SummaryAn analysis of incidence of Phytophthora spp. in 732 European nurseries producing forest transplants, larger specimen trees, landscape plants and ornamentals, plus 2525 areas in which trees and shrubs were planted, is presented based on work conducted by 38 research groups in 23 European countries between 1972 and 2013. Forty-nine Phytophthora taxa were recorded in 670 nurseries (91.5%); within these nurseries, 1614 of 1992 nursery stands (81.0%) were infested, although most affected plants appeared healthy. In forest and landscape plantings, 56 Phytophthora taxa were recovered from 1667 of 2525 tested sites (66.0%). Affected plants frequently showed symptoms such as crown thinning, chlorosis and dieback caused by extensive fine root losses and/or collar rot. Many well-known highly damaging host-Phytophthora combinations were frequently detected but 297 and 407 new Phytophthora-host associations were also observed in nurseries and plantings, respectively. On average, 1.3 Phytophthora species/taxa per infested nursery stand and planting site were isolated. At least 47 of the 68 Phytophthora species/taxa detected in nurseries and plantings were exotic species several of which are considered well established in both nurseries and plantings in Europe. Seven known Phytophthora species/taxa were found for the first For. Path. 46 (2016) 134-163 doi: 10.1111/efp.12239 © 2015 http://wileyonlinelibrary.com/ time in Europe, while 10 taxa had not been previously recorded from nurseries or plantings; in addition, 5 taxa were first detections on woody plant species. Seven Phytophthora taxa were previously unknown to science. The reasons for these failures of plant biosecurity in Europe, implications for forest and semi-natural ecosystems and possible ways to improve biosecurity are discussed.

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.

The influences of long-term flooding and Phytophthora alni subsp. alni infection on the growth and development of 4-year-old Alnus glutinosa (black alder) saplings were investigated. The black alder saplings were divided into four groups and then subjected to combinations of both factors-flooded and inoculated with pathogen, flooded non-inoculated, non-flooded inoculated, and control. The biomass of the living roots and actinorrhizae, increase in stem length, length of leaves, rate of chlorotic foliage, amount of foliage biomass and length of stem necrosis were assessed after seven weeks. Both factors, flooding and P. alni infection significantly affected the black alder. In addition, a significant effect of interaction was observed. The inoculated flooded group had a substantially lower biomass weight of living roots, actinorrhiza and leaves than the other groups. The necroses caused by the pathogen in the flooded group were more extensive than those in the non-flooded one. These findings demonstrate that the simultaneous incidence of stress caused by flooding and P. alni infection is highly dangerous for black alder.

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 β-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 β-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.

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.