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[EN] Grapevine trunk diseases (GTDs) are a big threat for global viticulture. Without effective chemicals, biocontrol strategies are developed as alternatives to better cope with environmental concerns. A combination of biological control agents (BCAs) could even improve sustainable disease management through complementary ways of protection. In this study, we evaluated the combination of Bacillus subtilis (Bs) PTA-271 and Trichoderma atroviride (Ta) SC1 for the protection of Chardonnay and Tempranillo rootlings against Neofusicoccum parvum Bt67, an aggressive pathogen associated to Botryosphaeria dieback (BD). Indirect benefits offered by each BCA and their combination were then characterized in planta, as well as their direct benefits in vitro. Results provide evidence that (1) the cultivar contributes to the beneficial effects of Bs PTA-271 and Ta SC1 against N. parvum, and that (2) the in vitro BCA mutual antagonism switches to the strongest fungistatic effect toward Np-Bt67 in a three-way confrontation test. We also report for the first time the beneficial potential of a combination of BCA against Np-Bt67 especially in Tempranillo. Our findings highlight a common feature for both cultivars: salicylic acid (SA)-dependent defenses were strongly decreased in plants protected by the BCA, in contrast with symptomatic ones. We thus suggest that (1) the high basal expression of SA-dependent defenses in Tempranillo explains its highest susceptibility to N. parvum, and that (2) the cultivar-specific responses to the beneficial Bs PTA-271 and Ta SC1 remain to be further investigated., Funding This work was supported by a French Grant from the Region GRAND-EST France and the City of GRAND-REIMS France through the BIOVIGNE Ph.D. program, whose functioning is supported by BELCHIM Crop Protection France. DG was supported by the Ramon y Cajal program, Spanish Government (RyC-2017-23098).

[EN] Fungal grapevine trunk diseases (GTDs) are some of the most pressing threats to grape production worldwide. While these diseases are associated with several fungal pathogens, Phaeomoniella chlamydospora and Phaeoacremonium minimum are important contributors to esca and Petri diseases. Recent research has linked grapevine xylem diameter with tolerance to Pa. chlamydospora in commercial rootstocks. In this study, we screen over 25 rootstocks for xylem characteristics and tolerance to both Pa. chlamydospora and Pm. minimum. Tolerance was measured by fungal incidence and DNA concentration (quantified via qPCR), while histological analyses were used to measure xylem characteristics, including xylem vessels diameter, density, and the proportion of the stem surface area covered by xylem vessels. Rootstocks were grouped into different classes based on xylem characteristics to assess the potential association between vasculature traits and pathogen tolerance. Our results revealed significant differences in all the analyzed xylem traits, and also in DNA concentration for both pathogens among the tested rootstocks. They corroborate the link between xylem vessels diameter and tolerance to Pa. chlamydospora. In Pm. minimum, the rootstocks with the widest xylem diameter proved the most susceptible. This relationship between vasculature development and pathogen tolerance has the potential to inform both cultivar choice and future rootstock breeding to reduce the detrimental impact of GTDs worldwide., Funding This research was founded by FEDER funding through a State Program of I + D + i oriented to the Challenges of Society (RTA2015-00015-C02-00), supported by The National Institute for Agricultural and Food Research and Technology (INIA). DG was supported by the Ramon y Cajal program, Spanish Government (RyC-2017-23098). This research has been developed as a result of a mobility stay funded by the Erasmus+ KA1 Erasmus Mundus Joint Master Degrees Programme of the European Commission under the PLANT HEALTH Project.

Fungal grapevine trunk diseases (GTDs) are some of the most pressing threats to grape production worldwide. While these diseases are associated with several fungal pathogens, Phaeomoniella chlamydospora and Phaeoacremonium minimum are important contributors to esca and Petri diseases. Recent research has linked grapevine xylem diameter with tolerance to Pa. chlamydospora in commercial rootstocks. In this study, we screen over 25 rootstocks for xylem characteristics and tolerance to both Pa. chlamydospora and Pm. minimum. Tolerance was measured by fungal incidence and DNA concentration (quantified via qPCR), while histological analyses were used to measure xylem characteristics, including xylem vessels diameter, density, and the proportion of the stem surface area covered by xylem vessels. Rootstocks were grouped into different classes based on xylem characteristics to assess the potential association between vasculature traits and pathogen tolerance. Our results revealed significant differences in all the analyzed xylem traits, and also in DNA concentration for both pathogens among the tested rootstocks. They corroborate the link between xylem vessels diameter and tolerance to Pa. chlamydospora. In Pm. minimum, the rootstocks with the widest xylem diameter proved the most susceptible. This relationship between vasculature development and pathogen tolerance has the potential to inform both cultivar choice and future rootstock breeding to reduce the detrimental impact of GTDs worldwide.

Plant roots support complex microbial communities that can influence nutrition, plant growth, and health. In grapevine, little is known about the impact of abiotic stresses on the below-ground microbiome. In this study, we examined the drought-induced shifts in fungal composition in the root endosphere, the rhizosphere and bulk soil by internal transcribed spacer (ITS) high-throughput amplicon sequencing (HTAS). We imposed three irrigation regimes (100%, 50%, and 25% of the field capacity) to one-year old grapevine rootstock plants cv. SO when plants had developed 2–3 roots. Root endosphere, rhizosphere, and bulk soil samples were collected 6-and 12-months post-plantation. Drought significantly modified the overall fungal composition of all three compartments, with the root endosphere compartment showing the greatest divergence from well-watered control (100%). The overall response of the fungal microbiota associated with black-foot disease (Dactylonectria and “Cylindrocarpon” genera) and the potential biocontrol agent Trichoderma to drought stress was consistent across compartments, namely that their relative abundances were significantly higher at 50–100% than at 25% irrigation regime. We identified a significant enrichment in several fungal genera such as the arbuscular mycorrhizal fungus Funneliformis during drought at 25% watering regime within the roots. Our results reveal that drought stress, in addition to its well-characterized effects on plant physiology, also results in the restructuring of grapevine root microbial communities, and suggest the possibility that members of the altered grapevine microbiota might contribute to plant survival under extreme environmental conditions., This work was partially supported by project No. TP01010018 and by the infrastructure obtained from project CZ.02.1.01/0.0/0.0/16_017/0002334.

Grapevine (Vitis vinifera L.) is one of the most economically important cultivated perennial plants worldwide and is susceptible to an array of diseases caused by pathogenic fungi, bacteria, viruses, and oomycetes. In recent decades, extensive efforts have been made to reduce the use of chemical pesticides in viticulture by developing and deploying more sustainable alternative solutions. This Research Topic covers the most recent advances on both basic and applied research on grapevinemicrobe interactions. It provides an overview on (i) the different resistance loci/genes evolved in grapevine species and their application for breeding new resistant cultivars; (ii) the molecular basis of grapevine susceptibility and resistance to pathogens; (iii) the biocontrol and integrated grapevine disease management including induced plant innate immunity by beneficial microbes and natural elicitors; and (iv) the functional properties of endophytic or rhizospheric microbial communities that promote plant health.

© 2020 by the authors., Citrus trees with cankers and dieback symptoms were observed in Bushehr (Bushehr province, Iran). Isolations were made from diseased cankers and branches. Recovered fungal isolates were identified using cultural and morphological characteristics, as well as comparisons of DNA sequence data of the nuclear ribosomal DNA-internal transcribed spacer region, translation elongation factor 1α, β-tubulin, and actin gene regions. Dothiorella viticola, Lasiodiplodia theobromae, Neoscytalidium hyalinum, Phaeoacremonium (P.) parasiticum, P. italicum, P. iranianum, P. rubrigenum, P. minimum, P. croatiense, P. fraxinopensylvanicum, Phaeoacremonium sp., Cadophora luteo-olivacea, Biscogniauxia (B.) mediterranea, Colletotrichum gloeosporioides, C. boninense, Peyronellaea (Pa.) pinodella, Stilbocrea (S.) walteri, and several isolates of Phoma, Pestalotiopsis, and Fusarium species were obtained from diseased trees. The pathogenicity tests were conducted by artificial inoculation of excised shoots of healthy acid lime trees (Citrus aurantifolia) under controlled conditions. Lasiodiplodia theobromae was the most virulent and caused the longest lesions within 40 days of inoculation. According to literature reviews, this is the first report of L. theobromae and N. hyalinum on citrus in Iran. Additionally, we report several Phaeoacremonium species, S. walteri, Pa. pinodella and C. luteo-olivacea on citrus trees for the first time in the world.

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. © 2018 Society of Chemical Industry.

Two hundred and twenty-five Diaporthe isolates were collected from 2005 to 2019 in almond orchards showing twig cankers and shoot blight symptoms in five different regions across Spain. Multilocus DNA sequence analysis with five loci (ITS, tub, tef-1α, cal and his), allowed the identification of four known Diaporthe species, namely: D. amygdali, D. eres, D. foeniculina and D. phaseolorum. Moreover, a novel phylogenetic species, D. mediterranea, was described. Diaporthe amygdali was the most prevalent species, due to the largest number of isolates (85.3%) obtained from all sampled regions. The second most frequent species was D. foeniculina (10.2%), followed by D. mediterranea (3.6%), D. eres and D. phaseolorum, each with only one isolate. Pathogenicity tests were performed using one-year-old almond twigs cv. Vayro and representative isolates of the different species. Except for D. foeniculina and D. phaseolorum, all Diaporthe species were able to cause lesions significantly different from those developed on the uninoculated controls. Diaporthe mediterranea caused the most severe symptoms. These results confirm D. amygdali as a key pathogen of almonds in Spain. Moreover, the new species, D. mediterranea, should also be considered as a potential important causal agent of twig cankers and shoot blight on this crop., Research funded by the Instituto Nacional de Investigación y Tecnología Agraria y Alimentaria (INIA), grants RTA2017-00009-C04-01, -02, -03 and -04 and with matching funds from the European Regional Development Fund (ERDF). G. Elena and C. Agustí-Brisach were supported by the Spanish post-doctoral grants “Juan de la Cierva-Formación” and “Juan de la Cierva-Incorporación”, respectively. J. Luque and X. Miarnau were partially supported by the CERCA program, Generalitat de Catalunya. D. Gramaje was supported by the Ramón y Cajal program, Spanish Government (RYC-2017-23098).

[Background] Black‐foot and Petri diseases are the main fungal diseases associated with young grapevine decline. Two field experiments were established to evaluate the preventive effect of two potential biocontrol agents (BCAs), that is Streptomyces sp. E1 + R4 and Pythium oligandrum Po37, and three BCA‐commercial products containing Trichoderma atroviride SC1, Trichoderma koningii TK7 and Pseudomonas fluorescens + Bacillus atrophaeus on fungal infection in grafted plants and plant growth parameters. [Results] The effectiveness of some BCA in reducing the incidence and severity of both diseases was dependent on the plant part analyzed and the plant age. No single BCA application was able to control both diseases. Streptomyces sp. E1 + R4 were able to reduce significantly the infection of the most prevalent black‐foot disease fungi while P. oligandrum Po37 and Trichoderma spp. were able to reduce significantly Phaeomoniella chlamydospora and Phaeoacremonium minimum (Petri disease) infection. BCA treatments had no effect on the shoot weight, and root weight was significantly lower in all BCA treatments with respect to the control. [Conclusions] The combination of the disease‐suppressive activity of two or more beneficial microbes in a biocontrol preparation is required to prevent infection by black‐foot and Petri disease fungi in vineyards.

Diaporthe species are important pathogens, saprobes, and endophytes on grapevines. Several species are known, either as agents of pre- or post-harvest infections, as causal agents of many relevant diseases, including swelling arm, trunk cankers, leaf spots, root and fruit rots, wilts, and cane bleaching. A growing body of evidence exists that a class of small non-coding endogenous RNAs, known as microRNAs (miRNAs), play an important role in post-transcriptional gene regulation, during plant development and responses to biotic and abiotic stresses. In this study, we explored differentially expressed miRNAs in response to Diaporthe eres and Diaporthe bohemiae infection in Vitis vinifera cv. Chardonnay under in vitro conditions. We used computational methods to predict putative miRNA targets in order to explore the involvement of possible pathogen response pathways. We identified 136 known and 41 new miRNA sequence variants, likely generated through post-transcriptional modifications. In the Diaporthe eres treatment, 61 known and 17 new miRNAs were identified while in the Diaporthe bohemiae treatment, 101 known and 21 new miRNAs were revealed. Our results contribute to further understanding the role miRNAs play during plant pathogenesis, which is possibly crucial in understanding disease symptom development in grapevines infected by D. eres and D. bohemiae.

[EN] Weeds were sampled in grapevine rootstock mother fields, open-root field nurseries, and commercial vineyards of Albacete, Alicante, Castellón, Murcia, and Valencia provinces in Spain between June 2009 and June 2010 and evaluated as potential hosts of black-foot and Petri disease pathogens. Isolations were conducted in the root system and internal xylem tissues for black-foot and Petri disease pathogens, respectively. Cylindrocarpon macrodidymum was successfully isolated from the roots of 15 of 19 weed families evaluated and 26 of 52 weed species. Regarding Petri disease pathogens, one isolate of Phaeomoniella chlamydospora was obtained from Convolvulus arvensis, and three isolates of Cadophora luteo-olivacea were obtained from Bidens subalternans, Plantago coronopus, and Sonchus oleraceus. Pathogenicity tests showed that Cylindrocarpon macrodidymum isolates obtained from weeds were able to induce typical black-foot disease symptoms. When inoculated in grapevines, isolates of Cadophora luteo-olivacea and Phaeomoniella chlamydospora were also shown to be pathogenic on grapevine cuttings. Our ability to recover grapevine pathogens from vineyard weeds and to demonstrate pathogenicity of recovered strains on grape suggests that these weeds may serve as a source of inoculum for infection of grapevine. © 2011 The American Phytopathological Society., This research was financially supported by the projects RTA2007-00023-C04-03 and RTA2010-00009-C03-03 (Instituto Nacional de Investigacion y Tecnologia Agraria y Alimentaria, INIA, Spain) and the European Regional Development Fund (ERDF). We thank A. Rustignoli and V. Garrigues for technical assistance and R. Raposo for providing valuable advice on the writing of this manuscript.

From 2007 to 2009, Cylindrocladiella-like isolates were recovered from grapevine (Vitis vinifera L.) roots with symptoms of black-foot disease in Spain, where the causal agents of this disease have been previously reported as Campylocarpon and Cylindrocarpon species (1,2). Three representative isolates were selected to confirm their identity: CPa1 and CPa2 from Asturias (northern Spain), and CPe523 from Cuenca (central Spain). Isolates were incubated on malt extract agar (MEA) and Spezieller Nährstoffarmer Agar (SNA) with carnation leaves (4) at 25°C for 10 days in darkness. On MEA, colonies developed light brown, cottony mycelium. On SNA, all three isolates produced chlamydospores in chains, and conidia were zero-to one-septate, but CPa1 and CPa2 produced longer conidia (10.4 to 18.9 [15.3] × 1.7 to 3.1 [2.4] μm) than CPe523 (6.4 to 12.3 [9.7] × 1.6 to 3.3 [2.4] μm). A fragment of the beta-tubulin gene from all isolates was sequenced with primers T1 and Bt2b (1) and deposited in GenBank (Accession Nos. JQ693133, JQ693134, and JQ693135). CPa1 and CPa2 showed high similarity (99%) to Cylindrocladiella parva (AY793486) and CPe523 showed high similarity (99%) to C. peruviana (AY793500), which is in agreement with the corresponding morphological features of these species (4). Pathogenicity tests were conducted with inoculum produced on wheat (Triticum aestivum L.) seed soaked for 12 h in 300 ml of distilled water and autoclaved three times. Inoculum was prepared by inoculating two fungal disks (8 mm in diameter) of a 2-week-old culture of each isolate grown on potato dextrose agar to wheat seed and incubation at 25°C for 4 weeks. One-month-old grapevine seedlings were planted individually in 220-cc pots filled with a potting medium of sterilized peat moss and 10 g of inoculum, and grown in the greenhouse at 25°C in a completely randomized design. Controls were inoculated with sterile, noninoculated wheat seed. There were six replicate plants per isolate, with an equal number of controls, and the experiment was repeated once. Symptoms developed in all plants by 20 days post-inoculation and consisted of reduced vigor, necrotic root lesions, and occasionally mortality, all of which resembled the symptoms from grapevines in the field from which the isolates were originally recovered. Mean shoot dry weights of inoculated plants (0.25, 0.16, and 0.28 g for CPa1, Cpa2, and CPa523, respectively) were significantly lower (P < 0.05) than that of the controls (0.74 g). Mean root dry weights of inoculated plants (0.28, 0.16, and 0.29 g for CPa1, Cpa2, and CPa523, respectively) were also significantly lower (P < 0.05) than that of the controls (0.68 g). Isolates recovered from the roots of inoculated plants were identical morphologically and molecularly to C. parva and C. peruviana, thereby satisfying Koch's postulates. No symptoms were observed on the control plants. These Cylindrocladiella spp. have been reported from nurseries or vineyards in South Africa and New Zealand (3). To our knowledge, this is the first report of C. parva and C. peruviana associated with black-foot disease of grapevine in Spain, and in Europe. References: (1) S. Alaniz et al. Plant Dis. 91:1187, 2007. (2) S. Alaniz et al. Plant Dis. 95:1028, 2011. (3) E. E. Jones et al. Plant Dis. 96:144, 2012. (4) L. Lombard et al. Mycol. Progress DOI 10.1007/s11557-011-0799-1, 2012.

[EN] Grapevine trunk diseases (GTDs) are one of the main biotic stress factors affecting this crop. The use of tolerant grapevine cultivars would be an interesting and sustainable alternative strategy to control GTDs. To date, most studies about cultivar susceptibility have been conducted under controlled conditions, and little information is available about tolerance to natural infections caused by GTD fungi. The objectives of this study were: (i) to identify tolerant cultivars to GTD fungi within a Spanish germplasm collection, based on external symptoms observed in the vineyard; and (ii) to characterize the pathogenic mycoflora associated with symptomatic vines. For this purpose, a grapevine germplasm collection including 22 white and 25 red cultivars was monitored along three growing seasons, and their susceptibility for esca foliar symptoms was assessed. Fungi were identified by using morphological and molecular methods. Cultivars such as, 'Monastrell', 'Graciano', 'Cabernet Franc', 'Cabernet Sauvignon', 'Syrah', 'Moscatel de Alejandria', 'Sauvignon Blanc', and 'Airen' displayed high susceptibility to GTDs, whereas others such as 'Petit Verdot', 'Pinot Noir', 'Chardonnay', and 'Riesling' were considered as tolerant. The prevalent fungal species isolated from symptomatic vines were Phaeomoniella chlamydospora (27.9% of the fungal isolates), Cryptovalsa ampelina (24.6%), and Dothiorella sarmentorum (21.3%)., This research was founded by FEDER funding through a State Program of I+D+i oriented to the Challenges of Society (RTA2015-00015-C02-00), supported by The National Institute for Agricultural and Food Research and Technology (INIA). D.G. and J.M. were supported by the Ramon y Cajal program, Spanish Government (RyC-2017-23098 and RyC-028404).

Esca and leaf stripe diseases are two of the most destructive grapevine trunk diseases which are caused by a complex of fungal species, being the Ascomycete Phaeomoniella chlamydospora the most frequently isolated species from affected vines. To date, no curative measures are known for control these diseases; therefore, planting disease-resistant cultivars is a time-tested and sustainable approach for disease management. The aim of this study was to identify sources of resistance to P. chlamydospora among minority and commercial grapevine germplasm collections in Spain. For this purpose, 15 and 38 cultivars from the ITACyL and the EVEGA collections, respectively, were selected based on the visual assessment of symptom expression in standing vines over the last 3 years, and inoculated with P. chlamydospora in a detached cutting assay under greenhouse conditions over two consecutive years. Seven months after inoculation, cuttings were collected and inspected for lesion length. The severity of internal wood symptoms caused by P. chlamydospora varied considerably amongst the cultivars. No foliar symptoms were observed during the experiment. All cultivars were susceptible to fungal necrotic infection, indicating that there is no evidence of qualitative resistance to this Ascomycete fungus., The research was financially supported by The National Institute for Agricultural and Food Research and Technology (INIA) under the project RTA2015-00015-C02-01. David Gramaje and David Ruano-Rosa were supported by the DOC-INIA program from the INIA, co-funded by the European Social Fund. María del Pilar Martínez-Diz was supported by the FPI-INIA program from the INIA. Marcos Andrés-Sodupe was supported by a grant from the Government of La Rioja

Diaporthe species are important pathogens, saprobes, and endophytes on grapevines. Several species are known, either as agents of pre-or post-harvest infections, as causal agents of many relevant diseases, including swelling arm, trunk cankers, leaf spots, root and fruit rots, wilts, and cane bleaching. A growing body of evidence exists that a class of small non-coding endogenous RNAs, known as microRNAs (miRNAs), play an important role in post-transcriptional gene regulation, during plant development and responses to biotic and abiotic stresses. In this study, we explored differentially expressed miRNAs in response to Diaporthe eres and Diaporthe bohemiae infection in Vitis vinifera cv. Chardonnay under in vitro conditions. We used computational methods to predict putative miRNA targets in order to explore the involvement of possible pathogen response pathways. We identified 136 known and 41 new miRNA sequence variants, likely generated through post-transcriptional modifications. In the Diaporthe eres treatment, 61 known and 17 new miRNAs were identified while in the Diaporthe bohemiae treatment, 101 known and 21 new miRNAs were revealed. Our results contribute to further understanding the role miRNAs play during plant pathogenesis, which is possibly crucial in understanding disease symptom development in grapevines infected by D. eres and D. bohemiae., This work was supported from ERDF “Multidisciplinary research to increase application potential of nanomaterials in agricultural practice” (No. CZ.02.1.01/0.0/0.0/16_025/0007314). This research was also supported by the project No. TJ02000096.

Species of Diaporthe are considered important plant pathogens, saprobes, and endophytes on a wide range of plant hosts. Several species are well-known on grapevines, either as agents of pre- or post-harvest infections, including Phomopsis cane and leaf spot, cane bleaching, swelling arm and trunk cankers. In this study we explore the occurrence, diversity and pathogenicity of Diaporthe spp. associated with Vitis vinifera in major grape production areas of Europe and Israel, focusing on nurseries and vineyards. Surveys were conducted in Croatia, Czech Republic, France, Hungary, Israel, Italy, Spain and the UK. A total of 175 Diaporthe strains were isolated from asymptomatic and symptomatic shoots, branches and trunks. A multi-locus phylogeny was established based on five genomic loci (ITS, tef1, cal, his3 and tub2), and the morphological characters of the isolates were determined. Preliminary pathogenicity tests were performed on green grapevine shoots with representative isolates. The most commonly isolated species were D. eres and D. ampelina. Four new Diaporthe species described here as D. bohemiae, D. celeris, D. hispaniae and D. hungariae were found associated with affected vines. Pathogenicity tests revealed D. baccae, D. celeris, D. hispaniae and D. hungariae as pathogens of grapevines. No symptoms were caused by D. bohemiae. This study represents the first report of D. ambigua and D. baccae on grapevines in Europe. The present study improves our understanding of the species associated with several disease symptoms on V. vinifera plants, and provides useful information for effective disease management., This research was supported by the European COST Action FA1303 on Sustainable control of grapevine trunk diseases (ManaGTD). Surveys and fungal collection performed in Hungary were supported by Szechenyi 2020 programme, the European Regional Development Fund, the Hungarian Government (GINOP-2.3.2-15-2016-00061) and EU H2020 project no. 652601. We are grateful to Arien van Iperen (cultures), Mieke Starink-Willemse and Elias Jonk (DNA isolation, amplification, and sequencing) for their technical assistance.

Póster presentado en el 11 th International Verticillium Symposium, celebrado en Göttingen (Alemania) del 5 al 8 de mayo de 2013., Early, specific and accurate in planta detection and quantification of Verticillium dahliae are essential to prevent the spread of Verticillium wilt in olive using certified pathogen-free planting material and development of resistance. We comparatively assessed the accuracy, specificity and efficiency of eight real-time quantitative PCR (qPCR) protocols published since 2002 for the specific detection and quantification of V. dahliae in various host plant species and in soil, using a background of DNAs extracted from olive roots, stems and leaves. Results showed that some of those protocols were not specific for V. dahliae and/or were inhibited when using backgrounds other than water. Ranking of protocols according to a weighted score system placed protocol TAQ (based on IGS rDNA target gene; Bilodeau et al . 2012) and SYBR-4 (based on β -tubulin 2 target gene; Atallah et al . 211) first in sensitivity and efficiency for the quantification of V. dahliae DNA in small amounts and different types of olive tissues (root and st em) tested. Use of TAQ and SYBR-4 protocol allowed accurate quantification of V. dahliae DNA regardless of the background DNA with a detection limit being fixed at a C T of 36 (approximately 18 fg for SYBR-4 and 15 fg for TAQ) of V. dahliae ). The amount of DNA from defoliating (D) and nondefoliating (ND) V. dahliae pathotypes was monitored in Verticillium wilt-resistant 'Frantoio' olive using the TAQ and SYBR-4 protocols. In the infection bioassay, higher amounts of D- V. dahliae DNA were measured in olive stems, whereas the aver age amount of fungal DNA in roots was higher for ND-infected plants than D-infected ones. Overall, V. dahliae DNA amount in all olive tissues tested tended to slightly decrease or remain stable by the end of the experiment (35 days). The SYBR-4 and TAQ protocols further enabled detection of V. dahliae in tissues of symptomless plants, suggesting that both techniques can be useful for implementing certification schemes of pathogen-free planting material as well as a helpful tool in breeding resistance to V. dahliae in olive., Financial support was provided by grants P10-AGR-03528 from CICE-Junta de An dalucía, Spain and AGL- 2012-37521 and AGL2008-00344 from Ministerio de Econom ía y Competitividad, Spai n, and co-financed with FEDER founds from the European Union.

This study aims to identify and characterize species of Phaeoacremonium associated with Petri disease of table grapes in three regions in the Northeastern Brazil, to investigate the distribution of the species in these regions and to evaluate their pathogenicity and aggressiveness in excised green shoots of table grapes. Fungal identifications were made using a combination of morphology together with a phylogenetic analysis based on portions of the β-tubulin (TUB2) and actin (ACT) genes. Three species of Phaeoacremonium (Pm.) were identified: Pm. minimum, Pm. nordesticola sp. nov. and Pm. parasiticum. Phaeoacremonium minimum and Pm. parasiticum had previously been reported in grapevine. Phaeoacremonium minimum was the most prevalent species. All species of Phaeoacremonium were pathogenic on detached shoots of table grape, with Pm. minimum being the most aggressive., This work was financed by Fundação de Amparo à Ciência e Tecnologia do Estado de Pernambuco - FACEPE (APQ 137-5.01/12) and Coordenação de Aperfeiçoamento de Pessoal de Nível Superior – CAPES (Project 203/2009 - International Cooperation CAPES-Brazil/DGU-Spain). M. P. S. Câmara and S. J. Michereff are recipients of Conselho Nacional de Desenvolvimento Científico e Tecnológico - CNPq research fellowships. D. Gramaje was supported by the DOC-INIA program from the National Institute for Agronomic Research (INIA), co-funded by the European Social Fund.

Three citrus scions were evaluated to determine seasonal changes in susceptibility to infections by Phytophthora citrophthora and Phytophthora nicotianae. In a period of 24 months, the Clementine mandarin cv. Hernandina, the hybrid Fortune mandarin and the sweet orange cv. Lane-Late were branch-inoculated under field and laboratory conditions. Field studies showed that the cultivars inoculated with P. citrophthora developed the highest lesion areas during March–June (spring) and September–October (autumn) and with P. nicotianae from June to August (summer). However, lesion areas on detached citrus branches did not show a definite pattern of infection because lesion sizes fluctuated irregularly during the study. The lesion area caused by P. nicotianae in different citrus scions correlated significantly with the monthly mean maximum values of temperature, relative humidity, and the percentage of the relative water content in the 24-month period of inoculations. In contrast, there was no correlation between these variables and the extent of colonisation by P. citrophthora. Nevertheless, a significant relationship was observed between lesion areas caused by P. citrophthora from October to May of each year and the same variables that were significant in inoculations with P. nicotianae. Seasonal changes in the susceptibility of citrus cultivars to P. citrophthora and P. nicotianae may facilitate timing of disease control measures to coincide with periods when disease development is greatest.

In this study, 31 almond orchards with trees showing severe decline symptoms were surveyed from 2009 to 2014 on the island of Mallorca (Spain). In all, 45 Botryosphaeriaceae isolates were collected and characterized based on phenotypical features and comparisons of DNA sequence data of the nuclear ribosomal DNA-internal transcribed spacer region and elongation factor 1-α gene. Five species were identified as Diplodia olivarum, D. seriata, Neofusicoccum luteum, N. mediterraneum, and N. parvum. Pathogenicity tests were performed on four cultivars (‘Pons’, ‘Vivot’, ‘Jordi’, and ‘Ferragnes’) under field conditions for two consecutive years (2013 to 2014), and confirmed that all five species cause canker and dieback of almond, with Neofusicoccum spp. more virulent than Diplodia spp. in both years. Jordi was less sensitive to fungal infection in 2013. First reports from almond in Spain include N. mediterraneum and N. luteum., D. Gramaje was supported by the DOC-INIA program from the National Institute for Agronomic Research (INIA), co-funded by the European Social Fund. We thank the researchers of ENOCIDA group for providing the incubators for the temperature growth assay and J. Moral for statistical advice.

Grapevine diseases caused by soilborne fungal pathogens can cause significant reductions of yield and quality of fruit or wine production. Among them, black-foot and petri diseases have received special attention by plant pathologists due to its implication on the young grapevine decline syndrome, which has dramatically increased worldwide since the early 1990s, causing substantial economic losses and compromising the stable production in viticulture and the wine industry. In this paper, specific attention is given to the genetic and virulence diversity of the causal agents of these diseases. This information represents a good basis for use by plant pathologists and grapevine breeders for future development of grapevine rootstocks resistant to black-foot and petri diseases.

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.

© 2015, Koninklijke Nederlandse Planteziektenkundige Vereniging. Cross infection of multiple hosts by fungal trunk pathogens has been reported to occur worldwide. It supports the hypothesis that infected hosts adjacent to vineyards may serve as sources of inoculum for these pathogens. Among the alternative hosts, different studies have shown that almond represents a rich catch-crop for many fungal trunk pathogens. The hyphomycetes Collophora hispanica, Phaeoacremonium (Pm.) amygdalinum, Pm. iranianum and Pleurostomophora richardsiae have recently been isolated from wood samples with internal necroses and brown to black vascular streaking of almond trees showing symptoms of decline in Spain, although their status as pathogens was so far unknown. We inoculated almond trees cv. Ferragnes with these fungal species in greenhouse experiments to determine their pathogenic potential. All of them infected, colonised and produced lesions statistically different from the control, Pl. richardsiae being the most aggressive. Therefore, Co. hispanica, Pm. amygdalinum, Pm. iranianum and Pl. richardsiae should be considered as trunk pathogens of almond trees.

Thirty-eight Phaeoacremonium isolates collected from pruning wounds of tropical sandalwood in Western Australia were studied with morphological and cultural characteristics as well as phylogenetic analyses of combined DNA sequences of the actin and p-tubulin genes. Three known Phaeoacremonium species were found, namely P. alvesii, P parasiticum, and P venezuelense. Phaeoacremonium venezuelense represents a new record for Australia. Two new species are described: P. luteum sp. nov. can be identified by the ability to produce yellow pigment on MEA, PDA, and OA, the predominance of subcylindrical to subulate type II phialides, and the mycelium showing prominent exudate droplets observed as warts; and P santali sp. nov. which can be separated from other species producing pink colonies on MEA by the predominance of type I and II phialides, the distinct brownish olive colonies in OA, and slow growth., Isolations were made under project PRJ-004677 ”Heartwood Rot Identification and Impact in Sandalwood (Santalum album)“ funded by the Rural Industries Research and Development Corporation of Australia.

In May 2008, symptoms of black foot disease were observed on 8-year-old grapevines (Vitis vinifera L.) cv. Garnacha in Albuñol (Granada Province, southern Spain). Affected plants showed delayed budding with low vigor. Roots showed black discoloration and necrosis of wood tissues. Root fragments were cut, washed under running tap water, surface sterilized for 1 min in a 1.5% sodium hypochlorite solution, and washed twice with sterile distilled water. Small pieces of discolored or necrotic tissues were plated onto potato dextrose agar (PDA) supplemented 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. A Cylindrocarpon-like fungus was consistently isolated from necrotic root tissues. Single conidial isolates were obtained and grown on PDA and Spezieller Nährstoffarmer Agar (SNA) and incubated at 25°C for 10 days in darkness. On PDA, the isolates developed white, thick, and cottony to felty abundant mycelium. On SNA, all isolates produced slightly to moderately curved one-septate (22.5-) 25.6 (-27.5) × (5-) 5.63 (-6.25) μm, two-septate (30-) 36.1 (-45) × (6.25-) 7.08 (-7.5) μm, three-septate (37.5-) 47.9 (-52.5) × (6.25-) 7.5 (-8.75) μm, four-septate (47.5-) 53.3 (-62.5) × (7.5-) 7.89 (-8.75) μm, and five-septate (52.5-) 61.8 (-67.5) × (7.5-) 8 (-8.75) μm macroconidia. Microconidia were not observed. DNA sequence of the rDNA internal transcribed spacer region (ITS) was obtained for isolate Cf-270 and deposited in GenBank (Accession No. HQ441249). This sequence showed high similarity (99%) to the sequence of Campylocarpon fasciculare Schroers, Halleen & Crous (GenBank Accession No. AY677303), in agreement with morphological features (1). Pathogenicity tests were conducted with inoculum produced on wheat (Triticum aestivum L.) seeds that were soaked for 12 h in flasks filled with distilled water. Each flask contained 300 ml of seeds that were subsequently autoclaved three times after excess water was drained. Two fungal disks of a 2-week-old culture of C. fasciculare (isolate Cf-270) grown on PDA were placed aseptically in each flask. The flasks were incubated at 25°C for 4 weeks and shaken once a week to avoid clustering of inoculum. Plastic pots (220 cm3) were filled with a mixture of sterilized peat moss and 10 g of inoculum per pot. One-month-old grapevine seedlings were planted individually in each pot and placed in a greenhouse at 25 to 30°C in a completely randomized design. Control plants were inoculated with sterile uninoculated seeds. Six replicates (each one in individual pots) were used, with an equal number of control plants. The experiment was repeated. Symptoms developed on all plants 20 days after inoculation and consisted in reduced vigor, interveinal chlorosis and necrosis of the leaves, necrotic root lesions with a reduction in root biomass, and plant death. The fungus was reisolated from the roots of affected seedlings and identified as C. fasciculare, completing Koch's postulates. No symptoms were observed on the control plants. Black foot disease of grapevines can be caused by different species of Cylindrocarpon and Campylocarpon. C. fasciculare was first reported in South Africa in 2004 (1). To our knowledge, this is the first report of C. fasciculare causing black foot disease of grapevine in Spain as well as other countries in Europe. Reference: (1) F. Halleen et al. Stud. Mycol. 50:431, 2004.

Eight Phaeoacremonium (Pm.) isolates from grapevines in Iran and Spain were studied with morphological and cultural characteristics as well as phylogenetic analyses of combined DNA sequences of the actin and beta-tubulin genes. Two new species are described. Pm. cinereum was isolated from a young vine in Spain and from older vines in Iran and can be identified by its distinct gray colonies on malt extract agar, an optimum growth temperature of 25 C and subulate type III phialides. Pm. hispanicum was isolated only once from a young vine in Spain and can be identified by the common occurrence of percurrently rejuvenating phialides, an optimum growth temperature of 20 C and predominant type II phialides.

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. © 2009 BSPP.

Since 2010, a new foliar and fruit disease was observed in pomegranate (Punica granatum L.) orchards in Alicante Province (eastern Spain). Symptoms included black spots on leaves and fruits, as well as chlorosis and premature abscission of leaves. Fungal isolates were obtained by surface-disinfecting small fragments of symptomatic leaf and fruit tissues in 0.5% NaOCl, double-rinsing in sterile water, and plating them onto potato dextrose agar (PDA) amended with 0.5 g/liter of streptomycin sulfate. Gray-to-black colonies were obtained, which were identified as Alternaria sp. based on the dark, brown, obclavate to obpyriform catenulate conidia with longitudinal and transverse septa tapering to a prominent beak attached in chains on a simple and short conidiophore (4). Conidia (n = 100) measured (12.2-) 20.2 (-27.6) × (5.7-) 9.2 (-12.0) μm, and had 3 to 6 transverse and 0 to 5 longitudinal septa. Single spore cultures were obtained and their genomic DNA was extracted. The internal transcribed spacer (ITS) region of rDNA and partial sequences of the beta tubulin gene were amplified and sequenced with primers ITS1-ITS4 and Bt1a-Bt1b, respectively (3). BLAST analysis of the sequences showed that they were 100% identical to a pathogenic A. alternata (Fr.) Keissl. isolate obtained from black spot disease of pomegranate in Israel (Accession No. JN247826.1, ITS and Accession No. JN247836.1, beta tubulin) (2). As all the sequences obtained showed 100% homology, ITS and beta tubulin sequences of a representative isolate (1516B) were submitted to GenBank (KF199871 and KF199872, respectively). In addition, a PCR reaction with specific primers (C_for/C_rev) designed to recognize highly virulent isolates of A. alternata causing black spot of pomegranate was used with all isolates (2). A characteristic fragment of ~950 bp was amplified in two isolates: 1552B and 1707B. Pathogenicity was assessed on plants and detached fruit of pomegranate cv. Mollar (1). Two-year-old pomegranate trees were inoculated with isolates 1552B and 1707B by spraying a conidial suspension (106 conidia/ml) onto the upper and lower leaf surfaces. Five plants per fungal isolate were used and five control plants were sprayed with sterile water. Plants were covered with transparent plastic bags and incubated in a growth chamber for 1 month at 25°C, with a 12-h photoperiod. One-month-old fruits were surface sterilized in 1.5% sodium hypochlorite solution for 1 min and rinsed twice in water. Two filter paper squares (5 × 5 mm) were dipped in the conidial suspensions and placed on the fruit surface. Inoculated fruit were incubated in a humid chamber in the dark at 25°C. Ten fruit per fungal isolate were used and 10 control fruit were inoculated with sterile water. Black spots were visible on inoculated leaves and fruit, 10 and 3 days after inoculation, respectively. Symptoms were not observed on controls. The fungus was re-isolated from leaf and fruit lesions, confirming Koch's postulates. Leaf black spot of pomegranate caused by A. alternata was first described in India in 1988, and later in Israel in 2010 affecting both fruit and leaves (1). To our knowledge, this is the first report of the disease in Spain, where it could represent a threat for pomegranate cultivation due to the increasing amount of area dedicated to this crop. References: (1) D. Ezra et al. Australas. Plant Dis. Notes 5:1, 2010. (2) T. Gat et al. Plant Dis. 96:1513, 2012. (3) N. L. Glass and G. C. Donaldson. Appl. Environ. Microbiol. 61:1323, 1995. (4) E. G. Simmons. Alternaria: An identification manual. CBS Fungal Biodiversity Center, Utrecht, Netherlands, 2007.