Studies on the epidemiology of black-foot disease of grapevine in Spain

Las enfermedades de la madera se encuentran entre las patologías más dañinas que afectan al cultivo de la vid. El pie negro es una de las más destacadas, afectando a las plantas en vivero y en plantaciones jóvenes. Los agentes causales están incluidos dentro de los géneros Campylocarpon, ¿Cylindrocarpon¿, Cylindrocladiella e Ilyonectria. Éstos se caracterizan por ser habitantes del suelo y, se ha demostrado que permanecen en él, infectando al material de propagación cultivado en campos de vivero. La presencia de hongos asociados al pie negro en vivero, así como sus fuentes potenciales de inóculo en suelos de vivero y de viñedos comerciales, no han sido nunca estudiados en España. En este sentido, el objetivo de esta Tesis ha sido estudiar la epidemiología de hongos que causan el pie negro de la vid en España. En primer lugar, las distintas fases del proceso viverístico se evaluaron como fuentes potenciales de inóculo de estos patógenos. Se tomaron muestras en cuatro fases del proceso de propagación de las que se extrajo el ADN, detectándose las especies causantes del pie negro de la vid mediante multiplex, nested PCR. En las fases estudiadas se detectaron I. liriodendri y el complejo I. macrodidyma. También se estudió la detección de especies de Ilyonectria en material de propagación de vid, antes y después de la fase de enraizamiento en campos de vivero, mediante técnicas de aislamiento y multiplex, nested PCR. Este estudio confirmó que el número de plantas infectadas con estos patógenos aumenta durante el proceso de enraizamiento en campos de vivero. Ilyonectria torresensis fue la única especie aislada de las plantas tras la inducción del callo. Sin embargo, las especies I. liriodendri, I. novozelandica e I. torresensis se aislaron frecuentemente tras el período de cultivo en campos de vivero. Respecto a la detección molecular, se detectaron un número elevado de muestras positivas en planta tras la inducción del callo y después del proceso de enraizamiento. Mediante el uso de cuatro técnicas diferentes, aislamiento fúngico a partir de raíces de plántulas de vid utilizadas como plantas trampa, aislamiento a partir de raíces de malas hierbas, multiplex, nested PCR y qPCR, se estudió el suelo de campos de plantas madre como fuente de inóculo de estos patógenos. De las raíces de plantas trampa se aislaron las especies I. alcacerensis, I. macrodidyma, I. novozelandica e I. torresensis. ¿Cylindrocarpon¿ macrodidymum fue la única especie aislada de las raíces de malas hierbas. En los análisis de suelos realizados mediante multiplex, nested PCR así como mediante qPCR se observó un elevado porcentaje de detección del complejo I. macrodidyma en muestras de ADN de suelo, mientras que el porcentaje de detección de I. liriodendri fue menor. Las mismas técnicas descritas para campos de plantas madre se utilizaron para estudiar los suelos de campos de vivero y de viñedos comerciales. Los resultados obtenidos en estos dos tipos de campos fueron similares a los obtenidos en campos de plantas madre. Finalmente, se estudió el efecto de la temperatura, pH y potencial osmótico (¿s) sobre el crecimiento miceliar, la esporulación y la producción de clamidosporas de ¿C.¿ liriodendri, ¿C.¿ macrodidymum y ¿C.¿ pauciseptatum, con el objetivo de mejorar el conocimiento de los factores que afectan al crecimiento, reproducción y supervivencia de estos patógenos. Todos los aislados estudiados crecieron en un rango de temperaturas comprendido entre 5 y 30ºC. Se observó crecimiento miceliar en un rango de pH comprendido entre 4 y 8. Respecto al efecto del ¿s, el crecimiento miceliar fue mejor en medio de cultivo PDA ajustado a -0,5, -1,0 y/o -2,0 MPa en comparación con el crecimiento miceliar observado en PDA sin ajustar a ningún ¿s (-0,3 MPA). La mayoría de los aislados de ¿Cylindrocarpon¿ esporularon a todas las temperaturas, pHs y valores de ¿s estudiados. En general, la producción de clamidosporas no se vio afectada por la temperatura, el pH y el ¿s.Agustí Brisach, C. (2013). Studies on the epidemiology of black-foot disease of grapevine in Spain [Tesis doctoral no publicada]. Universitat Politècnica de València. https://doi.org/10.4995/Thesis/10251/27598TESI

Evaluation of weeds as potential hosts of black foot and Petri disease pathogens on grapevine fields

Weeds were sampled in grapevine rootstock mother fields, openroot field nurseries and commercial vineyards in Spain between June 2009 and June 2010 and evaluated as potential hosts for black foot and Petri disease pathogens. Isolations were performed from the root system and internal xylem tissues for black foot and Petri disease pathogens, respectively. Cylindrocarpon macrodidymum was successfully isolated from the roots of fifteen families and twenty-six 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 C. macrodidymum isolates obtained from weeds were able to induce typical black foot disease symptoms and caused shoot and root mass reduction on grapevine seedlings. Isolates of Ca. luteo-olivacea and Pa. chlamydospora were also shown to be pathogenic on grapevine cuttings. These results indicate that weeds can serve as host for C. macrodidymum, Ca. luteo-olivacea and Pa. chlamydospora and may play roles in the epidemiology of black foot and Petri disease pathogens on grapevine.Agustí Brisach, C. (2010). Evaluation of weeds as potential hosts of black foot and Petri disease pathogens on grapevine fields. http://hdl.handle.net/10251/14216Archivo delegad

Biocontrol of Plant Diseases by Means of Antagonist Microorganisms, Biostimulants and Induced Resistance as Alternatives to Chemicals

Plant diseases are one of the biggest problems in conventional agriculture as they reduce both yield and crop value [...

Effects of temperature, pH and water potential on mycelial growth, sporulation and chlamydospore production in culture of Cylindrocarpon spp. associated with black foot of grapevines

[EN] The effects of temperature, pH and water potential (Ψs) on mycelial growth, sporulation and chlamydospore production of Cylindrocarpon liriodendri, C. macrodidymum and C. pauciseptatum isolated from grapevines was studied. Three isolates per species were incubated on potato dextrose agar (PDA) under different temperature, pH, and Ψs conditions. All isolates were able to grow over a range of temperatures from 5 to 30ºC, with an optimum temperature between 20 to 25ºC, but they did not grow at 35ºC. Active mycelial growth was observed over a range of pHs, from 4 to 8. Regarding the effect of Ψs, in general, mycelial growth was greater on amended media at -0.5, -1.0 or/and -2.0 MPa compared with that obtained on nonamended PDA (-0.3 MPa), and was reduced at Ψs values lower than -2.0 MPa. Most of the Cylindrocarpon spp. isolates were sporulated at all temperatures, pHs and water potentials tested. In all studied conditions, C. liriodendri had the greatest sporulation capacity compared with C. macrodidymum and C. pauciseptatum. In general, chlamydospore production was not much affected by temperature, pH and Ψs. Chlamydospores were observed in PDA cultures of all isolates at all pH values studied, while some isolates did not produce chlamydospores at 5 and 10ºC or -4.0 and/or -5.0 MPa. These results improve understanding of the biology of these important grapevine pathogens.This research was financially supported by the Project RTA2010-00009-C03-03 (Instituto Nacional de Investigación y Tecnología Agraria y Alimentaria, INIA, Spain) and the European Regional Development Fund (ERDF). We acknowledge M. A. GarcíaMartínez and V. Garrigues for technical assistance and Dr D. Gramaje for providing valuable advice on the writing of this manuscript.Agustí Brisach, C.; Armengol Fortí, J. (2012). Effects of temperature, pH and water potential on mycelial growth, sporulation and chlamydospore production in culture of Cylindrocarpon spp. associated with black foot of grapevines. Phytopathologia Mediterranea. 51(1):37-50. https://doi.org/10.14601/Phytopathol_Mediterr-9499S375051

Black-foot disease of grapevine: an update on taxonomy, epidemiology and management strategies

[EN] Black-foot is one of the most destructive grapevine trunk diseases in nurseries and young vineyards, causing necrotic root lesions, wood necrosis of the rootstock base, and a gradual decline and death of grapevines. Causal agents of the disease are included into the genera Campylocarpon, "Cylindrocarpon", Cylindrocladiella and Ilyonectria. Recent taxonomical studies of Neonectria and related genera with "Cylindrocarpon"-like anamorphs based on morphological and phylogenetic studies, divided Neonectria into five genera. Thus, the current taxonomical position and classification of the causal agents of black-foot disease, mainly "Cylindrocarpon"/Ilyonectria, comprises one of the main topics of this review. The review also provides an update on geographical distribution, epidemiology and management strategies of the disease.Agusti Brisach, C.; Armengol Fortí, J. (2013). Black-foot disease of grapevine: an update on taxonomy, epidemiology and management strategies. Phytopathologia Mediterranea. 52(2):380-387. doi:10.14601/Phytopathol_Mediterr-12662S38038752

Influence of Cultivar and Biocontrol Treatments on the Effect of Olive Stem Extracts on the Viability of Verticillium dahliae Conidia

The effect of olive (Olea europaea) stem extract (OSE) on the viability of conidia of Verticillium dahliae, the causal agent of Verticillium wilt of olive (VWO), is not yet well understood. Thus, the aim of this study was to determine the influence of the olive genotype (cultivar resistance) and the interaction between olive cultivars and biocontrol treatments on the effect of OSE on conidial germination of V. dahliae by in vitro sensitivity tests. To this end, OSE from cultivars Frantoio, Arbequina, and Picual, respectively tolerant, moderately susceptible, and highly susceptible to V. dahliae, were tested alone or after treatments with biological control agents (BCAs) and commercial products efficient at reducing the progress of VWO. Aureobasidium pullulans strain AP08, Phoma sp. strain ColPat-375, and Bacillus amyloliquefaciens strain PAB-24 were considered as BCAs. Aluminium lignosulfonate (IDAI Brotaverd®), copper phosphite (Phoscuprico®), potassium phosphite (Naturfos®), and salicylic acid were selected as commercial products. Our results indicate that the influence of biological treatments against the pathogen depends on the genotype, since the higher the resistance of the cultivar, the lower the effect of the treatments on the ability of OSE to inhibit the germination of conidia. In ‘Picual’, the BCA B. amyloliquefaciens PAB024 and copper phosphite were the most effective treatments in inhibiting conidia germination by the OSE. This work represents a first approach to elucidate the role of cultivar and biological treatments in modifying the effect on the pathogen of the endosphere content of olive plants

Almond Anthracnose: Current Knowledge and Future Perspectives

Almond anthracnose caused by Colletotrichum spp. has been described as one of the most important diseases of this nut crop in the main almond-growing regions worldwide, including California, Australia and Spain. Currently, almond anthracnose is considered a re-emerging disease in the countries across the Mediterranean Basin due to the shift of plantations from the original crop areas to others with climatic, edaphic and orographic conditions favoring crop growing and yield. The pathogen mainly affects fruit at the youngest maturity stages, causing depressed, round and orange or brown lesions with abundant gum. The affected fruits can fall prematurely and lead to the drying of branches, causing significant economic losses in years of epidemics. This review aims to compile the current knowledge on the etiology, epidemiology and management of this disease

Resistance Induction in Olive Tree (Olea europaea) Against Verticillium Wilt by Two Beneficial Microorganisms and a Copper Phosphite Fertilizer

Enhancement of the natural defenses of a plant by beneficial microorganisms, i.e., endophytic bacteria and fungi or fertilizers such as copper phosphonates, could result in a potential alternative strategy against verticillium wilt of olive tree (Olea europaea). In this study, two beneficial microorganisms (the fungus Aureobasidium pullulans AP08 and the bacterium Bacillus amyloliquefaciens PAB-024) and a phosphonate salt copper phosphite (CuPh) were evaluated for their effectiveness as host resistance inducers against Verticillium dahliae in olive. To this end, 6-month-old healthy olive plants of the susceptible cultivar Picual were treated by foliar or root applications by spraying 15 ml per plant or by irrigation with 350 ml per plant of the dilutions of each product (CuPh: 3 or 10 ml l–1, respectively; PAB-024: 108 UFC ml–1; AP08: 106 UFC ml–1). Treatments were conducted weekly from 2 weeks before inoculation to 10 days after inoculation. A cornmeal–water–sand mixture (1:2:9; w:v:w) colonized by V. dahliae was used for plant inoculation. Additionally, treated and noninoculated, nontreated and inoculated, and nontreated and noninoculated plants were included for comparative purposes. Disease severity progress and shoot fresh weight were assessed. Parameters involved in plant resistance were monitored through determination and quantification of reactive oxygen species (ROS) response (H2O2), and evaluation of hormones was done by gene expression analysis. Aureobasidium pullulans and CuPh were the most effective in disease reduction in planta by foliar or root application, respectively. Plants treated with CuPh showed significantly higher shoot fresh weight compared to the other treatments. ROS was significantly enhanced in plants treated with B. amyloliquefaciens PAB-024 compared to the rest of treatments and control. With regard to the evaluation of hormones, high levels of salicylic acid were detected on leaves from all treatment combinations, but without significant enhancements compared to the nontreated control. Regarding the gene expression related to salicylic acid, only the WRKY5 gene has shown a strong enhancement in the treatment with B. amyloliquefaciens. On the other hand, a strong accumulation of jasmonic acid and jasmonic acid-isoleucine in plants treated with A. pullulans was observed in all the tissues analyzed and also in the roots of plants treated with B. amyloliquefaciens and CuPh

Effect of latent and symptomatic infections by Colletotrichum godetiae on oil quality

Anthracnose, caused by Colletotrichum spp., is the main olive fruit disease. Colletotrichum can severely infect olive fruit with a negative impact on the oil quality. However, the relationship between visible infections of Colletotrichum spp. and olive oil quality is unclear and the influence of latent infections is unknown. This study considers Colletotrichum spp. latent infections and visible infections as factors affecting the quality of olive oil. IAbsorbance in UV (K232 and K270), free acidity, and peroxide index were evaluated in oils from fruit with latent and symptomatic infections by Colletotrichum godetiae of the cvs. Arbequina, Hojiblanca, and Picual. Olive oil samples from i) latent infected fruit at three maturity stages and after two incubation periods and from ii) sets of oils from mixtures of healthy and infected symptomatic fruit were used to determine the impact of the disease on oil quality. Oils from latent infected fruit of cv. Arbequina showed higher acidity than control oils (P = 0.012). Linear and exponential models were fitted to relate the oil quality parameters to the proportion of symptomatic fruit. Acidity was the most affected parameter, mainly in oils from cv. Arbequina. The thresholds of the percentage of affected fruits causing the loss of category in the quality of the oil varied greatly according to cultivar, with ‘Arbequina’ being the most sensitive

Diversity of Cellulolytic Microorganisms Associated with the Subterranean Termite Reticulitermes grassei

Reticulitermes grassei is a subterranean termite species that forages on woody structures of the Iberian Peninsula, and is often a building and crops pest. A total of 23 microorganisms associated with the activity of R. grassei were isolated from colonized ecosystems in southern Spain. They were morphologically and molecularly characterized, with fungi being the most prevalent ones. The fungi showed high values of optimum growth temperature, suggesting that they could be able to survive and develop in warm regions. Their cellulolytic activity was tested in carboxymethylcellulose (CMC) agar, concluding that all fungal isolates produce cellulases, and the enzymatic index (EI) was revealed in CMC agar with Gram’s iodine solution, with Penicillium citrinum showing the highest EI and Trichoderma longibrachiatum the highest mycelial growth rate on CMC. A preliminary microorganism dispersion assay was carried out with the termites, concluding that these insects may have a positive influence on fungal dispersion and the subsequent colonization of new substrates. Our study suggests that fungi associated with R. grassei may potentially be of interest in biotechnological fields such as biofuel production and the food industry