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.

First Report of Calosphaeria pulchella Causing Canker and Branch Dieback of Sweet Cherry Trees in Spain.

In autumn 2012, severe branch cankers and diebacks of sweet cherry trees (Prunus avium L.) were observed in orchards located in two different growing areas in Alicante Province (eastern Spain). In affected trees, leaves become dried without defoliation. Sectorial wood necrosis was also observed, occasionally associated with swollen bark and gum exudates. Isolations were made from diseased branches by surface-disinfecting small fragments of symptomatic tissue in 0.5% NaOCl, double-rinsing in sterile water, and plating them onto potato dextrose agar (PDA) amended 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. Pink to red colonies with white margins were consistently isolated. All isolates produced hyaline, allantoid to oblong-ellipsoidal conidia, 4 to 6 × 1.5 to 2 µm. The fungus was identified as Calosphaeria pulchella (Pers.: Fr.) J. Schröt (anamorph Calosphaeriophora pulchella Réblová, L. Mostert, W. Gams & Crous) based on morphology (1). Identification of C. pulchella isolates was confirmed by sequence comparison in GenBank database using the internal transcribed spacer region (ITS1-5.8S-ITS2) of the rDNA. Sequences showed 100% identity and 100% query coverage with C. pulchella reference isolate CBS 115999 (EU367451) (2). The ITS sequence of one of the isolates obtained in this study was deposited into GenBank (KJ396346). Two-year-old sweet cherry trees cv. Burlat were inoculated with two representative C. pulchella isolates from different orchards (1701 and 1702). A 5-mm cork borer was used to remove bark, and an agar plug from the growing margin of 20-day-old colonies was placed directly into the fresh wound, mycelium side down. Five trees were inoculated per isolate (five branches per tree) and 25 control branches were inoculated with non-colonized agar plugs. Inoculated tissue was covered with Vaseline and Parafilm to avoid the loss of water. Branches were taken to the laboratory 9 months after inoculation and thoroughly examined for canker development. The length of vascular discoloration was evaluated in each branch and resulting data were statistically analyzed. Length of vascular discoloration on the inoculated branches (6.6 ± 0.7) was significantly longer than in control plants (2.3 ± 0.3) at P < 0.001. Perithecia were neither observed on the artificially inoculated branches nor in the diseased sweet cherry trees from the sampled orchards. C. pulchella was re-isolated from the inoculated branches and no fungi were isolated from discolored tissue of the controls, confirming Koch's postulates. Canker of sweet cherry caused by C. pulchella is responsible for reducing yields and tree longevity in California and South Australia (3). Cultivated area of sweet cherry in Spain is around 25,000 ha. Hence, the potential economic loss from this pathogen could be substantial if left unchecked. To our knowledge, this is the first report of C. pulchella as a pathogen of sweet cherry trees in Spain. References: (1) M. E. Barr. Mycologia 77:549, 1985. (2) U. Damm et al. Persoonia 20:39, 2008. (3) F. P. Trouillas et al. Plant Dis. 96:648, 2012.

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.

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 Fusarium Wilt Caused by Fusarium oxysporum f. sp. basilici on Ocimum minimum in Portugal.

Ocimum minimum L. (fine-leaved basil) is cultivated along the Douro Litoral Region in Portugal. In June 2009, a disease was observed in fine-leaved basil fields in three geographically separated locations: Maia, Rio Tinto, and São Mamede Infesta. Affected plants showed wilt symptoms, chlorotic leaves, and stem necrosis. Discolored vascular tissue was observed when the stems were cut longitudinally. For isolation, crown and stem sections (10 cm long) were surface disinfected for 1 min in 1.5% NaOCl and washed twice with sterile distilled water. The sections were cut longitudinally and small pieces of discolored vascular tissue were plated onto potato dextrose agar (PDA) amended with streptomycin sulfate (0.5 g liter-1). Plates were incubated at 25°C in the dark. Fusarium colonies were consistently isolated from symptomatic plants sampled from the three different locations and transferred to PDA and Spezieller Nährstoffarmer agar (SNA) culture media for morphological species identification (2). After 10 days of incubation at 25°C, all isolates were identified as F. oxysporum. A PCR-based assay was conducted with nine single-spored isolates (F2, F3, F4, F7, F8, F9, F10, F11, and F13) using the F. oxysporum f. sp. basilici specific primer pair, Bik 1 and Bik 2 (1). A single DNA fragment of 382 bp was amplified in all isolates, which confirmed the identification of F. oxysporum f. sp. basilici. Pathogenicity of all nine isolates was determined on 2-month-old fine-leaved basil seedlings growing in sterile peat moss. Plants were inoculated by watering the roots with 20 ml of a conidial suspension (106 conidia ml-1) harvested from 3-week-old cultures grown on PDA. Thirty-six replicates (each one in individual pots) for each isolate were used, with an equal number of control plants. Plants were maintained in a greenhouse at 15 to 20°C. Within 2 weeks of inoculation, all inoculated plants wilted and exhibited severe leaf and stem necrosis. The fungus was reisolated from vascular tissues of the crown area and the stems of symptomatic seedlings, fulfilling Koch's postulates. Control plants remained healthy. To our knowledge, this is the first report of F. oxysporum f. sp. basilici infecting fine-leaved basil in Portugal. References: (1) A. Chiocchetti et al. Plant Dis. 85:607, 2001. (2) J. F. Leslie et al. The Fusarium Laboratory Manual. Blackwell Publishing, Ames, IA, 2006.

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.

Genetic Diversity and Host Range of Verticillium dahliae Isolates from Artichoke and Other Vegetable Crops in Spain.

Artichoke is severely affected by Verticillium wilt, caused by Verticillium dahliae, in eastern-central Spain, which is one of the most important vegetable-cropping areas in the country. To determine genetic and virulence variability in local populations of V. dahliae, 18 isolates collected from artichoke and other vegetable species cultivated in eastern-central Spain were selected to represent local vegetative compatibility groups (VCGs). Diversity in the isolates was characterized by molecular markers and virulence in 12 important hosts for that region. Recently developed microsatellite markers (simple-sequence repeats) and polymorphic sequences were used to assess the genetic variation among those isolates to reveal any association occurring among host source, VCG, and virulence. Although all isolates caused severe disease symptoms on artichoke, cardoon, eggplant, and watermelon, those from artichoke had a limited host range and isolates from watermelon, muskmelon, and eggplant were not pathogenic to some of the hosts tested. VCG diversity was related to differential virulence in certain hosts.

First Report of Verticillium Wilt of Faba Bean Caused by Verticillium dahliae in Spain.

Faba bean (Vicia faba L.) crops in eastern-central Spain are usually grown in rotation or double cropped with artichoke (Cynara cardunculus L. var. scolymus (L.) Fiori). In this region, artichoke is grown annually and is severely affected by Verticillium dahliae Kleb. (1). During February of 2007, wilt symptoms were observed at harvesting time on faba bean fields located in Castellón Province (eastern-central Spain). Symptoms consisted of leaf yellowing, wilting, and gradual death of the leaves while stems generally remained green except for severely affected plants. The vascular tissue in the stems showed a tan-to-light brown discoloration and plants were stunted. For isolation, crown and stem sections (10 cm long) were surface disinfected for 1 min in 1.5% NaOCl and washed twice with sterile distilled water. The sections were cut longitudinally and small pieces of discolored vascular tissue were plated onto potato dextrose agar (PDA) amended with streptomycin sulfate (0.5 g liter-1). Plates were incubated at 25°C in the dark. V. dahliae was consistently isolated and colonies transferred to PDA were identified on the basis of the presence of microsclerotia and conidiophore morphology. Identity of monoconidial isolates 3H, 4H, 7H, and 8H was confirmed by specific multiplex nested-PCR assays using primers NDf/NDr in the first PCR round and INTND2f/INTND3r/MCR2B in the second round (2). PCR markers amplified with these primers were originally developed for the detection and vegetative compatibility group (VCG) identification of V. dahliae isolates infecting artichoke plants. Isolates 3H, 4H, 7H, and 8H amplified the 688-bp and the 964-bp markers indicating that they belong to VCG2B. Recent studies identified VCG2B as the prevalent group in the population of V. dahliae affecting artichoke in Castellón Province (3). Pathogenicity of two selected isolates, 3H and 7H, was determined on faba bean (cv. Muchamiel) and artichoke seedlings (cv. Madrigal) at the two-true-leaf stage. Seedlings were inoculated by watering the roots with 25 ml of a conidial suspension (106 conidia ml-1) harvested from 3-week-old cultures grown on PDA. Ten replicates (each one in individual pots) for each isolate and plant species were used, with an equal number of control plants. Plants were maintained in a greenhouse at 23 to 25°C. Within 1 month of inoculation, symptoms developed on all inoculated plants as severe stunting, leaf necrosis, and wilting. The fungus was reisolated from vascular tissues of the crown area and the stems of inoculated seedlings, completing Koch's postulates. Symptoms were not visible in the control seedlings and V. dahliae was not isolated from them. To our knowledge, this is the first report of V. dahliae infecting faba bean in Spain. Verticillium wilt had been previously reported on V. faba in Greece (4). Verticillium wilt of faba bean may bear importance in the epidemiology of the disease in artichoke as an alternative host for inoculum increase and survival of V. dahliae under field conditions. References: (1) M. Berbegal et al. Plant Dis. 91:1131, 2007. (2) M. Collado-Romero et al. Online publication. doi:10.1111/j.1365-3059.2008.01981.x. Plant Pathol., 2008. (3) R. M. Jiménez-Díaz et al. Phytopathology 96:288, 2006. (4) E. K. Ligoxigakis and D. J. Vakalounakis. Plant Pathol. 43:755, 1994.

Effect of Cauliflower Residue Amendments and Soil Solarization on Verticillium Wilt Control in Artichoke.

The effect of fresh cauliflower residue amendment alone and with a low dose of metham sodium (MS) combined with soil solarization was investigated for the control of Verticillium wilt of artichoke in two commercial fields under artichoke-cauliflower rotation. Treatments were a factorial combination of three main plots (an unamended control, soil amended with cauliflower residue, and a combination of cauliflower residue and a low dose of MS) and two subplots (application of a plastic cover or uncovered). Inoculum densities of Verticillium dahliae were measured before and after soil treatments as well as disease incidence, symptom severity, and yield. Although soil solarization reduced inoculum of V. dahliae and the incidence of Verticillium wilt of artichoke, no added benefit was obtained when solarization was used with cauliflower residue amendments. In addition to toxic volatile compounds, other mechanisms could be involved in disease suppression because the effects of incorporating cauliflower residue were not enhanced by the plastic covering. The effect of cauliflower residues on populations of V. dahliae microsclerotia in soil was inconsistent, possibly due to varying pretreatment inoculum levels. Treatments with cauliflower residue amendments and low doses of MS maintained low inoculum densities in the fields until the end of the growing season and significantly reduced the percentage of infected plants.

Inoculum Density-Disease Development Relationship in Verticillium Wilt of Artichoke Caused by Verticillium dahliae.

The relationship between inoculum density of Verticillium dahliae in soil and disease development was studied in 10 commercial artichoke fields. Inoculum density of V. dahliae varied between 2.2 and 34.2 microsclerotia (ms) g-1 of soil near planting. Artichoke plants were monitored for disease at the beginning and the end of each growing season. There was a significant correlation, which was best described by negative exponential models, between inoculum density and disease incidence, symptom severity, and recovery of the pathogen from the plants. Inoculum densities ranging from 5 to 9 ms g-1 of soil were associated with a mean percentage of infected plants of about 50%. Additionally, three fields were monitored in two consecutive growing seasons to evaluate the population dynamics of V. dahliae microsclerotia in soil and disease development. Numbers of microsclerotia per gram of soil decreased significantly by the end of the first growing season but slightly increased at the end of the second growing season. In these fields, symptom severity was greatest during the second growing season when high percentages of infected plants also were recorded.

Outbreak of Pitch Canker Caused by Fusarium circinatum on Pinus spp. in Northern Spain.

During the winter of 2003-2004, dieback symptoms were observed on Pinus radiata and P. pinaster in pine nurseries in Asturias (northern Spain). Small groups of affected seedlings appeared randomly distributed throughout the nurseries. The seedlings died rapidly, showing basal needle dieback, stem lesions, resin exudations, and wilting. Isolations from infected material onto potato dextrose agar (PDA) supplemented with 0.5 mg/ml of streptomycin sulfate and Komada's medium consistently yielded Fusarium sp. cultures. The isolates were transferred to PDA and Spezieller Nährstoffarmer agar and incubated at 25°C for 10 days with a 12-h photoperiod. The cultures were identified as Fusarium circinatum Nirenberg & O'Donnell (= Fusarium subglutinans Wollenweb. & Reinking), causal agent of pitch canker disease, on basis of the presence of polyphialides and characteristic sterile, coiled, hyphae (2). To further confirm their identity, a polymerase chain reaction (PCR) and restriction fragment length polymorphism (RFLP) based on histone H3 gene sequences (4) and a test based on the F. circinatum-specific primers, CIRC1A-CIRC4A, which amplifies a 360-bp DNA fragment of the intergenic spacer region of the nuclear ribosomal operon (3), were used. Results obtained with both techniques confirmed the morphological identification of the cultures. A representative culture has been placed in the Centraalbureau voor Schimmelcultures (CBS 117843). The pathogen was isolated only from seedlings of P. radiata and P. pinaster. Other species such as P. nigra, P. sylvestris, and Pseudotsuga menziesii, which were also grown in these nurseries, did not show symptoms. Pathogenicity was confirmed by inoculating 6- to 9-month-old P. radiata and P. pinaster seedlings. Small strips of bark (10 × 1 mm) were cut from the stems and similar sized pieces of PDA colonized by F. circinatum were placed in contact with the open wounds and covered with parafilm. Basal needle dieback was observed 10 days after inoculation that resulted in wilting of the seedlings. F. circinatum was reisolated from the affected stems fulfilling Koch's postulates. Later in the year, symptoms of pitch canker were also observed on 20-year-old P. radiata in one forest plantation in Cantabria (northern Spain). Infected branches and shoots of the trees exudated abundant resin, resulting in resinous cankers. The needles, distal to branch tip infections, wilt, fade to yellow then red, and fall from the tree. Affected trees showed noticeable crown dieback. The isolations from the cankers also yielded F. circinatum cultures that were identified as described above. Although a nonrefereed report appeared in 1998 (1), to our knowledge, this is the first report of F. circinatum on P. radiata and P. pinaster in Spain and in Europe. References: (1) L. D. Dwinell et al. Int. Congr. Plant Pathol. 7th. 3:9, 1998. (2) H. I. Nirenberg and K. O'Donnell. Mycologia 90:434, 1998. (3) W. Schweigkofler et al. Appl. Environ. Microbiol. 70:3512, 2004. (4) E. T. Steenkamp et al. Appl. Environ. Microbiol. 65:3401, 1999.

Leprosy elimination campaign, Amazonas-Brazil 1997.

A leprosy elimination campaign (LEC) was carried out in 15 endemic areas of Amazonas State, Brazil, in 1997. The LEC concentrated effort to detect leprosy cases during a multi-vaccination national campaign for serious public health problems other than leprosy, such as polio, diphtheria, hepatitis, measles, etc. The national campaign involved intensive population mobilization, giving a valuable opportunity to examine people for leprosy. The LEC personnel included 2964 individuals (municipal and state health workers and community volunteers), distributed in 688 health units and 53 reference health centres. As a result of the LEC, 74,814 person-to-person communications in the community were given; 10,297 clinical skin examinations were conducted, and 40 new leprosy cases were detected on the day of the campaign in urban areas of the municipalities. This total was low, compared to results in other states of Brazil, possibly due to the development of health education activities and regular community services in the state of Amazonas since 1987 and to the early implementation of WHO multiple drug therapy (MDT) from 1982 onwards. Despite the fact that the LEC was carried out only in the urban areas of the municipalities, the finding of no cases of leprosy in 7 out of 15 of them was surprising and may indicate that the prevalence of hidden cases of leprosy is not all that high, at least in these areas of the Amazonas State.