FUNGI

Author: AVILA DE LA CALLE ARANZAZU.
Year: 2004.
University: CÓRDOBA [www.uco.es].
Place of defense: E.T.S.I. AGRONOMOS Y MONTES.
Place of preparation: E.T.S.I. AGRONOMOS Y MONTES.

Summary: The Emplomado olive caused by P. Cladosporioides is a specific disease olive causing premature falling of the leaves and hence a general weakening of the trees affected. The objectives of this thesis were known etiologic different aspects, epidemiológicosy control, in order to reach a wider knowledge of this disease. The study of morphological characterization, culturally and genetically diverse isolates of P. Cladosporioides showed a low variability of poblaciónde this patógenoen Spain, because of the absence of teleomorph and sparse, propágulosde this pathogen. The inoculations carried out on olive seedlings and separate sheets have allowed reproduce the symptoms observed in the field. However, the severidadde infections and homogeneidadfueron casualties. It has obtained a primer specific Pseudocercospora that could be used in future as a method to detect infections latentesde the enfermedadsobre hojasde olive. Based on an extensive epidemiological study, known in greater depth various aspects related to the development of the disease over time, the survival of the fungus, production and sources of inoculum and the effect of environmental factors on them. The Emplomado developed during the months with average temperatures between 10 and 20Â ° CY with relative humidity greater than 80%, so that the dates with increased risk for infections are occurring in October and the end of winter. The most important sources of inoculum are infected leaves that remain on the tree during the summer. The conidia exist in the leaves fall, pose a low risk of production of new infections. These studies have also yielded the degree of susceptibility to Emplomado of the major varieties grown in Spain. The systemic fungicides were more effective than organic or cúpricos on mycelial growth and germination of conidia. However, the trials of fungicides in the field have not helped draw definitive conclusions about disease control.

Author: BELTRÁN MARTÍNEZ ROBERTO.
Year: 2005.
University: POLITÉCNICA DE VALENCIA [www.upv.es].
Place of defense: UNIVERSIDAD POLITÉCNICA DE VALENCIA.
Place of preparation: UNIVERSIDAD POLITÉCNICA DE VALENCIA.

Summary: The ascomiceto Monosporascus cannonballus Pollack et Uecker is a major fungi associated with the syndrome of "collapse", which affects the growing cucurbits in Spain and other countries. The ascospsoras are the primary inoculum of the fungus, leaving on the floor after the decomposition of the affected roots, which can be extracted from the ground through a physical process, which allows their quantification over time. Based on this technique, there have been a number of epidemiological studies that have resulted in innovative results. It has studied the population dynamics of the ascosporas of M.cannonballus in soils with different water conditions and farming. In fields with cultivation of melon, it has been observed that the level of ascosporas reaches a maximum seven months after planting (3-4 months after the end of the crop), to go after declining gradually, reaching levels similar to the original one year after planting. In areas with ponding winter, there has been a progressive decrease in the level of ascosporas, noting that they can survive in soil for at least a period of three years without losing its emotional appeal. M.cannonballus has been regarded as a fungus termofilo typical of desert and semi-arid areas, this study has shown that it is able to survive in temperate zones and in a position to waterlogging. It has conducted a study to quantify soil ascosporas melon fields of various producing areas of the Valencia region, detected ascosporas of M.cannoballus in all of them. At the time of onset of symptoms of "collapse", there has been significant differences between the fields and the areas between symptomatic and asymptomatic, in favor of some other, depending on the fields. When comparing the initial levels of ascosporas soil with those obtained in two or three months after the end of the crop, have been contradictory situations, such as an increase in the final level of ascosporas per gram of soil, or a decline of this level. Thus, in order to study the population dynamics of the ascosporas of M.cannonballus in soil, it is necessary to carry out sequential sampling. Due to the complexity of its life cycle, the samples do not show off enough information about the level of inoculum of the fungus in the soil. There has been a study of the population of sequential ascosporas in soil M.cannonballus in several crops cucurbits. In melon and watermelon there was a decline in the population of ascosporas from the first sample, due to germination of ascosporas and colonization of roots, and that coincided with the observation of the first symptoms in the air and rising the isolation of the fungus in the roots of plants affected. Subsequently there was an increase in the number of ascosporas associated with the production of perithecia in the roots. This increase was significantly higher in the ranks than in entrelíneas associated with a higher density in those roots. In grafted watermelon, the level of ascosporas soil remained stable throughout the growing season, not observed symptoms in the air, nor perithecia in the roots, and the percentage of insulation M.cannonballus was very low throughout the study . In view of these results, it believes that the grafting watermelon on hybrid Cucurbita is a method of effective struggle against the "collapse" caused by M.cannonballus.

Author: COLLADO ROMERO MELANIA.
Year: 2005.
University: CÓRDOBA [www.uco.es].
Place of defense: FACULTAD DE CIENCIAS.
Place of preparation: FACULTAD DE CIENCIAS.

Summary: The Verticilosis caused by the fungus Deuteromyceto haploid Verticilium dahliae are one of the main problems phytopathological in many economically important crops globally. The wide range of host plants of the pathogen, its variability pathogenic, restriction in the xylem during parasitic, and prolonged persistence in the soil, considerably hamper the control of Verticilosis. Stocks of V.dahilae include vegetative compatibility groups (VCGs) in which an isolated loa VCG have the ability to establish genetically determined anastomosis hifal and heterocariontes stable one another but are incompatible with those of other VGCs. Therefore, V.dahliae isolates of VCG could be regarded as a subpopulation genetically isolated from those formed by other VCGs. Knowledge of the genetic diversity of populations V.dahliae is a key element for the proper handling of Verticilosis, particularly where such diversity can be correlated with phenotypes relevant to the pathogenesis of disease, such as the correlation between VCGs and patoenicidad and / or virulence demonstrated in earlier studies this Doctoral Thesis. Until the development of it through analysis of markers based on the length polymorphisms in DNA fragments amplified (AFLPs), had not been established whether or not there is correlation between genetic diversity in populations V.dahliae determined by its structure VCGs and the defined by molecular markers, and if that correlation can be influenced by the host of origin or geographical origin of those. In this Doctoral Thesis, AFLPs analysis of 250 isolates of VCG1A, 1B, 2A, 2B, 4A, 4B, and 6 of V.dahliae from different host plants and geographical origin, has shown that there is a close correlation between molecular groups and VCGs; being isolated from a molecularly VCG generally more similar to each other than with isolated from other VCGs, regardless of geographical origin or host isolates. Furthermore, the resolution provided by AFLPs has identified the existence of diversity intra-VCG, which was associated with the geographical origin of the isolates or the host plant of which were obtained. Analysis of AFLPs and conserved gene sequences indicated that VCGs are monophyletic origin, except in the VCG2B and helped elucidate the existence of three storylines in different VCGs. The use of resistant cultivars and plant material pathogen-free certificate preventive measures are well suited for the control of Verticilosis. In this Doctoral Thesis has been optimized a procedure for detecting V.dahliae in artichoke plants infected naturally or artificially by testing multiplex PCR and sequence DNA using as a mold total extracted from them, which is more efficient and informative that the pathogen Detection based on microbiological isolation growing, and that could be very useful for the certification of the health of plant material planting. Finally, the level of susceptibility of the genotypes Acebuche "Arbequina", and "Picual" Olea europaea L., infection with patotipo of V.dahliae defoliante and highly virulent, and patotipo not defoliante and moderately virulent, it could be positively correlated with the greatest amount of pathogen in the plant determined by quantitative PCR testing in real time, and the higher virulence of patotipo defoliante compared to the non defoiante was correlated with the existence of the infection in stem olive indicated by the DNA quantity of pathogen cuantifi 8 each at 292.

Author: Rodríguez Navarro Estefanía.
Year: 2006.
University: GRANADA [www.ugr.es].
Place of defense: Facultad de Ciencias.
Place of preparation: Facultad de Ciencias (UGR) - Estación Experimental del Zaidín (CSIC).

Summary: It is currently estimated that exist in the world around 1000 million trees, of which 98% are in the Mediterranean region. Andalusia maintained a clear leadership in the olive tree representing a third of the European olive grove. Grenada is the third Spanish province both in number and in production of olive oil behind Cordoba and Jaen. In Grenada there are 22 million trees of which 18 are productive and unproductive corresponding four are mostly young plantations. The regions with the largest areas planted with olive groves correspond to the Vega Montefrío and Iznalloz, followed Alhama Baza, Guadix, El Valle, Huéscar, and Alpujarra Coast. Over the past 10 years, the cultivation area has experienced an increase of more than 29%, a significant portion of which is under irrigation. In recent years the olive trees are being seriously affected by a relatively new disease called Verticilosis Olive Tree (VO)

Author: GUIJARRO DÍAZ-OTERO BELÉN.
Year: 2006.
University: POLITÉCNICA DE MADRID [www.upm.es].
Place of defense: E.T.S. INGENIEROS AGRÓNOMOS.
Place of preparation: INSTITUTO NACIONA DE INVESTIGACIONES AGRARIAS Y ALIMENTARIAS.

Summary: Monilinia spp. Because marchitez in peach buds and flowers, as well as the rotting of the fruit produce significant losses in recent years with climate favorable to the development of the disease. Biological control by applying antagonists (biofungicidas) can be an alternative to other methods of control and, in any case, is more a tool to be used in the integrated control of plant diseases. The biological control by introducing antagonists has had an extensive development at the pilot. But its practical application presents serious drawbacks referred mainly to the difficulty of obtaining commercial formulations user-friendly and effective in terms uncontrolled field, as well as their compatibility with other products. On the other hand, filosfera is a way unfavorable to get a biocontrol consistent and it is necessary to know the dynamics of populations of the biocontrol agent and the pathogen if we want to get a good control of the disease. The investigation of new alternatives for biological control of brown rot caused by fungi of the genus Monilinia began conducting a search of antagonists against the pathogen among microflora epiphyte annual peach outbreaks both in vitro and in vivo. It demonstrated the potential of antagonist Penicillium frequentans in biological control of the marchitez of outbreaks caused by Monilinia lax in experimental gardens under artificial inoculation of the pathogen. In this thesis is seeking to establish the foundations for the development of a practical method of biological control of brown rot caused by M. Laxa through Biocontrol Agent (CBA) Penicillium frequentans. This would have raised the following objectives: 1-Finding a culture medium leading to biomass of the CBA P. Frequentans viable and effective in a fast and economical. 2-Get a formulated to maintain the viability and effectiveness of CPF P. Frequentans over storage and during and after their implementation. 3-Ensure that the CPF P. Frequentans colonice the effective filosfera, to establish high levels of population and maintain the viability of their propagules in the areas of the plant in order to achieve maximum effectiveness against the pathogen. To achieve these objectives, first tested various systems of mass production of conidia P. Frequentans (fermented liquid and solid fermentation). It was subsequently sought a system design to keep the viability and stability of conidia well produced. It tested several systems cellular dehydration (lyophilization, atomization and fluidised bed) and identified the most favorable conditions for conservation (moisture content, temperature, the presence of oxygen and light). Finally, to improve its stability and efficiency were added substances stabilizers and supporters to ensure the permanence of the biocontrol agent on the surface plant treated, thus promoting interaction with the pathogen. Different formulations obtained from P. Frequentans were tested in controlling the rot of the fruit harvest through applications. It also tested the effectiveness of these formulations in treatments trees in orchards in bloom and precosecha. In these trials were also studied the population dynamics of P. Frequentans on the surface of flowers and fruit. The results obtained in this thesis show that presents the characteristics favorable CBA P. Frequentans for its commercial application as biofungicida. We have obtained the following results: 1-A method for mass production in solid substrate fermentation of conidia P. Frequentans. With this method are obtained 108-109 conidia / g of dry substrate, with a viability of 80-90% and an effective 48-65% in postharvest treatment. The cost of this medium is 2â ¬ / 8 drive d a3a and production (fermentation bag of 500g), which is considered low price. 2-Three formulated with high stability, adherence to the surface of the fruit and effectiveness in the conidia fresh P. Frequentans. These formulations have been obtained through: A. use an effective system of cellular dehydration: the fluid bed drying, which maintains the viability of the cells dehydrated and its effectiveness against the pathogen after one year storage at room temperature. B. adding substances stabilizers that keep viability and effectiveness of the CBA P. Frequentans more than 50% at room temperature for 12 months. C-adding acceding to improve the permanence of the biocontrol agent on the surface plant treated, thus promoting interaction with the pathogen. D.-adding silica gel as inert product that improves handling, and application of the formulations in the form of wettable powders, which are suspended in water to be applied on the surface plant. 3-Formulations obtained show an efficacy of 48-65% over the rotting of fruits in postharvest treatment. 4 - The implementation of the formulated obtained in biological treatments made in commercial orchards show that: A. The antagonist should be applied in early flowering, the petals fall and the tightening of endocarp and precosecha to maintain an adequate population to be effective in controlling brown rot. B. Through these biological treatments has increased the native population of P. Frequentans to levels adequate to control Monilinia spp: reducing the population of the pathogen on the surface of peach, thereby reducing the inoculum potential. C. - The population distribution of P. Frequentans depends on the environmental conditions (temperature and relative humidity) and the time and number of applications made.

Author: RUANO ROSA DAVID.
Year: 2006.
University: CÓRDOBA [www.uco.es].
Place of defense: ETSIAM.
Place of preparation: ETSIAM.

Summary: The white rot (PB), caused by Rosellinia nectrix Prilleux (anamorph Dematophora necatrix Harting), is one of the major diseases in trees Avocado (Persea americana) in the area of the coast of southern Spain. The fungus invades the tree's root system, giving under optimal conditions rapid death. To control the fungus have been proposed chemical methods and physical methods mainly. The selection in vitro and in vivo, isolated from Trichoderma able to be used in biological control strategies, identified the antagonistic activity of four isolated monoconídicos of Trichoderma atroviride (CH101, CH273, CH304.1 and CH314) and isolation monoconídico of Trichoderma virens (CH 303) against the pathogen isolated from the Rn 400. Among those highlighted the isolated T.atroviride CH 273 for high control capacity in vitro. In vitro Compatibility between 5 isolated from Trichoderma selected determined that there is an incompatibility between interspecific studied isolates of T.virens and T.atroviride, excluding the first Test in vivo which combined these isolated Trichoderma. In in vivo experiments using isolated CH101, CH273, CH304.1 and CH314, isolated CH 304.1 showed the best results of control, just as combined with the other three isolated. He also showed a synergistic effect on the in vitro growth of the other three isolated T.atroviride. The in vivo study of two isolated no-patogénicos of r.necatrizx (NP1 and NP2), as potential biocontrol agents, determined the effectiveness, inoculations with these two isolates, the delay in the start of the appearance of the first symptoms and reducing the progress of the epidemic disease. The optimum temperature of creiciento of isolated R.necatrix was 25 ° C, while within the range of optimum temperature for growth given the isolation of Trichoderma studied (20 ° C-25 ° C) and within the optimum temperature for optimal development of the disease in avocado (20 ° C-25 ° C). The incompatibility somatic studied for 57 isolates R.necatriz revealed incompatibility between most of the isolated when they came from different farms or different rings disease. But five isolates (R 12, R 29, R 49, R 108 and R 411) is not formed barrier incompatibility with the majority of the isolates with those who opposed. Isolates presented great variability in the morphological characteristics of its colonies, the majority being those who have registered mycelium thin air or low mycelium air edges of the colony lobulados high melanización. Instead Molecular analysis revealed a little differentiation of the isolated using RAPD, contrasting with the diversity observed on morphological characterization and the somatic incompatibility. The improved protocol purification of DNA samples of soil and plant tissue from avocado crops, possible to obtain DNA of better quality, which allowed detection of eliminating real-time PCR, nested PCR step. This removal decreases the possibility of obtaining false positives and the time for detection. This methodology increased accuracy, increasing the number of detections made positive achieved with regard to the methodology of conventional isolation and identification of the pathogen in culture medium. This work is an important initial step in the search for biocontrol agents of the PB avocados, which improves detection field with the application of a technique for early detection of the pathogen in trees "asymptomatic" or "escape", which can if carriers such agents (isolated from Trichoderma spp. OR non-pathogenic) responsible for the escape from the disease.

Author: MORALES VALLE HECTOR BRAULIO.
Year: 2006.
University: LLEIDA [www.udl.es].
Place of defense: E.T.S. DE INGENIEROS AGRONOMOS UDL.
Place of preparation: ESCUELA TECNICA SUPERIOR DE INGENIERIA AGRARIA UDL.

Summary: Accumulation of patulin in apples and pears and subsequently in their by-products is caused mostly by Penicillium expansum which causes blue mould. Patulin can damage organs and tissues in animals and some studies revealed carcinogenic and teratogenic effects. Process-grade apples are mostly cold stored apples (which may have developed blue rot during storage) and low quality apples (ground harvested or visibly wounded apples) and thus, patulin contamination of raw material occurs. The influence of cold storage and further ambient storage prior to processing on patulin accumulation in apples were studied in this thesis. A morfo-ecophysiological study revealed that almost 100% of P. expansum isolates assayed in this study presented strong aggressiveness to apples and were patulin producers. Different degrees of sensitivity to fungicides was observed and some isolates were resistant to fungicides Duration of cold storage influenced on patulin accumulation. However, when lesions development was minimized, patulin amounts detected in apples were smaller regardless of duration of cold storage. Inoculum size an intraspecific interactions affected rot development and patulin accumulation. Modified atmospheres controlled patulin accumulation in longer storages. Ripeness degree of apples when harvesting influences on fungicide efficiency, which could not avoid completely fruit spoilage. The treatment with biocontrol agents seemed to be an alternative to fungicides not only avoiding mould growth but also controlling patulin accumulation. Further storage at ambient temperature led to rapid rot development and strong patulin accumulation in all cases. The size of the lesion caused by P. expansum at the end of cold storage significantly influenced on patulin accumulated during ambient storage and postharvest treatments assayed in this study lost their efficiency at this stage too. Thus, efficiency of such postharvest treatments during ambient storage depended on their effect during cold storage. However, the use of modified atmospheres during cold storage led to significantly lesser patulin contamination during ambient storage. During ambient storage, a diffusion of patulin to sound apple flesh occurred. Thus, washing with high pressure water or trimming the rotten tissue is insufficient to completely remove patulin. The influence of apple variety on patulin accumulation during ambient storage was also studied. The differences observed in patulin accumulation seemed to be related to acidity content and pH of flesh. Influence of pH in pear juice was not as strong as in apple juice, in which a high accumulation of patulin was found at pH 3.5. Both cold and ambient storage are critical in patulin control. Duration of cold storage should be programmed depending on postharvest treatments and quality of apples. Further deck storage should be avoided. When not possible, quality state of apples at the end of cold storage should be assessed in order to state a critical limit for the duration of such storage. The implementation of the Hazard Analysis and Critical Control Points (HACCP) principles at these two stages may reduce significantly the risk of patulin contamination.

Author: VALERO RELLO ANA.
Year: 2006.
University: LLEIDA [www.udl.es].
Place of defense: E.T.S. DE INGENIERIA AGRARIA UDL.
Place of preparation: ESCUELA TECNICA SUPERIOR DE INGENIERIA AGRARIA.

Summary: Ochratoxin A (OTA) is a fungal secondary metabolite produced by Aspergillus and Penicillium species. It has been detected in a wide range of commodities, including cereals, coffee, grapes, raisins, must and wine. Within grape derivative products, the raisins, red wine and sweet wines have reported to contain the highest OTA levels. Aspergillus section Nigri (A. niger and A. carbonarius) are considered the OTA source in these commodities and they are commonly isolated among other fungi from grapes and raisins. Starting from this basis the objectives of this thesis were focused into three main aspects: (1) Evaluation of the food products: vine dried fruits and special wines, concerning the mycobiota and OTA occurrence and incidence; (2) Ecophysiological studies of the ochratoxigenic fungi and accompanying mycobiota as affected by environmental conditions; (3) Control and preventive methods such as the evaluation of residual activity of pre-harvest fungicides during grape dehydration and the use of modified atmospheres. Wine origin and winemaking procedure showed to be determinant for the final OTA content. All special wines analysed from northern European regions were negative for OTA while more than 50% of wines from warmer regions were positive for OTA contamination. The wines with higher OTA levels were fortified musts followed by those made from dried grapes. Acoholic and malo-lactic fermentations, biological 'crianza' (Flor yeast) and the action of Botrytis cinerea in noble rot of grapes may diminish the OTA levels in wine. In grapes, the presence of Aspergillus section Nigri became predominant at harvest and mainly during sun-drying. Prevalence of Aspergillus section Nigri can be explained by their adaptation to environmental conditions of sun-drying, and by their ability to dominate other fungal species involved when coming into contact with them. Among the Aspergillus section Nigri, A. niger aggregate was dominant, although A. carbonarius increased its incidence in dehydrated grapes and showed a higher potential for OTA production, close to 100% of strains were ochratoxigenic, it being optimal at 20ºC and high water activity. The complex fungal interactions studied in vitro and during grape dehydration may act as a control factor in some cases while other few fungal species stimulated OTA production. Integration of these findings on the effects of temperature, water activity and competitive abilities on the fungal spore resistance to ultraviolet radiation (UVC) provides a good explanation to the incidence of fungi found on both fresh and sun dried grapes. The ultraviolet light could also be considered as an alternative control treatment given that 10 min of UVC irradiation on fungal spores were enough to reduce almost the 100% of their viability. Regarding the residual effect of pre-harvest fungicides, it was observed that they remained active in grapes during further dehydration, so they could be effective in reducing OTA production by black aspergilli. The application of modified atmospheres packaging showed differential results in controlling fungal growth and OTA production, combination of 15% CO2 with 1% O2 being the most effective treatment. However, to minimise the risk of OTA contamination during grape storage, modified atmospheres should be used in combination with low temperatures or with other preventive measures. Finally, the application of chemical or biological treatments to grape products, other than those intrinsic to the winemaking process, should be considered as the last option. Attention should focus in preventive rather than in corrective actions. A suitable crop management followed by a smart post-harvest processing of grapes and also during winemaking should comprise enough cautions to obtain a safe final product.