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. References: (1) P. Crous et al. Stud. Mycol. 55:235, 2006. (2) B. Slippers et al. Mycologia 96:1030, 2004.
During the early spring of 2004, an estimated 20% of containerized nursery stocks of Rhododendron spp. in Asturias (northern Spain) were affected by a foliar disease that has reoccurred annually. Leaf spots were dark brown to almost black, generally oval to round, visible from both sides of the leaf, and expanded to affect the entire leaf including the petiole. Affected leaves abscised from the plant. A Phytophthora sp. was consistently isolated from symptomatic leaf tissues on PARBH medium (3) and hyphal tips were transferred onto potato dextrose agar (PDA). Colonies grown on PDA at 20°C were submerged, had a growth rate of 2.2 mm/day, and had lobes of compact mycelium. Sporangia were semipapillate and caducous with a pedicel (20.0-) 37.7 (-52.5) μm long. Sporangia were asymmetrical in shape with the broadest point near the apex: 25.2 to 40.4 μm long × 10.2 to 15.8 μm wide (average 33.1 × 12.6 μm), and length/width ratio was 2.8:1. Chlamydospores were not observed. Isolates were homothallic and oogonia ranged from 26.5 to 27.5 μm in diameter. Antheridia were mostly amphigynous but occasionally paragynous. Oospores were plerotic and 23.1 to 25.5 μm in diameter. These characteristics conformed to those of Phytophthora hibernalis Carne (2). Sequences of the internal transcribed spacer regions on the isolates and comparison with other sequences in GenBank showed that they were identical to P. hibernalis (Accession No. AY827556.1 from Citrus sp.). For pathogenicity tests, four isolates of P. hibernalis were used to inoculate detached leaves of Rhododendron hybrid Brigitte. The underside of five detached leaves was inoculated with a drop of 40 μL of a suspension of 104 zoospores/ml. Controls were inoculated with a 40-μL drop of sterile distilled water. Leaves were incubated in a moist chamber at 20°C in the dark. A quantification of the lesion area was made 8 days after inoculation using the software Assess-APS. All inoculated leaves developed necrotic lesions that ranged from 0.246 to 1.512 cm2. P. hibernalis was reisolated from infected tissue. Symptoms were not detected on the controls. The test was repeated twice and similar results were obtained each time. P. hibernalis has been described previously as causing brown rot on citrus in Spain (4) and was isolated from rhododendron plants in California and Oregon (1). To our knowledge, this is the first record of P. hibernalis causing foliar blight on Rhododendron species in Spain as well as in Europe. References: (1) C. Blomquist et al. Online publication. doi:10.1094/PHP-2005-0728- 01-HN. Plant Health Progress, 2005. (2) D. C. Erwin and O. K. Ribeiro. Phytophthora Diseases Worldwide. The American Phytopathological Society, St. Paul MN. 1996. (3) S. N. Jeffers and S. B. Martin. Plant Dis. 70:1038, 1986. (4) J. J. Tuset. An. Inst. Nac. Investig. Agrar. Ser. Prot. Veg. N.7, 1977.
Entre agosto de 2005 y septiembre de 2007 se realizó la prospección de enemigos naturales de áfidos en alfalfa (Medicago sativa L.), trébol rojo (Trifolium pratense L.) y lotus (Lotus corniculatus L.). La misma incluyó semilleros comerciales de cada leguminosa en La Estanzuela (Colonia) y un área de lotus de pastoreo en EEMAC (Paysandú). Los muestreos se realizaron con frecuencia quincenal y consistieron en corte de plantas en 30 cuadrados de 30 x 30 cm. En laboratorio, los áfidos muertos se separaron de las plantas y se conservaron individualmente hasta identificar la causa de mortalidad. Se identificaron los áfidos presentes y sus poblaciones se estimaron mediante método volumétrico. Las especies de áfidos registradas en los diferentes cultivos y situaciones, en el período, fueron: Therioaphis trifolii (Monnell), Acyrthosiphon pisum (Harris), Acyrthosiphon kondoi Shinji, Aphis craccivora Koch y Nearctaphis bakeri (Cowen). Los principales agentes de mortalidad fueron parasitoides y el hongo Pandora neoaphidis (Remaudière y Hennebert) Humber (Entomophthoromycotina: Entomophthorales). Tanto las poblaciones como la mortalidad natural de áfidos tuvieron variaciones amplias entre años y en las diferentes situaciones muestreadas. El hongo provocó mayor mortalidad que los parasitoides, aunque su acción estuvo restringida a los meses de otoño e invierno, en los dos años. Los parasitoides estuvieron presentes en todas las situaciones de muestreo. En lotus, en ambas regiones y zafras el total de parasitoides colectados fue igual o mayor que el de P. neoaphidis. Contrariamente, en trébol rojo y alfalfa la colecta total de este hongo fue siempre significativamente mayor que la de parasitoides.
The methodology of a spaced plant seed increase nursery to produce pre-basic seed of the subtropical forage legume Lotononis bainesii Baker cv. 'INIA Glencoe' is presented. Twenty grams of nucleus seed were grown for 2 months in a glasshouse in a forestry nursery. The best 30 000 plants were transplanted in September 2002 in a 1 × 1 m grid to a seed increase area at Glencoe research station of INIA Tacuarembó, Uruguay. After one multiplication cycle, 104.8 kg of seed was produced giving a multiplication rate of 5240 times and a saving in time of at least 3 years in the seed multiplication process.
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