Trees In Spain Research Articles

New Biomass Sources to Reduce Peat Dependence in Mediterranean Substrates: Validation of Morus alba L., Sorghum vulgare L., and Phoenix canariensis Pruning Wastes

The use of alternative materials to peat in soilless crop production, such as woody pruning wastes, has been reported by several researchers. In Mediterranean countries, several potential alternatives have been identified such as palm species or woody pruning wastes coming from different plant species. Mulberry (Morus alba L.) is a tree crop widely used in Europe for urban landscaping and to provide shade. Mulberry trees are pruned in sericulture two or three times a year to obtain a better yield of leaves. In an urban landscape, the trees are pruned once a year to form the canopy. Sorghum (Sorghum vulgare L.) is a cereal crop widely cultivated because it is considered the fourth most important summer crop in the world after soy, corn, and sunflower. It is one among the few resilient crops that can adapt well to future climate change conditions, particularly increasing drought, soil salinity, and high temperatures. After the harvest, the inedible parts of the plant, the stalks and leaves, are left over and must be disposed of, most of the time by burning. Palm trees in Spain, as in most of the European regions where palms are cultivated, are mainly used as ornamental plants in urban areas. One of the most commonly used palm tree in landscaping is the Canary Island date palm (Phoenix canariensis Hort. Ex Chavaud). These palm trees are pruned up to twice a year in this region, producing huge amounts of biomass that are disposed of in landfills. In this experiment, physical and chemical parameters were determined in the different particle sizes (<0.25, 0.25–1, 1–2, and 2–4 mm) of the raw residual materials from pruning activities of mulberry (Morus alba L.), sorghum (Sorghum vulgare L.), and palm (Phoenix canariensis) to evaluate their suitability for use as growing substrate. According to the results obtained, it can be concluded that the alternative materials for substrate preparation obtained from the pruning wastes of Morus alba (MAPW), Sorghum vulgare (SVPW), and Phoenix canariensis (PCPW) had suitable values of physical properties related to substrate porosity and wetting capacity. These materials also had high or very high total organic carbon (C) to total nitrogen (N) ratio values and low nutrient contents, except for potassium (K) in SVPW and PCPW.

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.

First Report of Phaeoacremonium venezuelense Associated with Wood Decay of Apricot Trees in Spain

In July 2011, a survey was conducted to evaluate the phytosanitary status of apricot trees (Prunus armeniaca L.) in an orchard in Binissalem (Mallorca Island, Spain). Fungal isolation was performed on a 40-year-old apricot trees (cv. Galta Vermella, double-grafted onto bitter almond and Japanese plum) showing a collapse of branches, chlorosis of leaves, and shoot dieback. These symptoms appeared in approximately 10% of the trees. Black spots and dark streaking of the xylem vessels were observed in cross- or longitudinal sections of the branches. Symptomatic branches were collected and wood sections (10 cm long) were cut, washed under running tap water, surface-disinfested 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 liter-1 of streptomycin sulfate. Dishes were incubated at 25°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 (more than 50% of the isolations). 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 brownish orange on PDA and MEA. Conidiophores were short and occasionally branched, and 26 to 35 (avg. 29) μm long. Phialides were terminal or lateral, mostly monophialidic. Conidia were hyaline, oblong-ellipsoidal or fusiform-ellipsoidal, 3 to 4 (avg. 3.9) μm long, and 1 to 1.5 (avg. 1.2) μm wide. Based on these characters, the isolates were identified as Phaeoacremonium venezuelense L. Mostert, Summerb. & Crous (2,3). DNA sequencing of a fragment of the beta-tubulin gene of the isolate 9.3 using primers T1 and Bt2b (GenBank Accession No. KF765487) matched P. venezuelense GenBank accession HQ605026. Pathogenicity tests were conducted using isolate 9.3. Ten 2-year-old apricot trees of cv. Galta Rotja grown in pots were wounded in two branches with a 8-mm cork borer. A 5-mm mycelium PDA plug from a 2-week-old culture was placed in the wound before being wrapped with Parafilm. Ten control plants were inoculated with 5-mm non-colonized PDA plugs. Plants were maintained in a greenhouse at 25 to 30°C. Within 5 months, shoots on all Phaeoacremonium-inoculated branches had weak growth with chlorosis of leaves and there were black streaks in the xylem vessels. The vascular necroses that developed on the inoculated plants were 5.5 ± 0.6 cm long, significantly greater than those on the control plants (P < 0.01). Control plants did not show any symptoms. The fungus was re-isolated from discolored tissue of all inoculated cuttings, completing Koch's postulates. P. venezuelense was reported as a pathogen of grapevines in Algeria (1) and South Africa (2) and, to our knowledge, this is the first report of P. venezuelense associated with wood decay of apricot trees in Spain or any country in the world.

First Report of 'Candidatus Phytoplasma pyri' Causing Peach Yellow Leaf Roll (PYLR) in Spain.

'Candidatus Phytoplasma prunorum,' which causes European stone fruit yellows (ESFY), is the prevalent phytoplasma affecting Prunus spp. in Europe. It is closely related to 'Ca. P. pyri,' which causes pear decline (PD) in pear trees. Both phytoplasma belong to the ribosomal group 16Sr-X and are naturally transmitted by different species of Cacopsylla spp. (4). In North America, 'Ca. P. pyri' is responsible for peach yellow leaf roll (PYLR), transmitted by Cacopsylla pyricola from pear to peach trees (1). In Spain, 'Ca. P. prunorum' is widespread on Prunus spp., but its occurrence on Prunus persicae is very low and 'Ca. P. pyri' is present in every pear orchard (3). During 2012, a previously unreported syndrome including early reddening, leaf curling, decline, abnormal fruits, and in some cases chlorosis and death of peach trees was reported on peach in Lleida, northern Spain. Symptoms were different to ESFY and PYLR, in that flowering disorders such as ESFY or yellows were not apparent, and reddening and decline were the most common symptoms. The disease was present in a wide range of varieties and rootstocks, suggesting insect transmission in an area where C. pruni, vector of 'Ca. P. prunorum,' was not previously reported, but C. pyri was abundant in pear orchards. Shoot samples from 20 symptomatic peach trees were collected in seven orchards within a 2 km2 area with an estimated incidence of 40%, which was higher in the borders. DNA was extracted from 1 g of leaf midribs and phloem tissue and amplified with ribosomal universal primers P1/P7 followed by nested PCR with R16F2n/R16R2 and specific primers fO1/rO1 that target the 16Sr-X group (3). The final PCR products were digested with RsaI enzyme. Amplifications with non-ribosomal specific primers, Imp ESFY, Imp PD A and Imp PD B that amplify sequences of gene Imp, that encode a phytoplasma membrane protein, were also carried out (2). Tissue samples with ESFY and PD and peach seedlings were used as positive and negative controls, respectively. Amplified PCR products were sequenced and compared to sequences deposited in GenBank. Phytoplasmas were detected in 18 of the 20 samples analyzed. No phytoplasmas were detected in negative peach controls. All digestions of fO1/rO1 PCR products from peach samples showed a PD profile, while no ESFY profile was detected. All samples were positive with specific primers Imp PD A and B. None of the peach samples were positive with the specific Imp-ESFY primers. Sequencing of R16 and Imp PDA and B amplicons revealed the presence of a stable isolate. The sequences were submitted to the European nucleotide archive (ENA) with the accession nos. HG737345 and HG737344. Based on the 16S rDNA sequence, this strain is 100% homologous to the reference strain PD1 (GenBank Accession No. AJ542543) and 99.55% homologous to strain PD 33 Lib (GenBank FN600725) based on the Imp gene sequence. This is the first report of PD phytoplasma in peach trees in Spain, and the first report in Europe of PD phytoplasma causing economically important outbreaks in peach orchards, following a pattern that could be similar to PYLR in North America. This strain is genetically closer to some European or Middle Eastern PDs than to North American PYLR.

NIR detection of non-recoverable collapse in sawn boards of Eucalyptus globulus

NIR predictions of cellulose content and stiffness (modulus of elasticity, MOE) from spectra collected from the radial longitudinal surface of Eucalyptus globulus wood were found to be reliable indicators of zones of non-recoverable collapse associated with the presence of tension wood. Radial sections from 25 quarter-sawn boards cut from plantation-grown E. globulus trees in Spain were scanned to generate radial profiles of NIR-predicted wood properties at 2 mm increments. These boards manifested a range of non-recoverable collapse features, from no collapse to one or more severe collapse bands. Collapse bands occurred where NIR-predicted cellulose content and MOE exceeded threshold levels of 50 % and 25 GPa, respectively for more than four consecutive millimetres. A non-recoverable collapse indicator provided a clear predictor of non-recoverable collapse. A NRCI value ≥100 for a consecutive interval of at least 4 mm successfully predicted all ten NRC bands with shrinkage ≥10 % of board thickness. The potential applications of this tension wood detection method are discussed.

Chryseobacterium oleae sp. nov., an efficient plant growth promoting bacterium in the rooting induction of olive tree (Olea europaea L.) cuttings and emended descriptions of the genus Chryseobacterium, C. daecheongense, C. gambrini, C. gleum, C. joostei, C. jejuense, C. luteum, C. shigense, C. taiwanense, C. ureilyticum and C. vrystaatense

A novel non-motile, Gram-staining-negative, yellow-pigmented bacterium, designated CT348T, isolated from the ectorhizosphere of an organic olive tree in Spain and characterised as an efficient plant growth promoting bacterium, was investigated to determine its taxonomic status. The isolate grew best in a temperature range of 5–35°C, at pH 5.0–8.0 and with 0–1% (w/v) NaCl. Chemotaxonomic and molecular characteristics of the isolate matched those described for members of the genus Chryseobacterium. The DNA G+C content of the novel strain was 38.2mol%. The strain contained a polyamine pattern with sym-homospermidine as the major compound and produced flexirubin-type pigments. MK-6 was the dominant menaquinone and the major cellular fatty acids were iso-C15:0, C17:1ω9c, iso-C17:0 3-OH and iso-C15:0 2-OH. The main polar lipids were phosphatidylethanolamine and several unidentified lipids and aminolipids. The 16S rRNA gene showed 92.2–97.8% sequence identity with the members of the genus Chyseobacterium. Based on the phenotypic traits and DNA–DNA hybridizations with the type strains of the most closely related species, the isolate is shown to represent a novel species, Chyseobacterium oleae, type strain CT348T (=DSM 25575 =CCUG 63020). Emended descriptions of the genus Chryseobacterium and C. daecheongense, C. gambrini, C. gleum, C. joostei, C. jejuense, C. luteum, C. shigense, C. taiwanense, C. ureilyticum and C. vrystaatense are also proposed.

Phytophthora niederhauserii sp. nov., a polyphagous species associated with ornamentals, fruit trees and native plants in 13 countries

A non-papillate, heterothallic Phytophthora species first isolated in 2001 and subsequently from symptomatic roots, crowns and stems of 33 plant species in 25 unrelated botanical families from 13 countries is formally described here as a new species. Symptoms on various hosts included crown and stem rot, chlorosis, wilting, leaf blight, cankers and gumming. This species was isolated from Australia, Hungary, Israel, Italy, Japan, the Netherlands, Norway, South Africa, Spain, Taiwan, Turkey, the United Kingdom and United States in association with shrubs and herbaceous ornamentals grown mainly in greenhouses. The most prevalent hosts are English ivy (Hedera helix) and Cistus (Cistus salvifolius). The association of the species with acorn banksia (Banksia prionotes) plants in natural ecosystems in Australia, in affected vineyards (Vitis vinifera) in South Africa and almond (Prunus dulcis) trees in Spain and Turkey in addition to infection of shrubs and herbaceous ornamentals in a broad range of unrelated families are a sign of a wide ecological adaptation of the species and its potential threat to agricultural and natural ecosystems. The morphology of the persistent non-papillate ellipsoid sporangia, unique toruloid lobate hyphal swellings and amphigynous antheridia does not match any of the described species. Phylogenetic analysis based on sequences of the ITS rDNA, EF-1α, and β-tub supported that this organism is a hitherto unknown species. It is closely related to species in ITS clade 7b with the most closely related species being P. sojae. The name Phytophthora niederhauserii has been used in previous studies without the formal description of the holotype. This name is validated in this manuscript with the formal description of Phytophthora niederhauserii Z.G. Abad et J.A. Abad, sp. nov. The name is coined to honor Dr John S. Niederhauser, a notable plant pathologist and the 1990 World Food Prize laureate.

Proposal to reclassify Brenneria quercina (Hildebrand and Schroth 1967) Hauben et al. 1999 into a new genus, Lonsdalea gen. nov., as Lonsdalea quercina comb. nov., descriptions of Lonsdalea quercina subsp. quercina comb. nov., Lonsdalea quercina subsp. iberica subsp. nov. and Lonsdalea quercina subsp. britannica subsp. nov., emendation of the description of the genus Brenneria , reclassification of Dickeya dieffenbachiae as

Bacterial isolates from oak trees in Spain and Britain, showing symptoms of bark canker and Acute Oak Decline (AOD), respectively, were examined by a polyphasic approach. Both 16S rRNA gene sequencing and multilocus sequence analysis (MLSA), based on partial sequences of gyrB, rpoB, infB and atpD genes, revealed that the isolates were separated into two genetic groups according to their origin. Their closest phylogenetic relative was Brenneria quercina, the causal agent of drippy nut disease of oak, which clustered distant to the other species of the genus Brenneria. MLSA data for species of the genera Brenneria, Pectobacterium, Dickeya, Erwinia, Pantoea and Samsonia confirmed the polyphyletic nature of the genus Brenneria and indicated synonymy of Dickeya dadantii and Dickeya dieffenbachiae. DNA-DNA hybridization experiments confirmed this synonymy and also revealed DNA-DNA relatedness values of 58-73% between the new oak isolates and B. quercina. Phenotypic and/or chemotaxonomic methods allowed B. quercina and the two genetic groups of new oak isolates to be discriminated from other recognized species of the genus Brenneria and from members of the closely related genera Dickeya, Pectobacterium and Samsonia. Based on the data obtained, the following taxonomic proposals are made: (1) reclassification of B. quercina as the type species of a novel genus, Lonsdalea gen. nov., as Lonsdalea quercina comb. nov. (type strain LMG 2724(T)=ATCC 29281(T)=CCUG 48867(T)=CFBP 3617(T)=CIP 105201(T)=DSM 4561(T)=ICMP 1845(T)), (2) classification of the oak isolates as Lonsdalea quercina subsp. iberica subsp. nov. (type strain LMG26264(T)=NCPPB 4490(T)) and Lonsdalea quercina subsp. britannica subsp. nov. (type strain LMG 26267(T)=NCPPB 4481(T)) and leading to the automatic creation of Lonsdalea quercina subsp. quercina subsp. nov. (type strain LMG 2724(T)=ATCC 29281(T)), (3) emendation of the description of the genus Brenneria, and (4) reclassification of Dickeya dieffenbachiae as Dickeya dadantii subsp. dieffenbachiae comb. nov. (type strain LMG 25992(T)=CFBP 2051(T)), with the automatic creation of Dickeya dadantii subsp. dadantii subsp. nov. (type strain LMG 25991(T)=CFBP 1269(T)).

Ceratonia siliqua(carob) trees as natural habitat and source of infection byCryptococcus gattiiin the Mediterranean environment

Recent Cryptococcus gattii infections in humans and animals, including several outbreaks in goats, were the basis of this environmental survey in six provinces of Spain. A total of 479 samples from 20 tree species were studied. Cryptococcus gattii was found for the first time in autochthonous Mediterranean trees in Spain. Fourteen isolates of this pathogen were obtained from seven trees of three different species: 12 from carob (Ceratonia siliqua), one from Mediterranean stone pine (Pinus halepensis) and another from eucalyptus (Eucalyptus camaldulensis). All C. gattii isolates were genotype AFLP4/VGI and mating type alpha, and were found to be genetically identical with C. gattii strains isolated from humans and animals in Spain. This supports the hypothesis that these trees may be a natural source for infection of humans and mammals in the Mediterranean area.

Characterization and Pathogenicity ofBotryosphaeriaceaeSpecies Collected from Olive and Other Hosts in Spain and California

Species in the family Botryosphaeriaceae are common pathogens causing fruit rot and dieback of many woody plants. In this study, 150 Botryosphaeriaceae isolates were collected from olive and other hosts in Spain and California. Representative isolates of each type were characterized based on morphological features and comparisons of DNA sequence data of three regions: internal transcribed spacer 5.8S, β-tubulin, and elongation factor. Three main species were identified as Neofusicoccum mediterraneum, causing dieback of branches of olive and pistachio; Diplodia seriata, causing decay of ripe fruit and dieback of olive branches; and Botryosphaeria dothidea, causing dalmatian disease on unripe olive fruit in Spain. Moreover, the sexual stage of this last species was also found attacking olive branches in California. In pathogenicity tests using unripe fruit and branches of olive, D. seriata isolates were the least aggressive on the fruit and branches while N. mediterraneum isolates were the most aggressive on both tissues. Isolates of B. dothidea which cause dalmatian disease on fruit were not pathogenic on branches and only weakly aggressive on fruit. These results, together with the close association between the presence of dalmatian disease symptoms and the wound created by the olive fly (Bactrocera oleae), suggest that the fly is essential for the initiation of the disease on fruit. Isolates recovered from dalmatian disease symptoms had an optimum of 26°C for mycelial growth and 30°C for conidial germination, suggesting that the pathogen is well adapted to high summer temperatures. In contrast, the range of water activity in the medium for growth of dalmatian isolates was 0.93 to 1 MPa, which was similar to that for the majority of fungi. This study resolved long-standing questions of identity and pathogenicity of species within the family Botryosphaeriaceae attacking olive trees in Spain and California.

Genetic variation and host specificity of Phytophthora citrophthora isolates causing branch cankers in Clementine trees in Spain

Considerable tree losses have been observed during the past few years in Spain due to Phytophthora branch canker of clementines caused by Phytophthora citrophthora. The emergence of this disease led to the speculation that either the pathogen has evolved increasing its aggressiveness or specificity to clementines. A total of 134 isolates of P. citrophthora collected from 2003 to 2005 in 135 citrus orchards in Spain and 22 reference isolates were analyzed genotypically and phenotypically to determine the structure of the population. Genotypic diversity was evaluated by means of Inter-Simple Sequence Repeat (ISSR) markers. Among the phenotypic characteristics examined, sporangial characters, sexual behavior, growth rates and colony morphology of the isolates at different temperatures were studied. The aggressiveness and host-specificity of selected isolates were evaluated by pathogenicity tests on sweet oranges and clementines under field conditions. Phytophthora branch canker of clementines was associated mainly with one genotype (P-1), which included 88% of the isolates obtained from branches. Strains isolated years before the first disease outbreak clustered also with this major genotype, thus it may be considered as a predominant population. Thirteen other minor genotypes were determined, but most contained only one isolate. Although there was wide variation in the morphological and physiological characters, all Phytophthora isolates obtained from branch cankers were sexually sterile and showed a characteristic petalloid colony pattern. As in previous greenhouse studies, pathogenicity tests under field conditions demonstrated that clementines and their hybrids were more susceptible to P. citrophthora than sweet oranges. However, no evidence was found to support the hypothesis that the emergence of the disease was associated with more aggressive or host-specific forms of P. citrophthora.

CHESTNUT IN SPAIN, FROM NUT AND TIMBER PRODUCTION TO INDUSTRY

Chestnut tree in Spain not only produces nuts and timber, but it is also part of the landscape and Spanish heritage. Chestnut is present in 169,572 ha, but only in 27,511 ha were orchards in 2006 (www.mma.es). Commercialised nut production is about 20,000 t according to the FAO while the Spanish Government estimates 59,907 t (www.mma.es). Main producing areas are, in decreasing order, Galicia, Castilla-Leon (El Bierzo), Central Spain (Salamanca, Caceres and Avila), Andalucia and Canary Islands (Tenerife, La Palma, Gomera and El Hierro). Timber production is important in Northern Spain (Asturias and Galicia), Extremadura and Cataluna. Nuts are sold fresh in Spain and abroad (50% approximately) and the rest are transformed after peeling and freezing. In Spain, there are six main industries that transform chestnuts, the biggest ones are located in Galicia, more than 20 chestnut traders and six cooperatives. They produce and sell frozen and peeled nuts, marron glace, marron natural, cream, marmalade, flour, bonbons and dried nuts. Worst nuts are used as fodder for pigs in Andalucia and Galicia. Nuts are collected by growers and sold to industries, central markets and platforms by intermediaries. Minimum postharvest treatments are disinfestations, cleaning and packaging. Cooperatives located in the main producing areas commercialize more than 10% of total production. Only big producers, mostly in Central Spain, have enough production to sell by themselves. Timber production is coming from coppices in Asturias, Central Spain and Cataluna, and from grafted trees in Galicia. Best timber quality is not only for furniture making, plain-slice veneer, but also for construction. Chestnut timber is also used for making barrels (Cataluna), fences (most of the regions) and baskets (Central Spain). Chestnut contributes to the landscape everywhere, some countryside paths have been improved in to visit our centenary trees and some rural festivals are based on chestnuts, as the so-called magostos. Some old trees are protected by authorities, since they are considered as natural monuments. Chestnut is referred to in Galician literature in medieval manuscripts since 10 th century and some painters, as Castelao, have reflected their relevance in our culture. These values have encouraged research programs to promote chestnut in our country, and the increase of chestnut plantations in North Spain is noticeable. The Protected Geographical Indication (PGI) Castana de Galicia has been approved recently to improve regional productions linked to the origin and the genetic singularity of chestnut cultivars. Contracts regulated by the Government, between growers and industrials, are being signed with prices ranging from 0.4 to 1.4 euros per kg depending on the nut size and the cultivar (in order of importance, 'Famosa', 'Negral', 'Longal', 'Garrida' and 'Parede'). Chestnut research in Spain begun in the fifties with hybridisation programs, which produced hybrids resistant to ink disease commercialized nowadays. During the last years, chestnut research has been focused in forestry and nut production, propagation, landscape and genetics.

Role of the Helix aspersa snail as a vector of Phytophthora citrophthora causing branch cankers on clementine trees in Spain

This study investigated the suspected role of invertebrate vectors in the transmission of phytophthora branch canker, a severe disease of clementine cultivars in Spain, caused by Phytophthora citrophthora. Ants (Lasius grandis) and snails (Helix aspersa and Rumina decollata) were collected in spring and autumn 2005 from 15 commercial citrus fields which were severely affected by the disease. Isolations made from L. grandis and R. decollata bodies did not yield positive results. However, P. citrophthora was isolated from 5·0% of bodies of H. aspersa and 4·8% of samples of their faeces. In one assay, after snails were allowed to feed for 5 h on citrus branches which had been artificially infected with P. citrophthora, the pathogen was isolated from 79% of their faeces. In another experiment, snails were infested by placing them in contact with a substrate colonized by P. citrophthora and then transferred to the base of potted 4‐year‐old trees of cvs Clemenules, Fortune and Nova in the glasshouse. One day after their release, infested snails were widely distributed throughout the tree canopies and 10 days later bark discoloration and gum exudations were observed on the trees. Phytophthora citrophthora was readily isolated from tissues showing symptoms.

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.

Branch cankers on citrus trees in Spain caused by Phytophthora citrophthora

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.

Avocado Dieback Caused by Neofusicoccum parvum in the Andalucia Region, Spain.

From 2002 to 2006, adult avocado trees, Persea americana Miller cv. Hass, located in the subtropical-fruit-producing area of Andalucia (southern Spain) developed symptoms of dieback characterized by death of twigs and branches in the tree canopy. Sections of surface-disinfested, necrotic branch tissues were plated on Difco potato dextrose agar (PDA) (Sparks, NV) and a Neofusicoccum-like fungus was isolated. On PDA, the isolates had white, appressed mycelium that turned dull gray as the colony aged, although conidia were not formed. Abundant pycnidia and conidia developed when isolates were cultured on 2% water agar with sterilized pine needles as substratum at 25°C under near-UV light for 2 weeks. Conidia were hyaline, unicellular, ellipsoid with an obtuse apex and subtruncate base, averaged 16.2 μm long by 5.8 μm wide and ranged from 12.0 to 20.0 by 4.0 to 8.0 μm, and becoming brown with one or two septa with age. Sequenced rDNA fragments (ITS1, 5.8S rDNA, and ITS2, amplified with ITS1 and ITS4 primers) of two avocado isolates were 100% homologous with Neofusicoccum parvum (Pennycook & Samuels) Crous, Slippers, & A.J.L. Phillips (1) (GenBank Accession Nos. AM410965 and AM410966). Morphological and molecular results confirmed this species as N. parvum, reported as the anamorph of Botryosphaeria parva (1). A pathogenicity test was conducted using two isolates on sets of five 2-year-old avocado plants produced from seeds of cv. Topa-Topa growing in 5-liter pots with soil. Unwounded and wounded plants were used for inoculations. Plants were wounded 2 to 3 cm below the apical tip with a lance (4 mm long and 1 mm deep). For inoculation, 4-mm 2-week-old PDA culture plugs were placed in contact with wounded tissues and covered with Parafilm. Five noninoculated plants treated similarly served as controls. Plants were maintained in the greenhouse with a temperature range of 18 to 26°C, and 1 month later, brown stem lesions, as much as 5 cm, originating from the inoculation site followed by dieback of branches were observed. Reisolations from necrotic branches were successful, and both isolates with identical morphology to those used for inoculations were recovered. Pathogenicity tests of seedlings using the same methods also caused stem lesions on unwounded plants and the pathogen was reisolated. To our knowledge, this is the first report of N. parvum causing dieback of avocado trees in Spain. Previously, B. parva has been reported causing stem-end rot of avocado fruit in New Zealand (2). In Spain, since diseased orchards are increasing rapidly, this pathogen could be efficiently distributed by pruning activities (tools and vegetal debris) as observed with other diseases (3). The presence of N. parvum in this subtropical area presents a serious disease problem not only to avocado but also to mango (Mangifera indica L.), which is another susceptible host (4).

Modelling height and diameter growth of dominant cork oak trees in Spain

Un nouveau plan d'amenagement pour la gestion durable des peuplements de chene-liege est necessaire. Cet objectif est realisable seulement si l'on dispose des modeles de croissance permettant de prevoir les consequences de differents traitements sylvicoles. On a developpe, sur la base des donnees d'analyses de tige, deux modeles pour estimer la croissance en hauteur et en diametre des chenes-lieges dominants dans deux des regions les plus productives d'Espagne. Les equations en differences des modeles de Lundqvist-korf, Mcdill-Amateis et Richards ont ete testees et ajustees en utilisant la methode des moindres carres generalisee. Les parametres des equations ont ete exprimes en fonction de caracteristiques des peuplements pour ameliorer les modeles. La fonction de McDill-Amateis a ete retenue comme modele de la croissance en hauteur tandis qu'on a choisi l'equation de Richards avec n dans l'exposant comme parametre libre pour modele de la croissance en diametre. Ces deux modeles ameliorent nos connaissances de la croissance du chene-liege et doivent nous permettre d' ameliorer la gestion des peuplements.

Factors related to the adoption of organic farming in Spanish olive orchards

The area given over to the cultivation of organic olive trees in Spain has greatly increased in the last ten years. The diffusion of organic farming is desirable since its principles are in line with the aim of the current EU Common Agricultural Policy to harmonise agrarian production and environmental protection, and to encourage the production of quality agricultural goods. This paper characterises organic olive growers and farms in the south of Spain and compares them to their conventional counterparts. Information was gathered in a survey of 322 olive growers. The organic growers differed from their conventional counterparts in that their orchards were less productive and in the more part-time nature of their dedication to agriculture. They were also younger, more involved in management and administration of the holding, attended more courses, were more commonly members of agricultural associations, received more information via the Andalusian Committee for Organic Agriculture (a control, certification and training organization), had a more negative opinion regarding the use of chemicals, and believed that organic agriculture required more time and effort but provided greater returns.

First Report of Botryosphaeria ribis as a Branch Dieback Pathogen of Olive Trees in Spain.

Botryosphaeria spp. are known to produce cankers and dieback in several woody hosts. Since 2001, a severe disease resulting in the dieback of branches was observed in olive (Olea europaea) orchards in Andalucía, southern Spain, where the only cultivar affected was Gordal de Sevilla. The affected trees showed abundant dead twigs, and wilted leaves remained attached. These symptoms were similar to those caused by Resseliella oleisuga, a bark borer insect common in the region. Closer inspection showed no evidence of insects, but revealed reddish brown bark lesions that girdled the affected branches. When the outer bark was removed, the affected tissue appeared dark brown, in contrast to the yellowish green of healthy inner bark. A Fusicoccum sp. was consistently isolated from the necrotic bark. Conidia produced in black pycnidia were hyaline, aseptate, fusoid, with a truncate base averaging 19.9 × 6.7 μm, becoming pale brown with all cells the same color, and developing one or two septa when germinated. These characteristics conform to Fusicoccum ribis (1), the anamorph of B. ribis. Pathogenicity of two isolates from symptomatic branches was determined by stem inoculations of 4-year-old cv. Gordal de Sevilla grafted on wild olive trees growing in plastic pots containing a sand/lime/peat soil mixture. Both isolates were inoculated by three techniques. In the first, 7-mm-diameter agar plugs bearing actively growing mycelium were applied to 7-mm-diameter bark wounds made with a cork borer on the middle point of stems (averaging 10 mm in diameter). In the second technique, the same type of inoculum was applied directly to the bark without any wounding. For the third technique, 100 μl of conidial suspension in water (2 × 105 conidia per ml) was applied to bark wounds. The inoculated stem sections were wrapped in Parafilm to retain moisture. All inoculated and control plants (only sterile agar medium or sterile water added) were kept in a greenhouse and watered as needed. There were three replicate plants per isolate and inoculation technique. After 3 months, all plants inoculated with mycelium applied to wounds showed girdling and apical death with browning and wilting of leaves above the inoculation point. In the treatments where mycelium was applied to unwounded bark or where conidial suspensions were applied to wounds, small bark cankers developed to 56 mm long, but neither girdling nor foliar wilting were observed. No symptoms developed in the control plants. There were no significant differences in pathogenicity between the two F. ribis isolates. F. ribis was recovered from necrotic bark from every inoculated plant. These results demonstrate that B. ribis is pathogenic on cv. Gordal de Sevilla olives, and they indicate that bark wounding favors lesion development but is not needed for infection. Reference: (1) B. Slippers et al. Mycologia 96:83, 2004.

Agromyces ulmi sp. nov., a xylanolytic bacterium isolated from Ulmus nigra in Spain

Two xylan-degrading bacterial strains were isolated from a decayed Ulmus nigra tree in Spain. The isolates were Gram-positive, non-motile, aerobic and formed substrate mycelium which fragmented into irregular rods. 16S rRNA gene sequence analysis indicated that the isolates form a separate branch within the genus Agromyces phylogenetic cluster, with Agromyces mediolanus DSM 20152(T) being their closest relative (97.7 and 97.6 % sequence similarity). Catalase, nitrate reduction and urease tests differentiated these strains from A. mediolanus. Cell-wall peptidoglycan composition, major menaquinone, predominant fatty acids and phospholipid pattern were typical of the genus Agromyces. The DNA G+C content determined for the type strain XIL01(T) was 72 mol%. Based on the data presented, a novel species Agromyces ulmi sp. nov. is proposed. The type strain is XIL01(T) (=LMG 21954(T)=DSM 15747(T)).