In Europe, chestnut blight caused by Cryphonectria parasitica (Murrill) Barr was first seen in Italy in 1938 (1). In Spain, the disease was first detected in Basque country in 1947 and later in other areas of northern Spain: Galicia, León, Navarra, and Catalonia, and in Trás-os-Montes in Portugal (2). In November 2012, in an orchard (2 ha) in Almonaster la Real (Huelva, Spain), approximately 20 cankered Castanea sativa (sweet chestnut) trees cv. Vazqueño, 40 to 50 years old, were observed. The trees were grafted 2 years before. In May and June 2013, six new disease focuses were detected near the first one. Five focuses were located in the same village and the other in Jabugo (a neighboring village). Diseased trees exhibited sunken cankers, cracked bark with mycelial fan spreads under the bark, and in some cases, orange fungal sporulation was visible on the bark. Samples were collected from two affected trees and symptom-bearing bark pieces were then placed in moist chambers at 20°C for up to 8 days to induce fungal sporulation. Cultures were made from spore masses extruding from the cankered bark and from the edge of necrotic lesions visible in the phloem of cankered bark tissue onto potato dextrose agar (PDA). Monoconidial fungal isolates were obtained from both trees. The morphological structure of two isolated fungi was identical to that described as C. parasitica (3). Species identity was confirmed by analysis of nucleotide sequences of the internal transcribed spacer (ITS) rDNA, using ITS1-ITS4 (4) as primer pairs, respectively. BLAST searches showed a high similarity between collected isolates' DNA sequences and C. parasitica sequences found on GenBank (96% coverage, 99% identity). Our isolates have been included in GenBank as KF220298 and KF220299. The pathogenicity assay of these two isolates was conducted using two cultivars of sweet chestnut (seedlings from Huelva and Granada nurseries). Isolate pathogenicity was tested on 3-year-old chestnut seedlings in a growth chamber at 25°C (day) and 20°C (night) with a 14-h photoperiod. The isolates were cultured on PDA at 25°C for 7 days. Stems were wounded at 10 cm height with a drill. Each isolate was inoculated to 25 replicates per cultivar by placing a mycelia agar plug (4 to 5 mm diameter) in the hole and wrapping the stem with Parafilm. Plants treated identically with sterile agar plugs were used as controls. Plants were then maintained at 100% relative humidity for 2 h. Both isolates induced diseases symptoms and death of seedlings of both cultivars at a mean time of 37.5 days after inoculation. No significant differences between isolates or between cultivars were detected. Twenty control plants similarly treated with sterile PDA discs did not display symptoms. C. parasitica was re-isolated from lesions, confirming Koch's postulates. Andalusia has 14,000 ha of chestnut crops with high commercial value due to their precocity. Dispersion of chestnut blight in this zone can reduce crop productivity. To our knowledge, this is the first report of C. parasitica causing chestnut blight in Andalusia (southern Spain), one of the few areas left in southwestern Europe free of chestnut blight. References: (1) A. Biraghi. Italia Agricola 7:1, 1946. (2) G. González-Varela et al. Eur. J. Plant Pathol. 131:67, 2011. (3) A. Sivanesan and P. Holliday. Cryphonectria parasitica. CMI Descriptions of Pathogenic Fungi and Bacteria. No. 704, Set. 71. Commonwealth Mycological Institute, Kew, UK, 1981. (4) T. J. White et al. Page 315 in: PCR Protocols: A Guide to Methods and Amplifications. M. A. Innis et al., eds. Academic Press, San Diego, CA, 1990.

In Europe, chestnut blight caused by Cryphonectria parasitica (Murrill) Barr was first seen in Italy in 1938 (1). In Spain, the disease was first detected in Basque country in 1947 and later in other areas of northern Spain: Galicia, León, Navarra, and Catalonia, and in Trás-os-Montes in Portugal (2). In November 2012, in an orchard (2 ha) in Almonaster la Real (Huelva, Spain), approximately 20 cankered Castanea sativa (sweet chestnut) trees cv. Vazqueño, 40 to 50 years old, were observed. The trees were grafted 2 years before. In May and June 2013, six new disease focuses were detected near the first one. Five focuses were located in the same village and the other in Jabugo (a neighboring village). Diseased trees exhibited sunken cankers, cracked bark with mycelial fan spreads under the bark, and in some cases, orange fungal sporulation was visible on the bark. Samples were collected from two affected trees and symptom-bearing bark pieces were then placed in moist chambers at 20°C for up to 8 days to induce fungal sporulation. Cultures were made from spore masses extruding from the cankered bark and from the edge of necrotic lesions visible in the phloem of cankered bark tissue onto potato dextrose agar (PDA). Monoconidial fungal isolates were obtained from both trees. The morphological structure of two isolated fungi was identical to that described as C. parasitica (3). Species identity was confirmed by analysis of nucleotide sequences of the internal transcribed spacer (ITS) rDNA, using ITS1-ITS4 (4) as primer pairs, respectively. BLAST searches showed a high similarity between collected isolates' DNA sequences and C. parasitica sequences found on GenBank (96% coverage, 99% identity). Our isolates have been included in GenBank as KF220298 and KF220299. The pathogenicity assay of these two isolates was conducted using two cultivars of sweet chestnut (seedlings from Huelva and Granada nurseries). Isolate pathogenicity was tested on 3-year-old chestnut seedlings in a growth chamber at 25°C (day) and 20°C (night) with a 14-h photoperiod. The isolates were cultured on PDA at 25°C for 7 days. Stems were wounded at 10 cm height with a drill. Each isolate was inoculated to 25 replicates per cultivar by placing a mycelia agar plug (4 to 5 mm diameter) in the hole and wrapping the stem with Parafilm. Plants treated identically with sterile agar plugs were used as controls. Plants were then maintained at 100% relative humidity for 2 h. Both isolates induced diseases symptoms and death of seedlings of both cultivars at a mean time of 37.5 days after inoculation. No significant differences between isolates or between cultivars were detected. Twenty control plants similarly treated with sterile PDA discs did not display symptoms. C. parasitica was re-isolated from lesions, confirming Koch's postulates. Andalusia has 14,000 ha of chestnut crops with high commercial value due to their precocity. Dispersion of chestnut blight in this zone can reduce crop productivity. To our knowledge, this is the first report of C. parasitica causing chestnut blight in Andalusia (southern Spain), one of the few areas left in southwestern Europe free of chestnut blight. References: (1) A. Biraghi. Italia Agricola 7:1, 1946. (2) G. González-Varela et al. Eur. J. Plant Pathol. 131:67, 2011. (3) A. Sivanesan and P. Holliday. Cryphonectria parasitica. CMI Descriptions of Pathogenic Fungi and Bacteria. No. 704, Set. 71. Commonwealth Mycological Institute, Kew, UK, 1981. (4) T. J. White et al. Page 315 in: PCR Protocols: A Guide to Methods and Amplifications. M. A. Innis et al., eds. Academic Press, San Diego, CA, 1990.

The Spanish Methyl Bromide (MB) Alternatives Project (INIA) has carried out experiments over ten-years in Huelva for strawberry production. More than 20 soil fumigant treatments have been evaluated on cv. 'Camarosa'. The last series has been carried out in two locations of Eastern coastal Huelva (Moguer and Palos de la Fra.) from 2002-2003 until now. Treatments: MB + chloropicrin, 1,3-dichloropropene + chloropicrin (1,3D:CP), chloropicrin (CP) alone, metam sodium (MS), dimethyl disulphide (DMDS), ethanedinitrile (EDN), propylene oxide (PO), sodium tetrathiocarbamate (ENZ), methyl iodide + chloropicrin (MI), essential oils (EO), were shank-applied, or drip-irrigated such as sodium azide (SEP), furfural (FUR) under mulched (VIF film) pre-formed beds, or incorporated into the soil: dazomet (DAZ) and calcium cyanamide (CC). Treatments were applied in September and cultivation cycle was between October and the end of May every year. Black root rot complex and nematodes Meloidogyne hapla and Pratylenchus penetrans appear as the main phytosanitary problems related to soil. Our results supported feasible alternatives to MB with 1,3D:CP, CP alone or DMDS plus CP (DMDS:CP). In coastal Huelva, significant amounts of alternative fumigants have been applied to the strawberry crop since 2005, mainly 1,3D:CP, CP alone, DAZ:1,3D and MS. This activity has allowed 100% MB replacement rates in 2007. However some concerns remain due to the implementing of the Directive 91/414/EEC.

Huelva in southern Spain is a major production area for strawberry (Fragaria × ananassa). At the end of the 2006 season (May–June) collapsed and dying strawberry plants were observed on several cultivars in four fields. Cut crowns of affected plants revealed dark brown necrotic areas on the margins and along the woody vascular ring. Roots of these plants were also shown to be necrotic. Macrophomina-like isolates developed from surface-disinfested affected tissues plated on potato dextrose agar amended with 250 mg L−1 of chloramphenicol. Dark, oblong-shaped sclerotia were observed in affected crown tissue and in culture after 5 to 7 days incubation at 25°C. They had an average length of 107 (217 to 62) µm and width of 71 (110 to 35) µm. Sequenced rDNA fragments of a single sclerotium isolate CH 724 (Spanish Type Culture Collection, CECT 20715; GenBank Accession No. AM410964) presented a 99% identity with Macrophomina phaseolina. Morphological and molecular results confirmed this species as M. phaseolina (Holliday & Punithalingam, 1970). Six single sclerotium isolates of M. phaseolina from strawberry were used for pathogenicity tests. Each isolate was used to inoculate six strawberry runner plants (cv. Camarosa) growing in pots of coconut fibre substrate for 5 weeks. Plants were inoculated by inserting a fungal colonised toothpick into each crown (Mertely et al., 2005). An equal number of uninoculated plants treated similarly were left as controls. After 58 days, the incidence of plant death ranged from 67 to 100% depending on isolate. Macrophomina phaseolina was reisolated from all plants showing symptoms. Uninoculated plants remained symptomless. Although M. phaseolina has been reported on other crops in Spain, this is the first report of the pathogen on strawberries in this important production area. Similarly, this pathogen has been reported recently from strawberry-growing areas of other countries using alternatives to methyl bromide (Mertely et al., 2005; Zveibil & Freeman, 2005). Crown and root rot in strawberries caused by M. phaseolina may be an emerging disease following the phase-out of methyl bromide treatment.

Since 2002, considerable losses of Clementine trees (Citrus clementina) have been observed in Spain due to Phytophthora branch canker of citrus caused by Phytophthora citrophthora. Due to the low efficiency of the available cultural and genetic control measures, application of fungicides is required for economic management of the disease. Fosetyl-Al, metalaxyl, and its enantiomer mefenoxam are the only systemic fungicides registered for control of Phytophthora diseases in Spain. However, their efficacy has not been tested against Phytophthora branch canker. Greenhouse and field experiments were conducted for 3 years in Spain to evaluate the ability of these fungicides and application methods to reduce lesion expansion. Nevertheless, with the inoculation technique used, it was not possible to evaluate the protective activity of fungicides, which can play an important role in their performance under field conditions. None of the fungicide treatments inhibited lesion expansion when applications were made on a curative basis. The residual effect was better on young than on mature trees. Paint treatments were generally more effective in reducing lesion expansion that drip chemigation or foliar sprays. However, this application method is laborious and becomes uneconomical in Spain. Trunk and branch sprays as well as long-term programs of foliar sprays or drip chemigation for control of spring and autumn infections are proposed as targets for future research.

Considerable losses of citrus trees have been observed in the major citrus‐growing areas of Spain. Samples were collected from 132 orchards, and isolations and pathogencity tests were conducted to determine the aetiology of a serious canker disease. Affected trees showed cankers on the scion that frequently began on the branches. Three Phytophthora species were identified based on their morphological, cultural, physiological and molecular profiles. Phytophthora citrophthora was the main species associated with this new syndrome in 114 orchards. Phytophthora nicotianae (syn. P. parasitica) was isolated from nine orchards as the sole Phytophthora species and in coinfection with P. citrophthora from another nine orchards. Phytophthora citricola was isolated only from one orchard. In stem‐inoculation studies conducted under greenhouse conditions, clementine mandarin cv. Hernandina and sweet orange cv. Navel Late were more susceptible to P. citrophthora than sour orange and Carrizo citrange rootstocks. Clementine cv. Hernandina was also highly susceptible in field inoculation experiments. In agreement with field surveys, clementine mandarin cultivars were the most affected, their rootstocks remaining healthy. Phytophthora citrophthora was found to be the predominant species in orchard soils; however, P. nicotianae was also isolated. This information changes the scenario of diseases caused by Phytophthora spp. in Spain and consequently, the present knowledge of epidemiology and the effectiveness of the current control measures should be reassessed.

The results obtained after six-year work (1997/98-2002/03), showed that the agronomic response of cv. 'Camarosa' to 1,3D-pic (61-35) and Pic alone was similar to that obtained with BM-pic (50-50), even with 50% dosage under black VIF film. The yield obtained with the application of Dazomet was a bit lower than for the other fumigants. These alternatives could represent an appropriate short and medium-term solution to MB ban in environments with low levels of lethal soil-borne strawberry pathogens, such as Huelva crop area. However, several challenges and question marks remain unsolved. In particular, EU policy on future 1,3D and/or Pic utilization is uncertain. For this reason, application for critical use exemption for the Spanish strawberry industry has been presented and recommended by MBTOC in 2003.