrocladium sp. was obtained. Four single conidial isolates were plated onto carnation leaf agar under near-UV light at 25°C for 7 days (2,3). Only conidiophores of the isolates growing on the surface of the carnation leaves were examined microscopically (1,3). Macroconidiophores were comprised of a stipe, a stipe extension, a terminal vesicle, and a penicillate arrangement of fertile branches (2). The stipe extension was septate, hyaline, and 90 to 165 × 2 to 4.5 µm (from the highest primary branch to the vesicle tip) (1) terminating in an ellipsoidal vesicle (6 to 11 µm in diameter) with a papillate apex. The widest part of the vesicle was above the middle. Primary branches were mainly aseptate or one septate (12 to 35 × 3 to 6 µm), secondary branches were aseptate (11 to 21 × 3 to 6 µm), and tertiary branches were rare. Each terminal branch produced two to five phialides (9 to 20 × 2.5 to 5 µm) that were reniform and aseptate. Conidia were cylindrical, straight, and one septate (56 to 75 × 4 to 6 µm). Chlamydospores were dark brown and aggregated to form microsclerotia. Cardinal temperatures of Cylindrocladium isolates on 2% malt extract agar ranged from 7 to 28°C (optimum 25°C). The 5' end of the ß-tubulin gene was amplified using primers T1 and Bt2b (3), and PCR products were sequenced directly and deposited in GenBank (Accession No. FJ696535). Comparison of the sequence with others available in GenBank showed 100% homology with those previously identified as C. buxicola (Accession Nos. AY078123 and AY078118). Pathogenicity of one representative isolate was confirmed by inoculating stems and leaves of four 3- to 4-year-old plants of B. sempervirens cv. Suffruticosa. Leaves were inoculated by spraying a spore suspension of the fungus (1 × 106 conidia per ml). For the stems, agar pieces of 1-week-old cultures grown on malt extract agar were placed and sealed with Parafilm. As a control, four plants were inoculated with agar malt plugs and sterile distilled water. Plants were incubated at 22°C and 95% humidity. Symptoms identical to ones previously described appeared 4 days after inoculation. C. buxicola was reisolated from inoculated plants but not from the controls. On the basis of morphological and physiological characteristics, pathogenicity, and the DNA sequencing of the ß-tubulin gene, the isolates obtained from B. sempervirens were identified as C. buxicola (3). To our knowledge, this is the first report of C. buxicola on B. sempervirens in Spain. References: (1) P. W. Crous. Taxonomy and Pathology of Cylindrocladium (Calonectria) and Allied Genera. The American Phytopathological Society, St. Paul, MN, 2002. (2) P. W. Crous and M. J. Wingfield. Mycotaxon 51:341, 1994. (3) B. Henricot and A. Culham. Mycologia 94:980, 2002. (4) B. Henricot et al. Plant Pathol. 49:805, 2000.

First Report of Phytophthora pseudosyringae on Chestnut Nursery Stock in Spain.

Phytophthora pseudosyringae causes stem necrosis and collar rot of deciduous tree species (Quercus spp., Fagus silvatica, and Alnus glutinosa) in several European countries (1,2). In November 2006, we received diseased Castanea sativa seedlings from a nursery in Galicia (northwest Spain). These plants had tongue-shaped necroses of the inner bark and cambium. Reddish, sunken lesions occurred on the surface of the bark, either in the stem base or higher on the stem. Tissue from the leading edge of the lesions was transferred to a selective V8 agar medium (4) and incubated for 7 days at 20°C in the dark. A Phytophthora sp. was isolated, transferred to cornmeal agar (CMA) and V8 agar, and incubated in the dark. Colonies were appressed with stellate to rosaceous growth patterns on CMA and stellate, limited aerial mycelium on V8 agar. Growth on V8 occurred from 2 to 25°C with an optimum at 20°C and a radial growth rate of 4.5 mm per day at 20°C. Chains of inflated spherical to deltoid hyphal swellings with radiating hyphae were abundantly produced in water (2). Chlamydospores were not observed on agar media. The deciduous, sympodial, semipapillate, rarely bipapillate sporangia with pedicels had a length/breadth average ratio of 1.55. Oogonia, antheridia, and oospores were produced within a single culture. Oogonia were spherical and smooth walled, antheridia were predominantly paraginous, but some were amphyginous, and oospores were plerotic that turned golden yellow with age (2). Internal transcribed spacer (ITS)-rDNA and mitochondrial DNA (mtDNA) regions were amplified by nested-PCR and sequenced with DNA extracted from mycelium. The amplicon sizes obtained were similar to those reported for P. pseudosyringae (2,3). DNA sequences showed 99 to 100% homology with those previously identified as P. pseudosyringae and deposited in GenBank. Pathogenicity of the isolate was confirmed by inoculating 10 C. sativa seedlings, as well as three detached leaves from each of another 10 young plants growing in containers. For the seedlings, one shallow cut was made into the bark on the main stem. A colonized agar plug was inserted beneath the flap that was sealed with Parafilm. Unwounded and wounded detached leaves of C. sativa were dipped into a zoospore aqueous suspension (1 × 105 zoospores ml-1) for 10 s., seedlings and leaves were incubated at 20°C and 95% humidity for 60 and 7 days, respectively. After 7 days, foliar lesions that developed exceeded 25 mm, and the pathogen was consistently reisolated. Leaves inoculated with sterile water did not develop symptoms. On inoculated seedlings, the external surface of the bark was reddish and sunken. Stem lesions progressed bidirectionally from the wound. P. pseudosyringae was recovered from inoculated seedlings but not from controls. On the basis of its unique combination of morphological and physiological characters, pathogenicity, and ITS and mtDNA sequences, the Phytophthora isolated from chestnut was identified as P. pseudosyringae. To our knowledge, this is the first report of P. pseudosyringae on C. sativa in Spain. References: (1) EPPO Reporting Service. Online publication. No. 10 2005/162, 2005. (2) T. Jung et al. Mycol. Res. 107:772, 2003. (3) F. N. Martin et al. Phytopathology 94:621, 2004. (4) C. Pintos Varela et al. Plant. Dis. 87:1396, 2003.

First Report of Phytophthora ramorum on Camellia japonica in Spain.

Phytophthora ramorum causes shoot and foliar blight on Rhododendron spp., Viburnum spp. (4), Pieris spp. (2), Kalmia latifolia, and Camellia spp. in several European countries (1-4). In December 2002, we received diseased C. japonica growing in containers from several nurseries in Galicia (northwestern Spain). These young camellia plants had leaves with brown-to-black, water-soaked lesions with diffuse borders that expanded into larger blotches resulting in dead leaves and necrotic lesions on the petioles. Eventually the entire plant wilted and died. Tissue from the leading edge of the lesions was transferred to a selective medium (V8 agar supplemented with pimaricin (10 µg/ml), rifampicin (25 µg/ml), hymexazol (5 µg/ml), and benomyl (5 µg/ml)) and incubated for 3 to 4 days at 20°C in the dark. A Phytophthora sp. was isolated, transferred to carrot piece agar (CPA) (4), and incubated in alternating light. Isolates exhibited coralloid mycelium with concentric rings and a radial growth of 2.5 to 3 mm per day at 20°C. The hyaline-to-yellowish chlamydospores were terminal and intercalary, occasionally lateral, and 24 to 74 µm in diameter. The caducous, sympodial, semipapillate sporangia had a length/breadth ratio of 1.8 to 2.1 and a short pedicel (<5 µm) or no pedicel. Oogonia, antheridia, and oospores were produced by pairing the isolates with P. cryptogea A2 tester BBA 63651 (3,4) provided by S. Werres. Oogonia were subspherical and smooth-walled, antheridia were amphyginous, and oospores were plerotic. The internal transcribed spacer-rDNA polymerase chain reaction (PCR) product obtained by using DNA extracted from mycelium and nested PCR with P. ramorum-specific primers was the size reported for P. ramorum (1). Pathogenicity of the isolates was confirmed by inoculating detached leaves of C. japonica. Five isolates were tested on leaves from 15 young plants growing in containers. Three leaves of each plant were detached and inoculated with each isolate. Leaves were dipped for 5 min into a suspension of sporangia and mycelial fragments and maintained at 20°C and 95% humidity. After 15 days, lesions that developed from the petiole base exceeded 25 mm, and the pathogen was consistently reisolated from the lesions. Leaves inoculated with water from sterile CPA plates did not develop symptoms. A C. japonica isolate has been deposited in the Spanish Type Culture Collection (CECT 20519). To our knowledge, this is the first report of P. ramorum on C. japonica in Spain, though the pathogen has been isolated from Rhododendron spp. and Viburnum tinus growing in several nurseries in Galicia. References: (1) J. M. Davidson et al. On-line publication doi:10.1094. Plant Health Progress, PHP, 2003-0707-01-DG, Plant Management Network. (2) A. J. Inman et al. First Report of Ramorum Dieback (Phytophthora ramorum) on Pieris in England. On-line publication. New Dis. Rep. Vol. 7, British Society for Plant Pathology, 2003. (3) E. Moralejo et al. Plant Dis. 86, 9:1052, 2002. (4) S. Werres et al. Mycol.Res.105:1155, 2001.