Worldwide forest surveys reveal forty-three new species in Phytophthora major Clade 2 with fundamental implications for the evolution and biogeography of the genus and global plant biosecurity.

During 25 surveys of global Phytophthora diversity, conducted between 1998 and 2020, 43 new species were detected in natural ecosystems and, occasionally, in nurseries and outplantings in Europe, Southeast and East Asia and the Americas. Based on a multigene phylogeny of nine nuclear and four mitochondrial gene regions they were assigned to five of the six known subclades, 2a-c, e and f, of Phytophthora major Clade 2 and the new subclade 2g. The evolutionary history of the Clade appears to have involved the pre-Gondwanan divergence of three extant subclades, 2c, 2e and 2f, all having disjunct natural distributions on separate continents and comprising species with a soilborne and aquatic lifestyle and, in addition, a few partially aerial species in Clade 2c; and the post-Gondwanan evolution of subclades 2a and 2g in Southeast/East Asia and 2b in South America, respectively, from their common ancestor. Species in Clade 2g are soilborne whereas Clade 2b comprises both soil-inhabiting and aerial species. Clade 2a has evolved further towards an aerial lifestyle comprising only species which are predominantly or partially airborne. Based on high nuclear heterozygosity levels ca. 38 % of the taxa in Clades 2a and 2b could be some form of hybrid, and the hybridity may be favoured by an A1/A2 breeding system and an aerial life style. Circumstantial evidence suggests the now 93 described species and informally designated taxa in Clade 2 result from both allopatric non-adaptive and sympatric adaptive radiations. They represent most morphological and physiological characters, breeding systems, lifestyles and forms of host specialism found across the Phytophthora clades as a whole, demonstrating the strong biological cohesiveness of the genus. The finding of 43 previously unknown species from a single Phytophthora clade highlight a critical lack of information on the scale of the unknown pathogen threats to forests and natural ecosystems, underlining the risk of basing plant biosecurity protocols mainly on lists of named organisms. More surveys in natural ecosystems of yet unsurveyed regions in Africa, Asia, Central and South America are needed to unveil the full diversity of the clade and the factors driving diversity, speciation and adaptation in Phytophthora. Taxonomic novelties: New species: Phytophthora amamensis T. Jung, K. Kageyama, H. Masuya & S. Uematsu, Phytophthora angustata T. Jung, L. Garcia, B. Mendieta-Araica, & Y. Balci, Phytophthora balkanensis I. Milenkovic, Z. Tomic, T. Jung & M. Horta Jung, Phytophthora borneensis T. Jung, A. Durán, M. Tarigan & M. Horta Jung, Phytophthora calidophila T. Jung, Y. Balci, L. Garcia & B. Mendieta-Araica, Phytophthora catenulata T. Jung, T.-T. Chang, N.M. Chi & M. Horta Jung, Phytophthora celeris T. Jung, L. Oliveira, M. Tarigan & I. Milenkovic, Phytophthora curvata T. Jung, A. Hieno, H. Masuya & M. Horta Jung, Phytophthora distorta T. Jung, A. Durán, E. Sanfuentes von Stowasser & M. Horta Jung, Phytophthora excentrica T. Jung, S. Uematsu, K. Kageyama & C.M. Brasier, Phytophthora falcata T. Jung, K. Kageyama, S. Uematsu & M. Horta Jung, Phytophthora fansipanensis T. Jung, N.M. Chi, T. Corcobado & C.M. Brasier, Phytophthora frigidophila T. Jung, Y. Balci, K. Broders & I. Milenkovic, Phytophthora furcata T. Jung, N.M. Chi, I. Milenkovic & M. Horta Jung, Phytophthora inclinata N.M. Chi, T. Jung, M. Horta Jung & I. Milenkovic, Phytophthora indonesiensis T. Jung, M. Tarigan, L. Oliveira & I. Milenkovic, Phytophthora japonensis T. Jung, A. Hieno, H. Masuya & J.F. Webber, Phytophthora limosa T. Corcobado, T. Majek, M. Ferreira & T. Jung, Phytophthora macroglobulosa H.-C. Zeng, H.-H. Ho, F.-C. Zheng & T. Jung, Phytophthora montana T. Jung, Y. Balci, K. Broders & M. Horta Jung, Phytophthora multipapillata T. Jung, M. Tarigan, I. Milenkovic & M. Horta Jung, Phytophthora multiplex T. Jung, Y. Balci, K. Broders & M. Horta Jung, Phytophthora nimia T. Jung, H. Masuya, A. Hieno & C.M. Brasier, Phytophthora oblonga T. Jung, S. Uematsu, K. Kageyama & C.M. Brasier, Phytophthora obovoidea T. Jung, Y. Balci, L. Garcia & B. Mendieta-Araica, Phytophthora obturata T. Jung, N.M. Chi, I. Milenkovic & M. Horta Jung, Phytophthora penetrans T. Jung, Y. Balci, K. Broders & I. Milenkovic, Phytophthora platani T. Jung, A. Pérez-Sierra, S.O. Cacciola & M. Horta Jung, Phytophthora proliferata T. Jung, N.M. Chi, I. Milenkovic & M. Horta Jung, Phytophthora pseudocapensis T. Jung, T.-T. Chang, I. Milenkovic & M. Horta Jung, Phytophthora pseudocitrophthora T. Jung, S.O. Cacciola, J. Bakonyi & M. Horta Jung, Phytophthora pseudofrigida T. Jung, A. Durán, M. Tarigan & M. Horta Jung, Phytophthora pseudoccultans T. Jung, T.-T. Chang, I. Milenkovic & M. Horta Jung, Phytophthora pyriformis T. Jung, Y. Balci, K.D. Boders & M. Horta Jung, Phytophthora sumatera T. Jung, M. Tarigan, M. Junaid & A. Durán, Phytophthora transposita T. Jung, K. Kageyama, C.M. Brasier & H. Masuya, Phytophthora vacuola T. Jung, H. Masuya, K. Kageyama & J.F. Webber, Phytophthora valdiviana T. Jung, E. Sanfuentes von Stowasser, A. Durán & M. Horta Jung, Phytophthora variepedicellata T. Jung, Y. Balci, K. Broders & I. Milenkovic, Phytophthora vietnamensis T. Jung, N.M. Chi, I. Milenkovic & M. Horta Jung, Phytophthora ×australasiatica T. Jung, N.M. Chi, M. Tarigan & M. Horta Jung, Phytophthora ×lusitanica T. Jung, M. Horta Jung, C. Maia & I. Milenkovic, Phytophthora ×taiwanensis T. Jung, T.-T. Chang, H.-S. Fu & M. Horta Jung. Citation: Jung T, Milenkovic I, Balci Y, Janousek J, Kudlácek T, Nagy ZÁ, Baharuddin B, Bakonyi J, Broders KD, Cacciola SO, Chang T-T, Chi NM, Corcobado T, Cravador A, Dordevic B, Durán A, Ferreira M, Fu C-H, Garcia L, Hieno A, Ho H-H, Hong C, Junaid M, Kageyama K, Kuswinanti T, Maia C, Májek T, Masuya H, Magnano di San Lio G, Mendieta-Araica B, Nasri N, Oliveira LSS, Pane A, Pérez-Sierra A, Rosmana A, Sanfuentes von Stowasser E, Scanu B, Singh R, Stanivukovic Z, Tarigan M, Thu PQ, Tomic Z, Tomsovský M, Uematsu S, Webber JF, Zeng H-C, Zheng F-C, Brasier CM, Horta Jung M (2024). Worldwide forest surveys reveal forty-three new species in Phytophthora major Clade 2 with fundamental implications for the evolution and biogeography of the genus and global plant biosecurity. Studies in Mycology 107: 251-388. doi: 10.3114/sim.2024.107.04.

Canker and decline diseases caused by soil- and airborne Phytophthora species in forests and woodlands.

Most members of the oomycete genus Phytophthora are primary plant pathogens. Both soil- and airborne Phytophthora species are able to survive adverse environmental conditions with enduring resting structures, mainly sexual oospores, vegetative chlamydospores and hyphal aggregations. Soilborne Phytophthora species infect fine roots and the bark of suberized roots and the collar region with motile biflagellate zoospores released from sporangia during wet soil conditions. Airborne Phytophthora species infect leaves, shoots, fruits and bark of branches and stems with caducous sporangia produced during humid conditions on infected plant tissues and dispersed by rain and wind splash. During the past six decades, the number of previously unknown Phytophthora declines and diebacks of natural and semi-natural forests and woodlands has increased exponentially, and the vast majority of them are driven by introduced invasive Phytophthora species. Nurseries in Europe, North America and Australia show high infestation rates with a wide range of mostly exotic Phytophthora species. Planting of infested nursery stock has proven to be the main pathway of Phytophthora species between and within continents. This review provides insights into the history, distribution, aetiology, symptomatology, dynamics and impact of the most important canker, decline and dieback diseases caused by soil- and airborne Phytophthora species in forests and natural ecosystems of Europe, Australia and the Americas.

Six new Phytophthora species from ITS Clade 7a including two sexually functional heterothallic hybrid species detected in natural ecosystems in Taiwan.

During a survey of Phytophthora diversity in natural ecosystems in Taiwan six new species were detected. Multigene phylogeny based on the nuclear ITS, ß-tubulin and HSP90 and the mitochondrial cox1 and NADH1 gene sequences demonstrated that they belong to ITS Clade 7a with P. europaea, P. uniformis, P. rubi and P. cambivora being their closest relatives. All six new species differed from each other and from related species by a unique combination of morphological characters, the breeding system, cardinal temperatures and growth rates. Four homothallic species, P. attenuata, P. flexuosa, P. formosa and P. intricata, were isolated from rhizosphere soil of healthy forests of Fagus hayatae, Quercus glandulifera, Q. tarokoensis, Castanopsis carlesii, Chamaecyparis formosensis and Araucaria cunninghamii. Two heterothallic species, P. xheterohybrida and P. xincrassata, were exclusively detected in three forest streams. All P. xincrassata isolates belonged to the A2 mating type while isolates of P. xheterohybrida represented both mating types with oospore abortion rates according to Mendelian ratios (4-33 %). Multiple heterozygous positions in their ITS, ß-tubulin and HSP90 gene sequences indicate that P. xheterohybrida, P. xincrassata and P. cambivora are interspecific hybrids. Consequently, P. cambivora is re-described as P. xcambivora without nomenclatural act. Pathogenicity trials on seedlings of Castanea sativa, Fagus sylvatica and Q. suber indicate that all six new species might pose a potential threat to European forests.

Nothophytophthora gen. nov., a new sister genus of Phytophthora from natural and semi-natural ecosystems.

During various surveys of Phytophthora diversity in Europe, Chile and Vietnam slow growing oomycete isolates were obtained from rhizosphere soil samples and small streams in natural and planted forest stands. Phylogenetic analyses of sequences from the nuclear ITS, LSU, ß-tubulin and HSP90 loci and the mitochondrial cox1 and NADH1 genes revealed they belong to six new species of a new genus, officially described here as Nothophytophthora gen. nov., which clustered as sister group to Phytophthora. Nothophytophthora species share numerous morphological characters with Phytophthora: persistent (all Nothophytophthora spp.) and caducous (N. caduca, N. chlamydospora, N. valdiviana, N. vietnamensis) sporangia with variable shapes, internal differentiation of zoospores and internal, nested and extended (N. caduca, N. chlamydospora) and external (all Nothophytophthora spp.) sporangial proliferation; smooth-walled oogonia with amphigynous (N. amphigynosa) and paragynous (N. amphigynosa, N. intricata, N. vietnamensis) attachment of the antheridia; chlamydospores (N. chlamydospora) and hyphal swellings. Main differing features of the new genus are the presence of a conspicuous, opaque plug inside the sporangiophore close to the base of most mature sporangia in all known Nothophytophthora species and intraspecific co-occurrence of caducity and non-papillate sporangia with internal nested and extended proliferation in several Nothophytophthora species. Comparisons of morphological structures of both genera allow hypotheses about the morphology and ecology of their common ancestor which are discussed. Production of caducous sporangia by N. caduca, N. chlamydospora and N. valdiviana from Valdivian rainforests and N. vietnamensis from a mountain forest in Vietnam suggests a partially aerial lifestyle as adaptation to these humid habitats. Presence of tree dieback in all forests from which Nothophytophthora spp. were recovered and partial sporangial caducity of several Nothophytophthora species indicate a pathogenic rather than a saprophytic lifestyle. Isolation tests from symptomatic plant tissues in these forests and pathogenicity tests are urgently required to clarify the lifestyle of the six Nothophytophthora species.

Fungal Planet description sheets: 214-280.

Novel species of microfungi described in the present study include the following from South Africa: Cercosporella dolichandrae from Dolichandra unguiscati, Seiridium podocarpi from Podocarpus latifolius, Pseudocercospora parapseudarthriae from Pseudarthria hookeri, Neodevriesia coryneliae from Corynelia uberata on leaves of Afrocarpus falcatus, Ramichloridium eucleae from Euclea undulata and Stachybotrys aloeticola from Aloe sp. (South Africa), as novel member of the Stachybotriaceae fam. nov. Several species were also described from Zambia, and these include Chaetomella zambiensis on unknown Fabaceae, Schizoparme pseudogranati from Terminalia stuhlmannii, Diaporthe isoberliniae from Isoberlinia angolensis, Peyronellaea combreti from Combretum mossambiciensis, Zasmidium rothmanniae and Phaeococcomyces rothmanniae from Rothmannia engleriana, Diaporthe vangueriae from Vangueria infausta and Diaporthe parapterocarpi from Pterocarpus brenanii. Novel species from the Netherlands include: Stagonospora trichophoricola, Keissleriella trichophoricola and Dinemasporium trichophoricola from Trichophorum cespitosum, Phaeosphaeria poae, Keissleriella poagena, Phaeosphaeria poagena, Parastagonospora poagena and Pyrenochaetopsis poae from Poa sp., Septoriella oudemansii from Phragmites australis and Dendryphion europaeum from Hedera helix (Germany) and Heracleum sphondylium (the Netherlands). Novel species from Australia include: Anungitea eucalyptorum from Eucalyptus leaf litter, Beltraniopsis neolitseae and Acrodontium neolitseae from Neolitsea australiensis, Beltraniella endiandrae from Endiandra introrsa, Phaeophleospora parsoniae from Parsonia straminea, Penicillifer martinii from Cynodon dactylon, Ochroconis macrozamiae from Macrozamia leaf litter, Triposporium cycadicola, Circinotrichum cycadis, Cladosporium cycadicola and Acrocalymma cycadis from Cycas spp. Furthermore, Vermiculariopsiella dichapetali is described from Dichapetalum rhodesicum (Botswana), Ophiognomonia acadiensis from Picea rubens (Canada), Setophoma vernoniae from Vernonia polyanthes and Penicillium restingae from soil (Brazil), Pseudolachnella guaviyunis from Myrcianthes pungens (Uruguay) and Pseudocercospora neriicola from Nerium oleander (Italy). Novelties from Spain include: Dendryphiella eucalyptorum from Eucalyptus globulus, Conioscypha minutispora from dead wood, Diplogelasinospora moalensis and Pseudoneurospora canariensis from soil and Inocybe lanatopurpurea from reforested woodland of Pinus spp. Novelties from France include: Kellermania triseptata from Agave angustifolia, Zetiasplozna acaciae from Acacia melanoxylon, Pyrenochaeta pinicola from Pinus sp. and Pseudonectria rusci from Ruscus aculeatus. New species from China include: Dematiocladium celtidicola from Celtis bungeana, Beltrania pseudorhombica, Chaetopsina beijingensis and Toxicocladosporium pini from Pinus spp. and Setophaeosphaeria badalingensis from Hemerocallis fulva. Novel genera of Ascomycetes include Alfaria from Cyperus esculentus (Spain), Rinaldiella from a contaminated human lesion (Georgia), Hyalocladosporiella from Tectona grandis (Brazil), Pseudoacremonium from Saccharum spontaneum and Melnikomyces from leaf litter (Vietnam), Annellosympodiella from Juniperus procera (Ethiopia), Neoceratosperma from Eucalyptus leaves (Thailand), Ramopenidiella from Cycas calcicola (Australia), Cephalotrichiella from air in the Netherlands, Neocamarosporium from Mesembryanthemum sp. and Acervuloseptoria from Ziziphus mucronata (South Africa) and Setophaeosphaeria from Hemerocallis fulva (China). Several novel combinations are also introduced, namely for Phaeosphaeria setosa as Setophaeosphaeria setosa, Phoma heteroderae as Peyronellaea heteroderae and Phyllosticta maydis as Peyronellaea maydis. Morphological and culture characteristics along with ITS DNA barcodes are provided for all taxa.

First Report of Alternaria Black Spot of Pomegranate Caused by Alternaria alternata in Spain.

Since 2010, a new foliar and fruit disease was observed in pomegranate (Punica granatum L.) orchards in Alicante Province (eastern Spain). Symptoms included black spots on leaves and fruits, as well as chlorosis and premature abscission of leaves. Fungal isolates were obtained by surface-disinfecting small fragments of symptomatic leaf and fruit tissues in 0.5% NaOCl, double-rinsing in sterile water, and plating them onto potato dextrose agar (PDA) amended with 0.5 g/liter of streptomycin sulfate. Gray-to-black colonies were obtained, which were identified as Alternaria sp. based on the dark, brown, obclavate to obpyriform catenulate conidia with longitudinal and transverse septa tapering to a prominent beak attached in chains on a simple and short conidiophore (4). Conidia (n = 100) measured (12.2-) 20.2 (-27.6) × (5.7-) 9.2 (-12.0) µm, and had 3 to 6 transverse and 0 to 5 longitudinal septa. Single spore cultures were obtained and their genomic DNA was extracted. The internal transcribed spacer (ITS) region of rDNA and partial sequences of the beta tubulin gene were amplified and sequenced with primers ITS1-ITS4 and Bt1a-Bt1b, respectively (3). BLAST analysis of the sequences showed that they were 100% identical to a pathogenic A. alternata (Fr.) Keissl. isolate obtained from black spot disease of pomegranate in Israel (Accession No. JN247826.1, ITS and Accession No. JN247836.1, beta tubulin) (2). As all the sequences obtained showed 100% homology, ITS and beta tubulin sequences of a representative isolate (1516B) were submitted to GenBank (KF199871 and KF199872, respectively). In addition, a PCR reaction with specific primers (C_for/C_rev) designed to recognize highly virulent isolates of A. alternata causing black spot of pomegranate was used with all isolates (2). A characteristic fragment of ~950 bp was amplified in two isolates: 1552B and 1707B. Pathogenicity was assessed on plants and detached fruit of pomegranate cv. Mollar (1). Two-year-old pomegranate trees were inoculated with isolates 1552B and 1707B by spraying a conidial suspension (106 conidia/ml) onto the upper and lower leaf surfaces. Five plants per fungal isolate were used and five control plants were sprayed with sterile water. Plants were covered with transparent plastic bags and incubated in a growth chamber for 1 month at 25°C, with a 12-h photoperiod. One-month-old fruits were surface sterilized in 1.5% sodium hypochlorite solution for 1 min and rinsed twice in water. Two filter paper squares (5 × 5 mm) were dipped in the conidial suspensions and placed on the fruit surface. Inoculated fruit were incubated in a humid chamber in the dark at 25°C. Ten fruit per fungal isolate were used and 10 control fruit were inoculated with sterile water. Black spots were visible on inoculated leaves and fruit, 10 and 3 days after inoculation, respectively. Symptoms were not observed on controls. The fungus was re-isolated from leaf and fruit lesions, confirming Koch's postulates. Leaf black spot of pomegranate caused by A. alternata was first described in India in 1988, and later in Israel in 2010 affecting both fruit and leaves (1). To our knowledge, this is the first report of the disease in Spain, where it could represent a threat for pomegranate cultivation due to the increasing amount of area dedicated to this crop. References: (1) D. Ezra et al. Australas. Plant Dis. Notes 5:1, 2010. (2) T. Gat et al. Plant Dis. 96:1513, 2012. (3) N. L. Glass and G. C. Donaldson. Appl. Environ. Microbiol. 61:1323, 1995. (4) E. G. Simmons. Alternaria: An identification manual. CBS Fungal Biodiversity Center, Utrecht, Netherlands, 2007.

Evidence for multiple introductions and clonality in Spanish populations of Fusarium circinatum.

Fusarium circinatum is thought to have been moved around the world with pine planting stock consisting, most probably, of infected seed. In this effort, we investigate the genetic structure of F. circinatum in Spain and globally. In total, 223 isolates were studied from five regions in northern Spain and eight countries. Eight microsatellite markers revealed 66 multilocus genotypes (MLGs). Minimum spanning network analysis of MLGs by region within Spain as well as globally, discriminant analysis of principal components, and analysis of molecular variance revealed that Spanish populations are significantly differentiated and structured into two distinct groups, each one including one of the dominant genotypes observed. This result suggests that two independent introductions occurred into Spain that subsequently underwent clonal divergence and admixture. This result is further supported by the linkage disequilibrium and clonality observed for F. circinatum populations in northern Spain. The maintenance of differentiation between the clusters could result from the lack of or rare sexual reproduction in Spain. Possible introduction pathways from other countries and subsequent routes of dispersion of F. circinatum in Spain are discussed.

Fungal trunk pathogens associated with wood decay of almond trees on Mallorca (Spain).

Severe decline of almond trees has recently been observed in several orchards on the island of Mallorca (Balearic Islands, western Mediterranean Sea). However, the identity of the causal agents has not yet been investigated. Between August 2008 and June 2010, wood samples from branches of almond trees showing internal necroses and brown to black vascular streaking were collected in the Llevant region on the island of Mallorca. Several fungal species were subsequently isolated from the margin between healthy and symptomatic tissue. Five species of Botryosphaeriaceae (namely Botryosphaeria dothidea, Diplodia olivarum, D. seriata, Neofusicoccum australe and N. parvum), Eutypa lata, Phaeoacremonium iranianum and Phomopsis amygdali were identified based on morphology, culture characteristics and DNA sequence comparisons. Neofusicoccum parvum was the dominant species, followed by E. lata, D. olivarum and N. australe. First reports from almond include D. olivarum and Pm. iranianum. Two species are newly described, namely Collophora hispanica sp. nov. and Phaeoacremonium amygdalinum sp. nov.

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 Sirosporium celtidis Causing a Foliar Disease of European Hackberry in Spain.

Hackberry (Celtis australis L.) is widely used for reforestation and as shade tree in parks and roadside plantings in southern Europe (4). In autumn 2011, a foliar disease was observed affecting several trees planted in a garden area located in Alzira (Valencia province, eastern Spain). Symptoms appeared on lower leaf surfaces as reddish to dark brown velvety irregular spots, later becoming grayish brown on the upper surface. Most of the infected trees were prematurely defoliated. Spots on lower leaf surfaces were covered by mycelium, conidiophores, and conidia. Fungal isolates were recovered directly from the structures present on the lesions and by surface-disinfecting small fragments of symptomatic leaf tissue in 0.5% NaOCl, double-rinsing the sections in sterile water, and plating the sections onto potato dextrose agar (PDA) amended with 0.5 g of streptomycin sulfate per liter. Single conidium cultures made onto PDA were maintained for 2 months at 25°C in darkness for morphological examination. Conidia were thick walled, dark reddish brown, often markedly curved or coiled, cylindrical to obclavate, smooth, wrinkled, or verrucose, typically multicellular, 2 to 40 transversely septate and occasionally with 1 to 3 longitudinal or oblique septa that were often constricted, 20 to 96 (44.9) × 6 to 9 (7.1) µm, with an inconspicuous scar at the base. Morphological characters corresponded to the description of Sirosporium celtidis (Biv. ex Spreng) M. B. Ellis published in 1963 (3). The internal transcribed spacer (ITS) region of the rDNA was amplified with the primers ITS1 and ITS4 from DNA extracted from the isolate AL1, and sequenced (GenBank Accession No. JX397963). The sequence was identical to that obtained from an isolate of S. celtidis from the Centraalbureau voor Schimmelcultures, Utrecht, The Netherlands (CBS 289.50). Pathogenicity tests were conducted on five 2-year-old hackberry trees by spraying onto the upper and lower leaf surfaces a conidial suspension of S. celtidis (approximately 50 ml/plant, 106 conidia/ml of water). Five control plants were sprayed with sterile water. Plants were covered with clear plastic bags and incubated in a growth chamber for 72 h at 25°C with a 12-h photoperiod. First leaf spots were visible on inoculated plants after 7 days, but symptoms were not observed on control plants. The fungus was reisolated from leaf lesions on inoculated plants, confirming Koch's postulates. S. celtidis was first described in Sicily in 1815 (3) and has been recorded on various hackberry species in Mediterranean countries and the USA (1,2). To our knowledge, this is the first report of the disease in Spain. The economic and ecological significance of the pathogen in natural ecosystems in Spain remains to be determined but it could certainly become a serious problem for nurseries and urban plantings. References: (1) S.O. Cacciola. 2000. Plant Dis. 84, 492. (2) D. H. Linder. 1931. Ann. Mo. Bot. Garden 18, 31. (3) M. B. Ellis. 1963. Mycological Papers, No. 87. Commonw. Mycol. Inst. Kew, England. (4) S. Pauleit et al., Urban For. Urban Green. 1:83, 2002.

First Report of Damping-Off Caused by Cylindrocarpon pauciseptatum on Pinus radiata in Spain.

In the fall of 2009, damping-off of Pinus radiata seedlings was observed in a pine nursery in Sant Feliu de Buixalleu, Girona Province, northeastern Spain. Plants exhibited needle blight, extensive root necrosis, and root death. Root sections of symptomatic plants were cut, washed under running tap water, surface disinfected for 1 min in a 1.5% sodium hypochlorite solution, and washed twice with sterile distilled water. Small fragments of discolored tissues were plated onto potato dextrose agar (PDA) supplemented with 0.5 g liter-1 of streptomycin sulfate. Plates were placed at 25°C in the dark for 10 to 14 days, and all fungal colonies were transferred to PDA. A Cylindrocarpon sp. was consistently isolated from necrotic root tissues. Single-conidial isolates were obtained and grown on PDA and Spezieller Nährstoffarmer agar (SNA) (2) at 25°C for 10 days with a 12-h photoperiod. On PDA, the isolates developed abundant mycelium, which varied from white-to-grayish brown or golden brown. On SNA, all isolates produced two-septate, (35-) 39.4 (-40) × (7.5-) 7.7 (-8.75) µm, and three-septate, (32.5-) 40.9 (-52.5) × (7.5-) 7.7 (-8.75) µm, macroconidia. Microconidia, one-septate macroconidia, and chlamydospores were not observed. Identity of these isolates was determined by a multiplex PCR system using a set of three pair of specific primers (Mac1/MaPa2, Lir1/Lir2, and Pau1/MaPa2) (1), which generated a 117-bp product that was characteristic of Cylindrocarpon pauciseptatum Schroers & Crous. Morphological characteristics also supported this identification (4). Internal transcribed spacers regions (ITS1 and ITS4) of rDNA were obtained for isolate 1052 and deposited in GenBank (Accession No. HQ441248). This sequence was identical (100%) with the sequence of C. pauciseptatum (GenBank Accession No. HM036590). Pathogenicity tests were conducted with inoculum produced on wheat kernels that were soaked in distilled water in flasks for 12 h. Each flask contained 200 ml of kernels that were subsequently autoclaved three times after excess water was drained. Two fungal disks from a 2-week-old culture of C. pauciseptatum (isolate 1052) grown on PDA were placed aseptically in each flask. Cultures in flasks were incubated at 25°C for 4 weeks and shaken once a week. A plastic pot (220 cm3) was filled with a mixture of sterilized peat moss and 10 g of inoculum. A 1-month-old seedling of P. radiata was planted in plastic pots and placed in a greenhouse at 25 to 30°C in a completely randomized design with six replications. Controls contained sterile wheat kernels. The experiment was repeated. Symptoms developed 20 days after inoculation and consisted of root lesions, a reduction in root biomass, needle blight, and the death of all seedlings. The fungus was reisolated from affected seedlings. Damping-off was not observed on the control plants. C. pauciseptatum causing black foot disease of grapevine (3) was first found in Spain in 2008, but to our knowledge, this is the first report of C. pauciseptatum causing damping-off of P. radiata in Spain. References: (1) S. Alaniz et al. Plant Dis. 93:821, 2009. (2) J. F. Leslie and B. A. Summerell. The Fusarium Laboratory Manual. Blackwell Publishing, Oxford, 2006. (3) M. T. Martin et al. Plant Dis. 95:361, 2011. (4) H. J. Schroers et al. Mycol. Res. 112:82, 2008.

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.

First Report of Circular Leaf Spot of Persimmon Caused by Mycosphaerella nawae in Spain.

Production of persimmon (Diospyros kaki L. f.) has increased significantly during the last decade in Spain as a profitable alternative for fruit growers. In August 2008, after a mild and rainy spring, symptoms of a new disease were observed in commercial persimmon fields located in Valencia Province (eastern-central Spain). Symptoms included circular necrotic spots on the leaves and defoliation. Early fruit maturation and premature abscission were associated with early symptom development in the trees. A fungus was consistently isolated from the margins of leaf lesions. All isolates obtained were hyphal-tipped twice and transferred to potato dextrose agar (PDA). The cultures grew slowly and reached a diameter of 21 to 29 (mean 26) mm within 4 weeks on PDA at 25°C in the dark. Mycelium was initially dark green and ultimately became dark gray to black. Several media and incubation conditions were tested to induce sporulation, but conidia formation was not observed. In April 2009, mature spherical pseudothecia were observed in lesions on fallen leaves that had remained in affected fields during the winter. Ascospores were uniseptate and mostly spindle shaped, 10 to 11.5 (mean 10.3) µm long, and 3 to 3.9 (mean 3.4) µm wide. Fungal colonies obtained from the ascospores were identical to those isolated from the leaf lesions. Morphological characters observed matched those described for the pathogen Mycosphaerella nawae Hiura & Ikata (1). In Korea, the circular leaf spot of persimmon caused by M. nawae was considered an economically important disease in the 1990s, especially in the southern regions (2). Sequences of the internal transcribed spacer (ITS) region of the rDNA were obtained for isolates MY2 and MY3 and deposited in GenBank (Accession Nos. GQ465767 and GQ465768). These sequences were identical to each other and to the sequence obtained from a Korean isolate of M. nawae. Symptoms of the disease were reproduced after inoculation of 2-year-old persimmon trees growing in individual pots. A ground mycelial suspension (5 × 105 CFU ml-1) of strain MY2 was sprayed onto 20 potted trees (200 ml per individual tree) in late May of 2009. Ten trees were sprayed with sterile distilled water as a control. Trees were incubated at 20°C in a growth chamber with a 12-h photoperiod and covered with a semitransparent plastic hood for the first 10 days after inoculation, after which the plastic was punctured for ventilation and trees were incubated at 22°C. The first symptoms (small circular spots on the leaves) appeared on inoculated trees 15 days after inoculation. One month after inoculation, all inoculated trees showed circular leaf spots and severe defoliation, whereas noninoculated trees remained healthy. M. nawae was successfully reisolated from the lesions. To our knowledge, this is the first report of M. nawae causing circular leaf spot of persimmon in Spain. References: (1) J. H. Kwon et al. Plant Dis. Agric. 1:18, 1995. (2) J. H. Kwon et al. Korean J. Plant Pathol. 14:397, 1998.

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.

First Report of Canker Disease Caused by Neofusicoccum australe on Eucalyptus and Pistachio in Spain.

In 2005 and 2006, dieback and branch cankers were observed in 12-year-old Eucalyptus globulus Labill. plantations in Gijón (northern Spain) and a 20-year-old pistachio (Pistacia vera L.) plantation in Constantí (northeastern Spain). Isolations were made from symptomatic branches. Small pieces of necrotic tissues were surface sterilized for 1 min in 1.5% NaOCl and plated onto malt extract agar amended with 0.5 g L-1 streptomycin sulfate. Plates were incubated at 25°C in the dark and all growing colonies were transferred to potato dextrose agar (PDA). A Neofusicoccum sp. was consistently isolated from necrotic tissues of both host species. On PDA at 25°C, isolates developed a moderately dense mycelium, initially with a pale yellow pigment diffusing into the medium but becoming olivaceous gray after 5 to 6 days. Pycnidia were produced on sterile eucalyptus and pistachio twigs placed on the surface of water agar after 1 month. Conidia were hyaline, fusiform, aseptate, with granular contents. Conidia from eucalyptus isolates measured (22.5-) 25.4 (-28.1) × (5-) 6.2 (-7.5) µm, (n = 40) and (20.0-) 23.6 (-28.0) × (6.5-) 7.1 (-8.0) µm, (n = 40) from pistachio isolates. Isolates were identified as Neofusicoccum australe (Slippers, Crous & M.J. Wingf.) Crous, Slippers & A.J.L. Phillips (1,2). DNA sequences of the rDNA internal transcribed spacer region (ITS), part of the beta-tubulin (BT2), and part of the translation elongation factor 1-alpha (EF1-α) genes from isolates CBS 122027 (pistachio) and CBS 122026 and CBS 122025 (eucalyptus) were used to confirm the identifications through BLAST searches in GenBank. Representative sequences of all studied regions were deposited in GenBank (ITS: EU375516 and EU375517; BT2: EU375520; EF1-α: EU375518 and EU375519). Pathogenicity tests were conducted on 8-month-old eucalyptus seedlings and 2-year-old pistachio plants with the three N. australe strains mentioned above. A mycelial plug taken from the margin of an actively growing colony of each isolate was put in a shallow wound (0.4 cm2) made with a scalpel on the stem of each plant. Inoculation wounds were wrapped with Parafilm. Controls were inoculated with sterile PDA plugs. Ten replicates for each isolate and plant species were used, with an equal number of control plants. Plants were maintained in a greenhouse at 25°C. After 3 weeks, all eucalyptus seedlings showed leaf wilting, stem canker, and pycnidia formation around the inoculation site. No foliar symptoms were observed in pistachio plants after 3 months, but depressed cankers variable in size and pycnidia formation developed around the inoculation site. Vascular necroses that developed on the inoculated plants were 10.2 ± 1.2 cm long in eucalyptus and 6.4 ± 1.6 cm long in pistachio, significantly greater than their respective controls (P < 0.01). There were no significant differences in necrosis lengths among the three N. australe isolate inoculations, irrespective of the inoculated host. These results point to a high susceptibility of eucalyptus to N. australe. No symptoms were visible in the control seedlings and no fungus was isolated from them. The pathogen was reisolated from all inoculated plants. To our knowledge, this is the first report of N. australe causing canker disease on eucalyptus and pistachio trees in Spain. References: (1) P. Crous et al. Stud. Mycol. 55:235, 2006. (2) B. Slippers et al. Mycologia 96:1030, 2004.

First Report of Leaf Spot and Twig Blight of Rhododendron spp. Caused by Phytophthora hibernalis in Spain.

During the early spring of 2004, an estimated 20% of containerized nursery stocks of Rhododendron spp. in Asturias (northern Spain) were affected by a foliar disease that has reoccurred annually. Leaf spots were dark brown to almost black, generally oval to round, visible from both sides of the leaf, and expanded to affect the entire leaf including the petiole. Affected leaves abscised from the plant. A Phytophthora sp. was consistently isolated from symptomatic leaf tissues on PARBH medium (3) and hyphal tips were transferred onto potato dextrose agar (PDA). Colonies grown on PDA at 20°C were submerged, had a growth rate of 2.2 mm/day, and had lobes of compact mycelium. Sporangia were semipapillate and caducous with a pedicel (20.0-) 37.7 (-52.5) µm long. Sporangia were asymmetrical in shape with the broadest point near the apex: 25.2 to 40.4 µm long × 10.2 to 15.8 µm wide (average 33.1 × 12.6 µm), and length/width ratio was 2.8:1. Chlamydospores were not observed. Isolates were homothallic and oogonia ranged from 26.5 to 27.5 µm in diameter. Antheridia were mostly amphigynous but occasionally paragynous. Oospores were plerotic and 23.1 to 25.5 µm in diameter. These characteristics conformed to those of Phytophthora hibernalis Carne (2). Sequences of the internal transcribed spacer regions on the isolates and comparison with other sequences in GenBank showed that they were identical to P. hibernalis (Accession No. AY827556.1 from Citrus sp.). For pathogenicity tests, four isolates of P. hibernalis were used to inoculate detached leaves of Rhododendron hybrid Brigitte. The underside of five detached leaves was inoculated with a drop of 40 µL of a suspension of 104 zoospores/ml. Controls were inoculated with a 40-µL drop of sterile distilled water. Leaves were incubated in a moist chamber at 20°C in the dark. A quantification of the lesion area was made 8 days after inoculation using the software Assess-APS. All inoculated leaves developed necrotic lesions that ranged from 0.246 to 1.512 cm2. P. hibernalis was reisolated from infected tissue. Symptoms were not detected on the controls. The test was repeated twice and similar results were obtained each time. P. hibernalis has been described previously as causing brown rot on citrus in Spain (4) and was isolated from rhododendron plants in California and Oregon (1). To our knowledge, this is the first record of P. hibernalis causing foliar blight on Rhododendron species in Spain as well as in Europe. References: (1) C. Blomquist et al. Online publication. doi:10.1094/PHP-2005-0728- 01-HN. Plant Health Progress, 2005. (2) D. C. Erwin and O. K. Ribeiro. Phytophthora Diseases Worldwide. The American Phytopathological Society, St. Paul MN. 1996. (3) S. N. Jeffers and S. B. Martin. Plant Dis. 70:1038, 1986. (4) J. J. Tuset. An. Inst. Nac. Investig. Agrar. Ser. Prot. Veg. N.7, 1977.

First Report of Leaf Spot, Blight, and Stem Lesions Caused by Cylindrocladium pauciramosum on Callistemon in Spain.

Callistemons (Callistemon citrinus (Curtis) Skeels), evergreen plants of the family Myrtaceae, are commonly grown in Mediterranean gardens for their red bottlebrush-like flowers. During November of 2006, 1-year-old potted plants of callistemon showed leaf spots and blight in commercial nurseries in Valencia, Spain. Symptoms consisted initially of minute brown spots on the leaves, developing into black-gray blotches that finally coalesced. Diseased plants also showed stem lesions and blight of young shoots. Approximately 30% of the plants were affected. A Cylindrocladium sp. was isolated consistently from the infected tissues. Six single conidial isolates were grown on carnation leaf agar (CLA) under near-UV light at 25°C for 7 days (1). The macroconidiophores comprised of a stipe, a sterile elongation, and a penicillated arrangement of primary, secondary, and tertiary branches. The stipes were septate, 110 to 175 (138) µm long, with a terminal obpyriform vesicle measuring 3.75 to 7.5 (5.8) µm wide. Phialides (12.5 × 3.6 µm) were hyaline, doliiform to reniform, with conidia 40 to 55 × 3.7 to 5 µm, cylindrical with rounded ends, aseptate or one septate. Chlamydospores were brown and formed microsclerotia. These features conformed to the description of Cylindrocladium pauciramosum (3). Further confirmation was obtained by sequence analysis. The 5' end of the ß-tubulin gene was amplified using primers T1 and ßt2b (2). Comparison with other sequences in GenBank revealed that the isolates described here were identical with C. pauciramosum (Accession No. AY880064) isolated from Ceanothus in the UK. To confirm pathogenicity, 1-year-old plants of callistemon were inoculated with two isolates by spraying with a spore suspension of the fungus (1 × 105 conidia per ml) obtained from 14-day-old single spore colonies on CLA. Control plants were treated with sterile distilled water. After inoculation, all plants were maintained in plastic bags and kept at 22 ± 2°C. Four days after inoculation, the plants developed symptoms similar to those observed in natural infections, and C. pauciramosum was reisolated, successfully completing Koch's postulates. No symptoms were observed on the control plants. C. pauciramosum has been recorded on several hosts, including Callistemon citrinus, in Italy (4). To our knowledge, this is the first report of C. pauciramosum on callistemon in Spain. References: (1) P. W. Crous and M. J. Wingfield. Mycotaxon 51:341, 1994. (2) B. Henricot and A. Culham. Mycologia 94:980, 2002. (3) C. L. Schoch et al. Mycologia 91:286, 1999. (4) C. L. Schoch et al. Plant Dis. 85:941, 2001.

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.

Bleeding Canker on Mesquite in Peru caused by Phytophthora syringae.

Mesquite (Prosopis pallida (Wildenow) Kunth) is a drought-tolerant tree widely distributed in the northern Pacific Coast of South America. This species prevents soil erosion, provides shade, conserves prairies, supports bee nutrition, and provides fruits for human and animal consumption. Since the spring of 2004, bark lesions and bleeding cankers were observed on trunks and branches of 70% of declining mesquite trees in some parks at Ica in southern Peru. Badly affected trees were killed by the disease. Isolations were made from the edge of necrotic lesions of the inner bark and roots using PARPH medium (2) and incubated at 22°C for 7 days. A Phytophthora species was consistently isolated from lesions of 10 mesquite trees, and six pure cultures (PS-87-PS-92) were obtained by transferring hyphal tips and characterized. Colonies were stellate on V8 juice agar (VJA; 2 g CaCO3, 200 ml of V8 juice, and 15 g of agar in 800 ml of distilled water), uniform to slightly radiate on corn meal agar (Oxoid Ltd., London, England), and knotty on PDA (Biokar Diagnostics, Beauvais, France). On VJA at 22°C, the average radial growth rate for the six isolates was 1.7 mm per day. Colonies grew slowly at 5 and 25°C with 0.4 and 0.7 mm per day growth rate, respectively. There was no growth at 30°C. Catenulate hyphal swellings formed on VJA and liquid media (1.5% sterile soil extract). Sporangia were persistent, ovoid to obpyriform, semipapillate with narrow exit pores (<5.0 µm in diameter), 32.3 to 39.7 × 21.0 to 27.2 µm, with a length/width ratio of 1.4:1 to 1.6:1. Sporangia were produced by cutting 5-mm disks from the advancing margin of a colony on VJA and adding disks to 10 ml of 1.5% sterile soil extract for 4 to 5 days at 22°C under fluorescent light. Isolates were homothallic with spherical oogonia, 32 to 35 µm in width with paragynous antheridia, and aplerotic oospores, 26 to 31 µm. These characteristics fit the descriptions of Phytophthora syringae (Kleb.) Kleb. (1). Sequences of the internal transcribed spacer regions on the isolates and comparison with other sequences in GenBank showed that they were identical to P. syringae (Accession No. AJ854297 from Citrus limon). In 2005, two methods were used to inoculate mesquite with two isolates. One method used two 20-mm-diameter branches of five 5-year-old mesquite trees where a 5-mm wound was made with a cork borer and a 5-mm block of the agar culture was placed under the bark and sealed with Parafilm. Another method used 10 4-month-old potted plants that received a 30-ml drench of a 104 zoospores/ml suspension per plant. Controls received clean agar blocks and a sterile water drench for 10 control pots. Two weeks after inoculation, black areas and resinosis were observed around inoculated wounds. Inoculated branches produced cankers of 4.7 to 6.8 cm2, 4 weeks after inoculations. Twenty days after inoculation of roots, wilting and root rots of seedlings occurred. No symptoms were found on the control plants. P. syringae was reisolated from the diseased branches and root rots and pure cultures were established. This test was repeated for both methods with similar results. To our knowledge, this is the first report of P. syringae in Peru and the first description of this pathogen on mesquite worldwide. References: (1) D. C. Erwin and O. K. Ribeiro. Phytophthora Diseases Worldwide. The American Phytopathological Society, St. Paul MN. 1996. (2) S. N. Jeffers and S. B. Martin. Plant Dis. 70:1038, 1986.