During the summer of 1995 and subsequent years, bark cankers were observed in walnut trees (cv. Hartley grafted on Juglans hindsii) imported from California in 1978 growing in Badajoz, Spain. Two foci were found in an orchard of 200 ha where 80 walnut trees were affected. Cankers were observed on trunks and branches, and dark exudates staining the bark appeared mainly in summer. Isolations were performed from affected tissue using King's B medium, and Brenneria (Erwinia)-like colonies (1) were purified and characterized. Gram reaction, Kovacs' oxidase, O/F metabolism, aesculin hydrolysis, urease activity, and levan production were assayed for five isolates (1). Biochemical characterization was performed by the miniaturized API 20E, API 20NE, and API 50CH systems (BioMérieux, Marcy-l'Etoile, France) as recommended, except for incubations that were made at 25°C for 48 h. Analyses of the cellular fatty acids of selected isolates were performed as described by Sasser (2). They were also tested in indirect enzyme-linked immunosorbent assay (ELISA) using antisera obtained against the reference strain CFBP 1284 and one Spanish isolate. When compared to the reference strain from California, isolates were identified as Brenneria rubrifaciens (1,3) on the basis of physiological and biochemical characteristics, fatty acid profiles, and ELISA. Pathogenicity of two selected Spanish isolates was confirmed using three 2-year-old walnut trees per bacterial isolate by inoculating 108 CFU of each isolate in deep wounds made in the trunk at 40 and 80 cm from the crown. The reference strain and water were also inoculated as controls. Two months later, removal of the outer bark of walnut revealed typical dark lesions in the inner bark at all the inoculation sites on trees inoculated with the Spanish and reference strains, but no external cankers were observed. Four years later, these plants showed internal lesions (20 to 80 cm), from which B. rubrifaciens was reisolated far away from the inoculation site. To our knowledge this is the first report of this bacterium in Europe. References: (1) L. Hauben et al. Syst. Appl. Microbiol 21:384, 1998. (2) M. Sasser. Pages 199–204 in: Methods in Phytobacteriology. Budapest, Hungary, 1990. (3) E. Wilson et al. Phytopathology 57:618, 1966.

In 2008, four isolates of Phaeoacremonium, morphologically and genetically different from known Phaeoacremonium spp. in Spain, were isolated from rootstocks of young grapevine (Vitis vinifera) plants showing Petri disease symptoms including low vigor, reduced foliage, and dark streaking of the xylem in Badajoz Province (western Spain; cv. Syrah on SO4 rootstock), Tarragona Province (eastern Spain; cv. Garnacha on 161 49 C rootstock), and Balearic Islands (eastern Spain; cv. Tempranillo on Rupestris de Lot rootstock). Single-conidial isolates were obtained and grown on potato dextrose agar (PDA) and malt extract agar (MEA) at 25°C for 2 to 3 weeks in the dark until colonies sporulated (3). Identification was based on morphological characteristics (1–3). Phaeoacremonium inflatipes W. Gams, Crous & M. J. Wingf. and P. iranianum L. Mostert, Gräf., W. Gams & Crous were detected in Badajoz Province and P. sicilianum Essakhi, Mugnai, Surico & Crous in Tarragona Province and Balearic Islands. Colonies of P. inflatipes were gray on PDA and gray-brown on MEA. Conidiophores were branched, 15 to 37 (mean 25) μm long. Conidia were hyaline, oblong-ellipsoidal or obovoid, 3 to 5.5 (mean 4) μm long, and 1.2 to 1.9 (mean 1.6) μm wide. Colonies of P. iranianum were brownish gray on PDA and pale brown on MEA. Conidiophores were unbranched and 18 to 47.5 (mean 29) μm long. Conidia were hyaline, oblong-ellipsoidal, 3 to 5 (mean 4) μm long, and 1 to 1.8 (mean 1.5) μm wide. Colonies of P. sicilianum were pale brown on PDA and brown to pale orange on MEA. Conidiophores were branched and 13 to 55 (mean 32.5) μm long. Conidia were hyaline, allantoid, 3 to 8.5 (mean 6) μm long, and 1.5 to 2 (mean 1.8) μm wide. Identity of isolates Pin-2, Pir-4, Psi-1, and Psi-2 was confirmed by sequencing a fragment of the beta-tubulin gene with primers T1 and Bt2b (P. inflatipes, isolate Pin-2: GenBank Accession No. FJ872407, 100% similarity to Accession No. AY579323; P. iranianum, isolate Pir-4: GenBank Accession No. FJ872406, 99% similarity to Accession No. EU128077; P. sicilianum isolates Psi-1 and Psi-2: GenBank Accession Nos. FJ872408 and No. FJ872409, 100% similarity to Accession No. EU863489). Pathogenicity tests were conducted using Pin-2, Pir-4, and Psi-1 isolates. One-year-old callused and rooted cuttings of 110 R rootstock cultivated in sterile peat were wounded at the uppermost internode with an 8-mm cork borer. An 8-mm mycelium plug from a 2-week-old culture was placed into the wound. Wounds were wrapped with Parafilm. Ten cuttings per fungal isolate were used. Ten control plants were inoculated with 8-mm noncolonized PDA plugs. Plants were maintained in a greenhouse at 25°C. Within 2 months, all Phaeoacremonium-inoculated cuttings exhibited shoots with poor growth, small leaves, short internodes, and black streaks in the xylem. The mean shoot weight per plant was 1.8 g in P. inflatipes-inoculated plants, 1.9 g in P. iranianum-inoculated plants, and 1.6 g in P. sicilianum-inoculated plants, all lower than the control treatment (6.8 g). Control plants did not show any symptoms. All fungal species were reisolated from wood of all inoculated cuttings, completing Koch's postulates. Their identity was confirmed with the methods described above. To our knowledge, this is the first report of P. inflatipes, P. iranianum, and P. sicilianum causing Petri disease in Spain. References: (1) P. W. Crous et al. Mycologia 88:786, 1996. (2) S. Essakhi et al. Persoonia 21:119, 2008. (3) L. Mostert et al. Stud. Mycol. 54:1, 2006.

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In 2006, a serious outbreak of bacterial leaf and flower spot disease was observed on zinnia ( Zinnia elegans ) cvs Capricio and Mondo) grown in several parks in Budapest, Hungary. The disease first appeared on leaves as diffuse, translucent, circular spots surrounded by large chlorotic haloes. The lesions enlarged and became reddish brown in the centre and angular in shape. On the flowers, at high humidity, small brown spots appeared, and the flower heads were disfigured and decayed completely. Bacteria were isolated on modified Tween medium (Schaad et al ., 2001). All isolates were Gram-negative rods, aerobic, and produced yellow, xanthomonadin pigments identified by thin-layer chromatography (Schaad et al ., 2001). Isolates were positive for catalase, negative for oxidase, hydrolised starch, gelatine, casein, and aesculin; hydrogen sulphide was produced from cysteine. In medium C of Dye (Dye, 1968) acids were produced from arabinose, glucose, maltose and sucrose, but not from sorbitol. The sequence of the 16S-23S rDNA spacer region of the bacterial strain (GenBank Accession No. EF514223) was determined. The sequence shared 99·7% identity with other X. campestris pv . zinniae strains available in GenBank.Pathogenicity was confirmed by artificial inoculation of healthy, four to six-leaf stage zinnia plants with a suspension of an isolated bacterial strain (1 × 10 7 CFU per mL). Sterile distilled water was used as a negative control. The inoculated plants were incubated in a mist chamber (95% relative humidity) for 3 days, and then transferred to a greenhouse at 21-27 ° C. Characteristic leaf spot symptoms were observed on inoculated zinnia plants 8 days after inoculation. No symptoms were observed on control plants. The original pathogen strain was reisolated from diseased leaves.Bacterial leaf spot of zinnia was first reported in 1929, in Italy (Nanizzi, 1929), and the pathogen was named later as X. campestris pv. zinniae (Dye, 1978). Since 1929 no report describing the disease has been published in Europe. This is the first report on the occurrence of this bacterium on zinnia in Hungary. Agrobacterium -like colonies were recovered from New and Kerr's medium from a crown gall of grapevine rootstock 41B ( Vitis vinifera × V. berlandieri ) from a nursery located in Badajoz province. After colony purification and tomato and tobacco plant inoculations, three Agrobacterium isolates, tumorigenic in both plant species, were characterized. On the basis of biovar classification, all isolates were identified as belonging to biovar 2 of Agrobacterium (also called A. rhizogenes ). Because this biovar is not common in grapevine, all isolates were also inoculated onto grapevine plants of cv. Tempranillo, which developed typical tumors one-month after inoculations. DNA from all isolates yielded the expected amplification product when using FGP tmr 530-FGP tmr 701', vir B/G and VCF/VCR primer sets (Cubero et al ., 1999), confirming their pathogenic nature, but not when using virA or pehA primer sets (Eastwell et al ., 1995), ...

Two begomovirus species, Tomato yellow leaf curl Sardinia virus (TYLCSV) and Tomato yellow leaf curl virus (TYLCV), have been identified as causal agents of tomato yellow leaf curl disease (TYLCD) in Spain. TYLCSV was reported in Spain in 1992 and TYLCV in 1997 on tomato crops (3). TYLCV was also reported in common bean (Phaseolus vulgaris L.) and pepper (Capsicum annuum L.) crops in southern Spain in 1997 and 1999, respectively. During the summer of 2004, symptoms of yellowing, crumpling, and necrosis of new leaves were observed sporadically in young, field-grown tobacco (Nicotiana tabacum L.) plants in the Badajoz Province. These tobacco plants were next to tomato crops where TYLCV was detected for the first time in Badajoz in 2003. In September 2004, four symptomatic tobacco plants were selected for double antibody sandwich enzyme linked immunosorbent assay (DAS-ELISA) and polymerase chain reaction (PCR) identification analyses. Serological analyses were carried out in two repetitions and with the following polyclonal antisera: Potato virus Y (PVY) (Loewe Biochemica, Sauerlach, Germany); Tobacco mild green mosaic virus (produced in our laboratory); Tobacco mosaic virus (BIO-RAD, Marnes-La-Coquette, France); and Tomato spotted wilt virus (Loewe Biochemica). A simplified method of duplex PCR was used for a rapid, sensitive, and simultaneous detection of TYLCSV and TYLCV (2). Mixed infections of PVY and TYLCV were detected in all four tobacco samples tested. TYLCV infection was confirmed using the primer pair TY-1/TY-2 specific for the coat protein (CP) gene of begomoviruses (1). The CP fragment was digested with the restriction enzyme AvaII, and the pattern obtained corresponded to that obtained from TYLCV-infected tomato that served as a positive control. Two PCR products from different tobacco samples were sequenced and both showed 100% identity with the corresponding region (Almería) of TYLCV (GenBank Accession No. AJ489258) and 99% with TYLCV-Mild (Spain) (GenBank Accession No. AJ519441), confirming the diagnosis. The symptoms observed in the tobacco plants can not be attributed solely to TYLCV since the virus was present in a mixed infection with PVY. However, tobacco infected with TYLCV may serve as an important alternate host for TYLCV in the tomato cropping system. To our knowledge, this is the first report of N. tabacum as a natural host of TYLCV in Spain. References: (1) G. P. Accotto et al. Eur. J. Plant Pathol. 106:179, 2000. (2) P. Martínez-Culebras et al. Ann. Appl. Biol. 139:251, 2001. (3) J. Navas-Castillo et al. Plant Dis. 81:1461, 1997.