J. A. Torés

Bacterial apical necrosis of mango in southern Spain: a disease caused by Pseudomonas syringae pv. syringae

ABSTRACT A necrotic bacterial disease of mango trees (Mangifera indica) in Spain affecting buds, leaves, and stems is described for the first time. Necrosis of flower and vegetative buds on commercial trees during winter dormancy was the most destructive symptom of the disease. The apical necrosis is caused by Pseudomonas syringae, which was always isolated from mango trees with disease symptoms. Of 95 bacterial strains isolated from symptomatic tissues and characterized from 1992 to 1997, over 90% were identified as P. syringae pv. syringae. Additional strains were isolated from healthy mango trees, and they were identical to the isolates from diseased tissues. Pathogenicity tests on mango plants showed that P. syringae pv. syringae incited the apical necrosis, but that climatic conditions determined the onset of disease development. Populations of total bacteria and of P. syringae and the number of active ice nuclei were monitored over a 3-year period. The largest populations of P. syringae were associated with cool, wet periods that coincided with the highest disease severity, whereas P. syringae was only occasionally detected on healthy trees. The median effective dose was estimated from infectivity titration assays.

First Report of Mango Malformation Disease Caused by Fusarium mangiferae in Spain

Mango (Mangifera indica L.) malformation disease (MMD) is one of the most important diseases affecting this crop worldwide, which causes severe economic losses because of the reduction of productivity. Symptoms of MMD in Spain were observed for the first time in April of 2006 in three mango orchards in the Axarquia Region (southern Spain). Symptoms included an abnormal development of vegetative shoots with shortened internodes and dwarfed leaves and hypertrophied short and thickened panicles. In the years of 2006, 2009, and 2010, isolates of Fusarium were obtained from vegetative shoots and floral tissue of symptomatic mango trees from 21 different orchards of cvs. Keitt, Kent, Osteen, Tommy Atkins, and a variety of minor commercial cultivars, all showing typical symptoms of MMD. Different Fusarium-like strains were isolated from infected tissues. Colonies from single-spored isolates possessed dark purple-to-salmon-colored mycelium when grown on potato dextrose agar medium. On fresh carnation leaf agar medium, mycelium contained aerial conidiophores possessing three- to five-celled macroconidia and abundant microconidia in false heads from mono- and polyphialides; while cream-orange-colored sporodochia were produced on the surface of the medium, typical for Fusarium mangiferae. The identification of 37 isolates was confirmed as F. mangiferae by species-specific PCR analysis with the primer pair 1-3 F/R that amplified a 608-bp DNA fragment from all Spanish isolates as well as a representative Israeli control strain, Fus 34, also designated as MRC7560 (2). Pathogenicity using four representative isolates, UMAF F02, UMAF F10, UMAF F17, and UMAF F38 of F. mangiferae from Spain as well as isolate MRC7560, was tested on 2-year-old healthy mango seedlings cv. Keitt by inoculating 15 buds from three different trees with a 20-μl conidial suspension (5 × 107 conidia per ml) per isolate (1). This experiment was conducted twice with two independent sets of plants and at different times (March and November 2010). Typical mango malformation symptoms were detected after bud break in March 2011, 5 and 12 months after inoculation. Symptoms were observed for 60% of the inoculated buds with the four F. mangiferae Spanish isolates and 75% with the MRC7560 control strain, but not with water-inoculated control plants. Recovered isolates from the infected floral and vegetative malformed buds were identical morphologically to those inoculated, and the specific 608-bp fragment described for F. mangiferae was amplified with specific-PCR, thus fulfilling Kochs postulates. To our knowledge, this is the first report of mango malformation disease caused by F. mangiferae in Spain and Europe. References: (1) S. Freeman et al. Phytopathology 89:456, 1999. (2) Q. I. Zheng and R. C. Ploetz. Plant Pathol. 51:208, 2002.

Analysis of Genetic Diversity of Fusarium tupiense, the Main Causal Agent of Mango Malformation Disease in Southern Spain

Mango malformation disease (MMD) has become an important global disease affecting this crop. The aim of this study was to identify the main causal agents of MMD in the Axarquía region of southern Spain and determine their genetic diversity. Fusarium mangiferae was previously described in the Axarquía region but it represented only one-third of the fusaria recovered from malformed trees. In the present work, fusaria associated with MMD were analyzed by arbitrary primed polymerase chain reaction (ap-PCR), random amplified polymorphic DNA (RAPD), vegetative compatibility grouping (VCG), a PCR screen for mating type idiomorph, and phylogenetic analyses of multilocus DNA sequence data to identify and characterize the genetic diversity of the MMD pathogens. These analyses confirmed that 92 of the isolates were F. tupiense, which was previously only known from Brazil and Senegal. In addition, two isolates of a putatively novel MMD pathogen were discovered, nested within the African clade of the Fusarium fujikuroi species complex. The F. tupiense isolates all belonged to VCG I, which was first described in Brazil, and the 11 isolates tested showed pathogenicity on mango seedlings. Including the prior discovery of F. mangiferae, three exotic MMD pathogenic species have been found in southern Spain, which suggests multiple independent introductions of MMD pathogens in the Axarquía region.

First Occurrence of Cucurbit Powdery Mildew Caused by Race 3-5 of Podosphaera fusca in Spain

A new race of cucurbit powdery mildew was observed for the first time on melon (Cucumis melo) in three research greenhouses in the Axarquia area of southern Spain during the spring of 2008. Fungal growth appeared as white powdery colonies initially restricted to the upper leaf surfaces. Morphological characteristics of colonies, conidiophores, conidia, germ tubes, and appressoria indicated that the powdery mildew fungus was Podosphaera fusca (also known as P. xanthii) (3), a fungal pathogen extensively reported in the area (1). However, the fungus developed on plants of melon cv. PMR 6, which is resistant to races 1 and 2 of P. fusca, suggesting that the fungus could belong to race 3, a race of P. fusca not yet reported in Spain. Race determination was carried out by inoculating the third true leaf of a set of differential melon genotypes that were maintained in a greenhouse. Symptoms and colonization observed on cvs. Rochet, PMR 45, PMR 6, and Edisto 47 indicated that the isolates belonged to race 3-5 of P. fusca. Fungal strains of races 1, 2, and 5 of P. fusca (all present in Spain) were used as controls. Pathotype designation was determined by inoculating different cucurbit genera and species (2). In addition to melon, the isolates were pathogenic on zucchini (Cucurbita pepo) cv. Diamant F1, but failed to infect cucumber (C. sativus) cv. Marketer and watermelon (Citrullus lanatus) cv. Sugar Baby; therefore, the isolates were pathotype BC (2). Races 1, 2, 4, and 5 of P. fusca have been previously reported in the area (1). The occurrence of race 3-5 represents another challenge in the management of cucurbit powdery mildew in Spain. References: (1) D. del Pino et al. Phytoparasitica 30:459, 2002. (2) E. Křístková et al. Sci. Hortic. 99:257, 2004. (3) A. Pérez-García et al. Mol. Plant Pathol. 10:153, 2009.

Sensitivity of Podosphaera xanthii populations to anti-powdery-mildew fungicides in Spain

BACKGROUND Cucurbit powdery mildew caused by Podosphaera xanthii limits crop production in Spain, where disease control is largely dependent on fungicides. In previous studies, high levels of resistance to QoI and DMI fungicides were documented in south-central Spain. The aim of this study was to investigate the sensitivity of P. xanthii populations to other fungicides and to provide tools for improved disease management. RESULTS Using a leaf-disc assay, sensitivity to thiophanate-methyl, bupirimate and quinoxyfen of 50 isolates of P. xanthii was analysed to determine discriminatory concentrations between sensitive and resistant isolates. With the exception of thiophanate-methyl, no clearly different groups of isolates could be identified, and as a result, discriminatory concentrations were established on the basis of the maximum fungicide field application rate. Subsequently, a survey of P. xanthii resistance to these fungicides was carried out by testing a collection of 237 isolates obtained during the 2002-2011 cucurbit growing seasons. This analysis revealed very high levels of resistance to thiophanate-methyl (95%). By contrast, no resistance to bupirimate and quinoxyfen was found. CONCLUSION Results suggest that thiophanate-methyl has become completely ineffective for controlling cucurbit powdery mildew in Spain. By contrast, bupirimate and quinoxyfen remain as very effective tools for cucurbit powdery mildew management.

Powdery Mildew of Dill (Anethum graveolens): A New Disease Caused by Erysiphe heraclei Detected in Spain

Powdery mildew was observed for the first time on dill (Anethum graveolens L.) in several commercial greenhouses in Almería (southern Spain) during the spring and summer of 2002. Fungal growth appeared as typical white, dense, persistent powdery mildew colonies on leaves, inflorescences, and stems. Hyphae were 6 to 10 μm wide (mean = 6.88, standard deviation [SD] = 1.22, and n = 50). Conidia were produced singly on unbranched three-celled conidiophores, were cylindrical to ovate, and ranged in length from 26 to 42 μm (mean = 33.7, SD = 4.33, n = 55) and width from 12 to 18 μm (mean = 14.4, SD = 1.46). No fibrosin bodies were observed. Germ tubes were formed from the ends of conidia. Appressoria from mycelia were lobed. Conidiophores were 64 to 154 μm long (mean = 110, SD = 19.86, n = 30) with straight foot cells 24 to 42 μm long (mean = 33.8, SD = 6.17) and 6 to 10 μm wide (mean = 8.4, SD = 1). No cleistothecia were found so an accurate identification of the species was not possible. However, on the basis of morphological characteristics of the imperfect state, this powdery mildew corresponds with Erysiphe heraclei, the powdery mildew of umbelliferous crops (1) that was previously reported on dill from France and Portugal (2) and recently from Turkey (3). In Spain, the disease has been previously reported in other umbelliferous plants, such as carrot and celery (2), which are common crops in southern Spain. When infected with E. heraclei, these plants can serve as potential sources of inoculum. References: (1) U. Braun. Page 216 in: A Monograph of Erysiphales (Powdery Mildew). Nova Hedwigia. J. Cramer Berlin-Stuttgart, 1987. (2) U. Braun. Pages 116-117 in: The Powdery Mildew (Erysiphales) of Europe. Gustav Fischer-Verlag, Jena, Germany, 1995. (3) E. M. Soylu and S. Soylu. Plant Pathol. 52:423, 2003.

First Report of Powdery Mildew Elicited by Podosphaera fusca (Synonym Podosphaera xanthii) on Euryops pectinatus in Spain

Euryops pectinatus Cass. is an evergreen shrub that is planted extensively in Spain for landscape use. In 2007, powdery mildew outbreaks were observed on E. pectinatus in several nurseries located in the Axarquia area (Malaga, southern Spain). Fungal growth appeared as typical, white, powdery mildew colonies that were restricted to upper leaf surfaces. Initially, individual colonies were small and nearly circular, but later enlarged and coalesced to cover the whole leaf surface. With progress of the disease, all green parts (leaves, stems, and petioles) were covered with a white mycelium. Newly developed leaves especially became rapidly infected. Diseased leaves ultimately dried up and senesced, making nursery plants aesthetically unattractive and unsaleable. Conidiophores were erect, had crenate edge lines, cylindrical foot cells that measured 37.5 to 45.0 × 10.0 to 12.5 μm, and were followed by one to three shorter cells. Conidia were hyaline, ellipsoid to ovoid, borne in chains, and measured 27.5 to 35.0 × 12.5 to 17.5 μm. Conidial length-to-width ratios ranged from 1.6 to 2.4. Conidia possessed conspicuous fibrosin bodies and from their sides produced short germ tubes. No chasmothecia were found. The nuclear rDNA internal transcribed spacer (ITS) regions were amplified by PCR and sequenced (GenBank Accession No. EU424056). On the basis of morphological characteristics of the imperfect state and ITS sequence data, this powdery mildew was identified as Podosphaera fusca (Fr.) U. Braun & N. Shishkoff (1), this isolate belonging to ITS haplotype 15 (group III) (3); this group is considered a separate species, P. xanthii (Castagne) U. Braun & N. Shishkoff by some authors (2). Pathogenicity was confirmed by gently pressing diseased leaves onto leaves of healthy E. pectinatus plants. Plants were incubated in a growth chamber at 25°C, and after 14 days, powdery mildew colonies developed. A similar disease of E. pectinatus was observed in 1999 in California (4). P. fusca parasitizes a large number of asteraceous species including field marigold (Calendula arvensis) and fleabane (Erigeron sp.) weeds, as previously reported in the same area, and ornamentals such as Calendula officinalis, Chrysanthemum spp., and Gerbera spp., which are also grown in the same nurseries and frequently attacked by powdery mildew. References: (1) U. Braun and S. Takamatsu. Schlechtendalia 4:1, 2000. (2) U. Braun et al. Schlechtendalia 7:45, 2001. (3) T. Hirata et al. Can. J. Bot. 78:1521, 2000. (4) G. S. Saenz et al. Plant Dis. 84:1048, 2000.