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[Aims] The study aimed to assess comparatively the accuracy and efficiency of three culture media protocols for estimating black-foot disease pathogens populations in soils and to examine how shifts in the abundance and composition of black-foot pathogens correspond to changes in specific soil properties. [Methods] Firstly, culture media were compared by evaluating the mycelial growth of selected black-foot pathogens and by estimating the population of Dactylonectria torresensis from artificially infested soils. Secondly, the most efficient culture medium was selected for estimating the viable propagules of black-foot disease pathogens in eight naturally infested soils. An analysis of the soil physicochemical properties was conducted. Data were statistically analyzed in order to explore possible relationships between the studied variables. [Results] Glucose-Faba Bean Rose Bengal Agar (GFBRBA) was selected as the most efficient culture medium. All naturally infested soils tested positive for the presence of black-foot pathogens. D. torresensis was the most frequently isolated species, followed by Dactylonectria alcacerensis and Ilyonectria liriodendri. A positive relationship between calcium carbonate and the Colony-Forming Units (CFUs) level of black-foot pathogens in soil was obtained. [Conclusions] In this study, we provide an early, specific, and accurate detection of viable propagules of black-foot pathogens in soil, which is critical to understand the ecology of these fungi and to design effective management strategie, The research was funded by CAR (Government of La Rioja, Spain), under the project “Characterization, epidemiology and control of fungal trunk pathogens of grapevine in La Rioja” (project number R-03-16). We thank “Instituto Navarro de Tecnologías e Infraestructuras Agroalimentarias” (INTIA), Spain, which collected the soil samples from the field in Olite for further use in the experiment. We thank P. Yécora and M. Andrés for technical assistance. David Gramaje was supported by the DOC-INIA program from the National Institute for Agronomic Research (INIA), co-funded by the European Social Fund. Carmen Berlanas was supported by the FPI-INIA program from the INIA. Beatriz López was supported by the PhD program from the Government of La Rioja.

In this study, 3,426 grafted grapevines ready to be planted from 15 grapevine nursery fields in Northern Spain were inspected from 2016 to 2018 for black-foot causing pathogens. In all, 1,427 isolates of black-foot pathogens were collected from the asymptomatic inner tissues of surface sterilized secondary roots and characterized based on morphological features and DNA sequence data of the nuclear ribosomal DNA-internal transcribed spacer region, histone H3, translation elongation factor 1-alpha and β-tubulin genes. Eleven species belonging to the genera Dactylonectria, Ilyonectria, Neonectria, and Thelonectria were identified, including Dactylonectria alcacerensis, D. macrodidyma, D. novozelandica, D. pauciseptata, D. torresensis, Ilyonectria liriodendri, I. pseudodestructans, I. robusta, Neonectria quercicola, Neonectria sp. 1, and Thelonectria olida. In addition, two species are newly described, namely D. riojana and I. vivaria. Twenty-four isolates representing 13 black-foot species were inoculated onto grapevine seedlings cultivar ‘Tempranillo’. The pathogenicity tests detected diversity in virulence among fungal species and between isolates within each species. The most virulent species was D. novozelandica isolate BV-0760, followed by D. alcacerensis isolate BV-1240 and I. vivaria sp. nov. isolate BV-2305. This study improves our knowledge on the etiology and virulence of black-foot disease pathogens, and opens up new perspectives in the study of the endophytic phase of these pathogens in grapevines., CAR (Government of La Rioja, Spain), under the project ‘Characterization, epidemiology and control of fungal trunk pathogens of grapevine in La Rioja’ Grant/Award Number: R-03-16

[Background] Black‐foot disease is one of the main soilborne fungal diseases affecting grapevine production worldwide. Two field experiments were established to evaluate the effect of white mustard cover crop residue amendment and chemical fumigation with propamocarb + fosetyl‐Al combined with Trichoderma spp. root treatment on the viability of black‐foot inoculum in soil and fungal infection in grafted plants and grapevine seedlings used as bait plants. [Results] A total of 876 black‐foot pathogen isolates were collected from grafted plants and grapevine seedlings used as bait plants in both fields. White mustard biofumigation reduced inoculum of Dactylonectria torresensis and the incidence and severity of black‐foot of grapevine, but no added benefit was obtained when biofumigation was used with Trichoderma spp. root treatments. The effect of white mustard residues and chemical fumigation on populations of D. torresensis propagules in soil was inconsistent, possibly because of varying pretreatment inoculum levels. [Conclusion] Biofumigation with white mustard plants has potential for improving control of black‐foot disease in grapevines. This control strategy can reduce soil inoculum levels and protect young plants from infection, providing grape growers and nursery propagators with more tools for developing integrated and sustainable control systems., The research was funded by CAR (Government of La Rioja, Spain), under the project ‘Characterization, epidemiology and control of fungal trunk pathogens of grapevine in La Rioja’ (project number R‐03‐16). David Gramaje was supported by the DOC‐INIA programme from the National Institute for Agronomic Research (INIA), co‐funded by the European Social Fund. Carmen Berlanas was supported by the FPI‐INIA programme from the INIA. This study could be co‐financed by the European Regional Development Fund, through a grant to the Autonomous Community of La Rioja, within the ERDF Operational Program La Rioja 2014‐2020.

Trabajo presentado en el X Congreso Nacional de Entomología Aplicada y XVI Jornadas Científicas de la Sociedad Española de Entomología Aplicada (SEEA), celebrado en Logroño (España), del 16 al 20 de octubre de 2017

In May 2015, a survey was conducted to evaluate the phytosanitary status of almond (Prunus dulcis) propagating materials in a commercial nursery in La Rioja Province (northern Spain). Fungal isolation was performed on 7 plants (2-year-old almond ‘Guara’ grafted onto rootstock GF 677) showing collapsed branches, leaf chlorosis, and shoot dieback in approximately 20% of the trees. Black spots and dark streaking of xylem vessels were observed in cross- or longitudinal sections of the stems. Symptomatic branches and stems were collected and wood sections (10 cm long) were cut, washed in 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 of streptomycin sulfate. Plates 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 isolation points). 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 (Mostert et al. 2006). Colonies were pale brown on PDA and yellowish white on MEA. Conidiophores were short and usually unbranched, 17 to 41 (mean 28) µm long. Phialides were terminal or lateral, mostly monophialidic. Conidia were hyaline, oblong-ellipsoidal or cylindrical, 3 to 4 (mean 3.9) µm long, 1 to 2 (mean 1.5) µm wide. Based on these characters, the isolates were identified as P. minimum (Tul. & C. Tul.) D. Gramaje, L. Mostert & Crous (Mostert et al. 2006; Gramaje et al. 2015). DNA sequencing of a fragment of the beta-tubulin gene of the isolates BV-008 and BV-009 using primers T1 (O’Donnell and Cigelnik 1997) and Bt2b (Glass and Donaldson 1995) (GenBank Accession Nos. KU094046 and KU094047) matched P. minimum Accession No. HQ605013. Pathogenicity tests were conducted using isolates BV-008 and BV-009. Two-year-old almond trees of cv. Ferragnes grown in pots were wounded in the stem with a 8-mm cork borer. A 8-mm mycelium PDA plug from a two-week-old culture was placed in the wound before being wrapped with Parafilm. Control plants were inoculated with 8-mm noncolonized PDA plugs. There were 10 replicate plants per isolate and ten controls, and the experiment was repeated once. Inoculated plants were immediately planted in a field site in Logroño (La Rioja). Within 4 months, shoots on all Phaeoacremonium-inoculated stems had weak growth with chlorosis of leaves and there was black streaking in the xylem vessels. The vascular necroses that developed on the inoculated plants were 5.4 ± 0.5 cm long (isolate BV-008) and 5.6 ± 0.5 cm long (isolate BV-009), significantly greater than those on the control plants (P < 0.01). Control plants did not show any symptoms. The fungus was reisolated from discolored tissue of all inoculated stems, completing Koch’s postulates. To our knowledge, this is the first report of P. minimum associated with wood decay of almond nursery stock in Spain or any country in the world. Phaeoacremonium minimum is the most common Phaeoacremonium species associated with esca and Petri disease in grapevines, thereby causing untenable economic losses to the grapevine industry worldwide (Mostert et al. 2006). This study demonstrates the risk for the almond industry posed by the potential spread of P. minimum using contaminated planting material.