BIOLOGICAL CONTROL OF PLANT DISEASES

Author: BARAJAS RAMÍRES DANIEL.
Year: 2004.
University: COMPLUTENSE DE MADRID [www.ucm.es].
Place of defense: FACULTAD DE BIOLOGÍA.
Place of preparation: CENTRO DE INVESTIGACIONES BIOLÓGICAS (CSIC).

Summary: Most viruses are capable of inducing in the plants that infect a response from gene silencing, who served on the host plant as a natural defense mechanism against the virus. At the same time, many viruses able to fight back by producing proteins that interfere with the mechanism of gene silencing. The balance between the induction of the antiviral defense of the plant, based on gene silencing, and the deletion of this defense by viruses, is probably one of the factors that determine the course of a viral infection. In this thesis has been used transgenic plants expressing the protein sequences for the silencing suppressor gene postranscripcional (PTGS) HC-Pro virus the sharka or Plum pox virus (PPV), in order to study the process of gene silencing induced meet this transgene and the possible effect of the protein HC-Pro, encoded by the transgene, on the gene silencing and resistance to the virus. With our system we wanted to provide information about the potential balance between induction and suppression of gene silencing by viruses. It transformed plants Nicotiana benthamiana constructs derived from gene HC-Pro of PPV. It employed a version translatable (HCt), designed to produce protein HC-Pro, and another version is not translatable (HCn). It determined the levels of expression of protein HC-Pro in several lines HCt obtained, as well as the ability of the protein HC-Pro expressed suppress silencing génico.La protein HC-Pro expressed in several lines HCt had no ability to interfere the gene silencing tested in a number of systems. We studied the PPV resistance in transgenic plants HC-Pro. We identified two lines HCt and two other HCn with a high proportion of resistant plants PPV and PVX-HCT, a chimeric virus derived from PVX which carries the gene HC-Pro of PPV. Resistance to PVV and PVX-HCT, a chimeric virus derived from PVX which carries the gene HC-Pro of PPV. Resistance to PPV correlates with low levels of transgene mRNA accumulation in the presence of small interfering RNAs (siRNAs) specific transgene and the transgene silencing of a gene gusA, which has homology with the portion 3 'of transgene HC-Pro. All this suggests that resistance to PPV is a result of silencing gene postranscripcional induced by the transgene HC-Pro part of transgenic plants. We studied genetic factors that determine gene silencing of transgene HC-Pro in a proportion of plants line HCt2 resistant PPV. Resistance is genetic and probably determined by the presence of a locus specific transgene HC-Pro among multiple loci in the línes HCt2, as suggested by the proportion of resistant plants PPV close to 3 / 4 of the total and the presence of up two loci of the transgene in the non-resistant plants PPV. It also studied the suppression of gene silencing of the transgene HC-Pro by PVY infection with the virus, or CMV, and its effect on the resistance PPV. Both viruses were capable of suppressing gene silencing of transgene HC-Pro. Moreover, in the case of infection with CMV, we were able to prove the removal of resistance PPV. Results 8 obtenid 453 years, are discussed from the point of view of the possible role of the protein HC-Pro on gene silencing suffered by the transgene that encodes. As part of the additional work, tested the capability of removing gene silencing by the protein p29 virus fungi CHV1 in plants. This protein shared some of the typical characteristics of the suppressor gene silencing in plants and animals. Meidante several tests failed to demonstrate suppressor activity of silencing géncio by protein p29 in plants, but this fact does not eliminate the possibility that p29 function as a suppressor gene silencing in yeast.

Author: GÓMEZ VIDAL SONIA DOLORES.
Year: 2005.
University: ALICANTE [www.ua.es].
Place of defense: FACULTAD DE CIENCIAS.
Place of preparation: DEPARTAMENTO CIENCIAS DEL MAR Y BIOLOGÍA APLICADA.

Summary: The Doctoral Thesis includes the study of colonization endofítica of entomopathogenic fungus Beauveria bassiana, Lecanicillium dimorphum and L. Cf. Psalliotae, leaves Phoenix dactylifera L. It has been assessed at histologic, biochemical and molecular level, the impact of such conduct endofítico on the defense mechanisms of P. Dactylifera, for application in biological control. The entomopathogenic fungi survived endofíticamente in inoculated leaves of P. Dactylifera floods field for at least 30 days. The fungi were isolated up to three centimeters instead of inoculation, without symptoms or abnormalities seen in the appearance of leaves and the rest of the plant. The fungi colonized mainly tissue parenchymatous, growing in the international and intracellular spaces, and the vascular bundles without causing damage at the cellular level. However, P. Dactylifera responded to colonization endofítica through the accumulation of phenols, deposition of protein and modifying the walls of cells parenquimáticas colonized. Colonization endofítica also altered levels of expression of proteins of P. Dactylifera. By proteomic studies, comparing proteomas tissues inoculated with the entomopathogenic fungi, proteomas tissue not inoculated, noting that the colonization endofítica possibly promotes responses defense P. Dactylifera. In the study, has developed a model of interaction between non-pathogenic fungi and the only family of monocots with carrying tree and of great importance globally. This model has applications in the biological control of pests and diseases, and studies of interactions planta-microorganismo at the cellular level, biochemical and molecular level.

Author: COTXARRERA VILAPLANA MARIA LURDES.
Year: 2005.
University: BARCELONA [www.ub.es].
Place of defense: FACULTAT DE BIOLOGÍA.
Place of preparation: UNITAT DE FISIOLOGIA VEGETAL. DEPARTAMENT DE BIOLOGIA VEGETAL.

Summary: The use of plant protection and herbicide has been and still is today, a very useful tool in agriculture. However, it presents many difficulties, and therefore the legislation governing their use is becoming increasingly restrictive. In this scenario it is necessary to develop new techniques to achieve effective control of diseases with a lower risk, and more in line with sustainability criteria. In this paper we analyzed the efficiency of the use of growing media formulated composts and Trichoderma asperellum T34 for the control of podredura campus cucumber caused by Rhizoctonia solani and the fusariosis vascular tomato caused by Fusarium oxysporum f.sp. Lycopersici. On the other hand, took place the development of molecular tools for tracking specific biological control agents of the genus Trichoderma spp.en substrates crop. We analyzed the ability of removing waste compost produced with market sewage sludge of sewage and waste pruning (METROCOMPOST) of compost depleted substrate for growing mushrooms, compost alperjuo, compost byproduct from the manufacture of cork , and compost byproduct of the industry produced alcohol (compost vine). In the tests of efficiency control of the podredura campus cucumber rated capacity removal of composts to aged between six months and one year (composts youth), and aged between one and a half and three years (composts mature). The development of molecular tools for tracking specific T34 was from the discovery of a fragment of DNA characteristic of T34 by random amplification by RAPD a collection of 51 isolates of Trichoderma spp., And the design of primers specific for the fragment characteristic detected. Moreover, for the quantification of the specific strain Trichoderma hmatum th382 in growing media developed a procedure based on the technique for real-time PCR amplification with primers specific. The fusariosis vascular tomato could be controlled effectively through the use of composted METROCOMPOSTS, and the inoculation of biological control agent for T34. The compost METROCOMPOST resulted in a high fugistasis pathogen. Their ability to removal was related to the following biotic and abiotic factors: slightly high pH, high electrical conductivity, abundant populations of bacteria and microbiological activity high. The agent of biological control T34 resulted in a significant inhibition of growth of FOL. Especially to be implemented two weeks anteriorida the pathogen inoculation. In terms of podredura campus cucumber, only composts young METROCOMPOST and cork helped obtain a level of control of significant disease, whereas all composts more than a year and a half old analyzed filed a control capacity disease highly effective. Inoculation of T34 improved capacity removal of composts young mushroom and cork, and those composts mature in which the incidence of the disease had been higher. The primers designed for T34 allowed to differentiate the earlier strain of a collection of 9 strains of Trichoderma spp., Demonstrating the high degree of specified obtained. Finally, the procedure for real-time PCr developed for the quantification of Th382 yielded equivalent results to the technical suspension dilution in substrate samples inoculated with densities on the order of 10 6 conidia / ml substrate.

Author: PUJOL ABAJO MARTA.
Year: 2006.
University: GIRONA [www.udg.es].
Place of defense: FACULTAT DE CIÈNCIES ECONÒMIQUES I EMPRESARIALS.
Place of preparation: UNIVERSITAT DE GIRONA.

Summary: Erwinia amylovora is a bacterium fitopatógena causing significant economic losses in the production of pear and apple globally. Traditionally, the control of fire blight has been conducted by chemicals, mostly made with copper compounds, or antibiotics depending on the law in each country. However, the repeated use of chemical pesticides has led to undesirable effects such as the emergence of resistant strains and the presence of residues in food and the environment. For this reason, we have developed alternative methods of control as a biological control. In the group of Plant Pathology at the University of Girona was selected and is developing the agent of biological control of fire blight Pseudomonas fluorescens EPS62e for its high efficacy in controlling infections caused by E. Amylovora in immature fruits, flowers and plants pear. With the aim of helping the development of the biocontrol agent P. Fluorescens EPS62e, this work was designed a method of traceability molecular based on real-time PCR and assessed colonization and survival of the strain EPS62e after their inoculation in apple and pear field. The development of a method for real-time PCR involved (i) the detection of polymorphisms in the genome of EPS62e, (ii) the selection of fragments amplified that discriminate EPS62e against other strains for the design of primers SCAR (Sequence Characterized Amplified Regions), (iii) the assessment of the specificity of SCAR primers, and (iv) the design of a real-time PCR in a region of internal sequence SCAR. To detect specific sequences occur naturally in the genome of EPS62e used two methods based on PCR that do not require prior knowledge of genome amplification multiple arbitrary (RAPD) and the PCR nonspecific (U-PCR). Through both technical amplification profiles were obtained that allowed discrimination EPS62e against other strains of the same species. It secuenciaron three pieces of specific amplification of EPS62e obtained using RAPD and U-PCR and a pair of primers designed in each sequence for obtaining SCAR markers. The specificity of the primers SCAR was evaluated versus 161 strains within the same species, 75 strains within related species and 61 samples of vegetable commercial farms. Of the three pairs of primers designed SCAR, two couples were quite specific for the detection of EPS62e, while the third pair of SCAR primers showed not be specific as additions were obtained in other strains of the same species. Since the two markers were designed specific SCAR two real-time PCR probes type TaqMan ®. The two designs in real time PCR were specific EPS62e, with a detection level of 102 CFU / g fresh weight, and a standard curve linear in the range of five logarithmic units. During the development and optimization of real-time PCR was carried out a study of traceability of arable population of EPS62e, pear leaves at ambient controlled by two classical techniques coupled with PCR. Both techniques are based on the counting of viable plate (RVP) and the most probable number (MPN) in selective medium, followed by PCR with primers SCAR. Both techniques were useful for tracing EPS62e, since there were no significant differences between the values estimated by the two techniques. The level of EPS62e decreased arable population gradually from 107 to 105-104 CFU / g fresh weight during the first 17 days, and then remained stable for 11 days until the end of the trial. Despite the suitability of both techniques for tracing 8 EPS62e, 10c5 was selected the RVP in selective medium for the following tests. The real-time PCR was validated method of how traceability of EPS62e through simultaneous use with the method of RVP in various experiments in flowers and leaves of apple, and the results were compared and contrasted statistically. The experiments were conducted in greenhouse conditions and field in the region of Maine (France) during the spring of 2004. The results obtained with flowers Apple showed a good correlation of population levels estimated by real-time PCR and RVP. The agent biocontrol EPS62e was an efficient colonizer of flowers Apple, reaching high population levels ranging between 107 and 108 CFU / corimbo. This population remained during the cuajado and development of fruit, 54 days after treatment, where up to 99% of the population in the area was restricted calyx of the fruit. However, the results obtained with apple leaves were completely different, as the population levels of EPS62e decreased over time. At leaves Apple, the values estimated by real-time PCR were significantly different from the values obtained by RVP. Overall, the values of real-time PCR was superior to the values of RVP. This difference between methods was attributed to two possible causes: entry into a viable state but not arable (VBNC) of a portion of the population of EPS62e may cause an underestimation by RVP, and the presence of non-degraded DNA from dead cells may cause an overestimation by real-time PCR. The combined use of molecular methods and cultivo-dependientes showed that EPS62e was under optimal conditions of colonization flowers Apple and was presumably under stress and with difficulty surviving on leaves of apple. Adaptability epiphytic of EPS62e in apple and pear was evaluated under field conditions in Girona during the spring of 2005. We evaluated various factors that may affect its future and its eco-efficiency after being released in the field, the influence of climatic conditions, the species of host plant, the influence of the indigenous microbiota, and its ability to spread from trees treated up untreated trees. There was no remarkable difference between the pattern of colonization and population levels of EPS62e in apple and pear flowers. The biocontrol agent successfully colonized flowers belonging to both species, with high population levels as had been observed in previous studies carried out in France, reaching the ability of microbial load of flowers to 107-108 CFU / corimbo. Climatic conditions appeared to be optimal as it is not observed any sudden change in population levels during the tests. EPS62e was able to colonize and dominate the microhabitat of flowers, representing up to 100% of the total arable microbiota few days after treatment. In addition, EPS62e was detected in all the flowers untreated located between 15 and 35 meters from the site of inoculation, showing a good ability to spread. Even so, the population of EPS62e leaf Apple under field conditions declined over time to be below the detection limit of the technique 30 days after treatment. This work shows that the strain EPS62e is well suited to the colonization of flowers in the field, encouraging their use in a biological control strategy of fire blight.

Author: SACRISTÁN PÉREZ MINAYO GONZALO.
Year: 2006.
University: BURGOS [www.ubu.es].
Place of defense: FACULTAD DE CIENCIAS.
Place of preparation: FACULTAD DE CIENCIAS.

Summary: The bacteria which promote plant growth, known as PGPR (Plant Growth Promoting Rhizobacteria), are characterized by rizobacterias that colonize plant roots and stimulate plant growth significantly both directly and indirectly. The overall objective of this dissertation is the characterization and identification and molecular microbiological of PGPR and the study of their involvement in health and crop production. The aim is to evaluate the use of these rizobacterias in agricultural production of sugar beets, squash and tomatoes, as well as contribute to the study of methods of controlling plant diseases, in particular the ability of biological control of these PGPR face plant pathogens. It also seeks to study and evaluate control capability compared to Rhizoctonia solani, and Ralstonia solanacearum Meloidogyne incognita, all these pathogens rizosféricos. The strain used in this dissertation was Pseudomonas fluorescens PfO-1 exercising beneficial effects on sugar beet, tomato and pumpkin, not only in the stimulation of plant growth, but also in biological control versus plant pathogens. The production of sucrose in sugar beet crop was increased and optimized with the inoculation of PGPR. Using Pseudomonas fluorescens PfO-1 was effective against Rhizoctonia solani and it appeared that stimulated some mechanism for systemic acquired resistance to Meloidogyne incognita in front tomato. With the results seem reasonable to consider Pseudomonas fluorescens PfO-1 as a PGPR in these crops. The use of PGPR in tomatoes, sugar beets and squash, feasible and beneficial, not only in the increase of agricultural production, but also in overall performance, and possibly lower economic costs. In addition represents an alternative to the use of plant protection products, as they sometimes are used improperly. These techniques of biological control are more compatible with the development of a more rational and sustainable agriculture.

Author: ROMERO HINOJOSA DIEGO FRANCISCO.
Year: 2006.
University: MÁLAGA [www.uma.es].
Place of defense: FACULTAD DE CIENCIAS.
Place of preparation: FACULTAD DE CIENCIAS.

Summary: The mildew is the most common fungal disease in cucurbits crops worldwide. In southern Spain Podosphaera fusca has been described as the sole causative agent of the disease. The development of resistance by the pathogen many of the fungicides and demand for commercial agriculture more environmentally friendly, located at biological control among the most interesting alternative control strategies to keep in mind. In this thesis we plant assess the possibility of controlling the mildew by using different biocontrol agents and elucidating the mechanisms through which conducted its inhibitory action. In the first part of this study we selected two fungi microparásitos, Ampelomyces quisqualis and Lecanicillium lecanii and 4 strains identified as Bacillus subtilis by its ability to successfully control the disease in experiments in vitro. Later, in tests with seedling melon held in camera cropping, we see as relative humidity values higher than 90% favored the bio-activity of these biological agents. Finally, the ability antogonista of these biocontrol agents was confirmed in tests held melon plants in the greenhouse, where levels were obtained control of the disease are similar to those obtained by a chemical fungicide used as a control. With regard to mechanisms of action employees, using different microscopy techniques look like A.quisqualis behaved as endoparásito and biótrofo strict, while l.lecanii acted as a ectoparásito, also showing a phase of life saprofita. To confirm the role of antibiosis in the bio-activity of the strains of B.subtilis, experiments were conducted with filtered free of bacterial cells obtained reductions in disease around 90%. As the same time we see is reduction in the symptoms of the disease are associated with a similar reduction in the rate of germination of conidia of P.fusca due to the induction of damage morphological and cytological ultimately responsible for their inability to germinate. Through the use of different analytical techniques identify the production of antibiotics lipoptídicos. The ability of these purified compounds to inhibit the germination of conidia of P.fusca supported his involvement in the activity of biocontrol. Finally, by directed mutagenesis to disrupt the production of these compounds, we confirm that lipopéptidos the family of ituínas and genficinas are essential in the bio-activity of these strains B.subtilis. The results obtained in this thesis suggests that biocontrol agents selected are good candidates for inclusion in integrated control programs against mildew of curcurbitáceas.