Optimización del Abonado Nitrogenado en el Melón (Cucumis Melo L.) Tipo Piel de Sapo

Spain is the fifth-largest producer of melon (Cucumis melo L.) and the second exporter in the world. To a national level, Castilla-La Mancha emphasize and, specifically, Ciudad Real, where is cultivated 27% of national area dedicated to this crop and 30% of melon national production. Melon crop is cultivating majority in Ciudad Real and it is mainly located in the Alto Guadiana, where the major aquifers of the region are located, the aquifer 23 or Mancha Occidental and the aquifer 24 or Campo de Montiel, both declared overexploited and vulnerable zones to nitrate pollution from agricultural sources. The problem is exacerbated because in this area, groundwater is the basic resource of supply to populations, and even often the only one. Given the importance of melon in the area, recent research has focused on the irrigation of melon crop. Unfortunately, scant information has been forthcoming on the effect of N fertilizer on melon piel de sapo crop, so it is very important to tackle in a serious study that lead to know the N requirements on the melon crop melon by reducing the risks of contamination by nitrate leaching without affecting productivity and crop quality. In fact, the recommended dose is often subjective and practice is a N overdose. In this situation, the taking of urgent measures to optimize the use of N fertilization is required. To do it, the effect of N in a melon crop, fertirrigated and on plastic mulch, was studied. The treatments consisted in different rates of N supply, considering N fertilizer and N content in irrigation water, so the treatment applied were: 30 (N30), 85 (N85), 112 (N112) and 139 (N139) Kg N ha-1 in 2005; 93 (N93), 243 (N243) and 393 (N393) kg ha-1 in 2006; and 11 (N11), 61 (N61), 95 (N95) and 148 (N148) kg ha-1 in 2007. A randomized complete-block design was used and each treatment was replicated four times. The results showed a significant effect of N on dry biomass and two patterns of growth were observed. On the one hand, a gradual increase in vegetative biomass of the plant, leaves and stem, with increasing N, and on the other hand, an increase of fruit biomass also with increasing N up to a maximum of biomass corresponding to the optimal dose determined in 90 kg ha-1 of N applied, corresponding to 160 kg ha-1 of N available for melon crop, since this optimum dose, the fruit biomass suffers a decline. A significant effect was observed in concentration and N uptake in leaf, steam, fruit and whole plant, increasing in all of them with increasing of N doses. Fast N uptake occurred from 30-35 to 70-80 days after transplanting, coinciding with the fruit development. The N had a clear influence on the melon yield, its components, skin thickness and flesh ratio. The melon yield increased, as the mean fruit weight and number of fruits per m2 with increasing N until achieve an above 95% of the maximum yield when the N applied is 90 kg ha-1 or 160 kg ha-1 of N available. When N exceeds the optimal amount, there is a decline in yield, reducing the mean fruit weight and number of fruits per square meter, and was also observed a decrease in fruit quality by increasing the skin thickness and decrease the flesh ratio, which means an increase in fruit hollowed with excessive N doses. There was a trend for all indexes of N use efficiency (NUE) to decline with increasing N rate. We observed two different behaviours in the calculation result of the NUE; on the one hand, all the efficiency indexes calculated with N applied and N available had an exponential trend, and on the other hand, all the efficiency indexes calculated with N uptake has a linear trend. The linear regression cuts the exponential curve, delimiting a range within which lies the optimum quantity of N. The N leaching as nitrates increased exponentially with the amount of N. The increase of N doses was affected on the N mineralization. There was a negative exponential effect of N available on the mineralization of this element that occurs in the soil during the growing season, calculated from the balances of this element. The study of N leaching for each N rate used, allowed to us to establish several environmental indices related to environmental risk that causes the use of such doses, a simple way for them to be included in the code of Best Management Practices.

Ecotoxicology of pesticides on natural enemies of olive groves. Potential of ecdysone agonists for controlling Bactrocera oleae (Rossi) (Diptera: Tephritidae)

Pesticide applications are still one of the most common control methods against the main olive grove pests and diseases: the olive fruit fly, Bactrocera oleae (Rossi), the olive moth, Prays oleae (Bernard), the black scale, Saissetia oleae (Olivier), and the olive leaf spot, caused by the fungus Spilocaea oleagina Fries. However, and because the new pesticide legislation is aimed at an integrated pest and disease management, it is still important to evaluate and to know the ecotoxicology of pesticides on the natural enemies of the different agrosystems. A part of this work has been focusses on evaluating the direct and indirect effects of kaolin particle films and two copper-based products (Bordeaux mixture and copper oxychloride) through different laboratory, extended laboratory and semi-field experiments. Two natural enemies have been chosen: Psyttalia concolor (Szèpligeti), a parasitoid of the olive fruit fly, and Chilocorus nigritus (F.), predator of Diaspididae. This predator has been used instead of C. bipustulatus (L.), which is the species found in olive orchards. Kaolin mainly acts as a repellent of insects and/or as an oviposition deterrent. It is used in olive groves to control the olive fruit fly and the olive moth. Copper is applied against fungal and bacterial diseases. In olive groves it is used against the olive leaf spot and other diseases. No statistical differences were found in any of the experiments performed, compared to the controls, except when the oral toxicity of the products was evaluated on P. concolor females. In this case, kaolin and copper oxychloride caused a higher mortality 72 hours after the treatments, and both kaolin and the two copper formulations decreased females’ life span. Reproductive parameters were only negatively affected when kaolin was ingested. Apart from these experiments, due to the uncommon mode of action of kaolin, two extra experiments were carried out: a dual choice and a no-choice experiment. In this case, both P. concolor females and C. nigritus adults showed a clear preference for the untreated surfaces when they had the possibility of choosing between a treated surface and an untreated one. When there was no choice, no statistical differences were found between the treatments and the controls. Furthermore, the efficacy and the selectivity of three insect growth regulators (methoxyfenozide, tebufenozide and RH-5849) on B. oleae and P. concolor, respectively, have also been evaluated. In addition to laboratory experiments to evaluate the toxicity of the insecticides, also molecular approaches were used. RNA of both insects was isolated. cDNA was subsequently synthesized and the complete sequences of the ligand biding domain (LBD) of the ecdysone receptor of each insect were then determined. Afterwards the three dimensional structures of both LBDs were constructed. Finally, the docking of the insecticide molecules in the cavity delineated by the 12 α-helix that composed the LBD was performed. Both toxicity assays and molecular docking approaches showed that either methoxyfenozide or tebufenozide had no negative effects nor on B. oleae nor on P. concolor. In contrast, RH-5849 had no deleterious effect to the parasitoid but decreased olive fruit fly adults’ life span, especially when they were in contact with the fresh residue of the insecticide applied on a glass surface. The docking study of RH-5849 molecule has shown a very light hindrance with the wall of the LBD pocket. This means that this molecule could more or less adjust in the cavity. Thus, searching of new insecticides for controlling the olive fruit fly could be based on the basic lead structure of RH-5849 molecule.

Determination of the uptake and translocation of nitrogen applied at different growth stages of a melon crop (Cucumis melo L.) using 15N isotope.

In order to establish a rational nitrogen (N) fertilisation and reduce groundwater contamination, a clearer understanding of the N distribution through the growing season and its dynamics inside the plant is crucial. In two successive years, a melon crop (Cucumis melo L. cv. Sancho) was grown under field conditions to determine the uptake of N fertiliser, applied by means of fertigation at different stages of plant growth, and to follow the translocation of N in the plant using 15N-labelled N. In 2006, two experiments were carried out. In the first experiment, labelled 15N fertiliser was supplied at the female-bloom stage and in the second, at the end of fruit ripening. Labelled 15N fertiliser was made from 15NH415NO3 (10 at.% 15N) and 9.6 kg N ha−1 were applied in each experiment over 6 days (1.6 kg N ha−1 d−1). In 2007, the 15N treatment consisted of applying 20.4 kg N ha−1 as 15NH415NO3 (10 at.% 15N) in the middle of fruit growth, over 6 days (3.4 kg N ha−1 d−1). In addition, 93 and 95 kg N ha−1 were supplied daily by fertigation as ammonium nitrate in 2006 and 2007, respectively. The results obtained in 2006 suggest that the uptake of N derived from labelled fertiliser by the above-ground parts of the plants was not affected by the time of fertiliser application. At the female-flowering and fruit-ripening stages, the N content derived from 15N-labelled fertiliser was close to 0.435 g m−2 (about 45% of the N applied), while in the middle of fruit growth it was 1.45 g m−2 (71% of the N applied). The N application time affected the amount of N derived from labelled fertiliser that was translocated to the fruits. When the N was supplied later, the N translocation was lower, ranging between 54% at female flowering and 32% at the end of fruit ripening. Approximately 85% of the N translocated came from the leaf when the N was applied at female flowering or in the middle of fruit growth. This value decreased to 72% when the 15N application was at the end of fruit ripening. The ammonium nitrate became available to the plant between 2 and 2.5 weeks after its application. Although the leaf N uptake varied during the crop cycle, the N absorption rate in the whole plant was linear, suggesting that the melon crop could be fertilised with constant daily N amounts until 2–3 weeks before the last harvest.

El injerto en pepino corto tipo español (Cucumis sativus L.) recomencaciones para su empleo en la zona central española

El injerto en hortalizas es uno de los temas de más actualidad en el panorama hortícola, no solo español, sino occidental, y recalcamos occidental, pues en muchos países que no corresponden a ese ámbito, sobre todo asiáticos: Japón, Corea, China, Filipinas, etc., esta es una técnica que cuenta con una gran difusión desde hace décadas, siendo, por ejemplo en Japón, la mayoría de sus cultivos de cucurbitáceas y solanáceas realizados con planta injertada. A finales de los noventa quedó claro que el empleo de bromuro de metilo tenía una fecha de caducidad y que las zonas que tenían una fuerte dependencia de este desinfectante de suelo debían de buscar alternativas a un plazo lo más corto posible, con un punto añadido sobre etapas anteriores, debían ser alternativas lo más respetuosas posible con el medio ambiente y que no incrementaran, de forma importante, los costes de producción. En la zona centro y concretamente en los invernaderos de la Comunidad de Madrid y zonas cercanas de Toledo y Guadalajara el pepino era y es el cultivo predominante, los horticultores empleaban el bromuro de metilo de forma sistemática para desinfectar sus suelos y la desaparición de este producto les planteaba una gran incertidumbre, lo que llevó a que desde diferentes instancias se buscaran diferentes alternativas. Tras analizar las posibilidades que se podían implementar y conocido el buen resultado que había dado el injerto en sandía en Almería, se decidió acometer los trabajos que conforman esta Tesis Doctoral, planteando en la zona, diferentes ensayos con la idea de conocer, si el injerto en pepino, con los cultivares empleados habitualmente, podía ser una alternativa real para los horticultores, tanto de Madrid, como los de las zonas cercanas de Toledo y Guadalajara. Se pretendía conocer sobre todo las repercusiones agronómicas y si esta técnica podría emplearse en solitario o era necesario complementarla con otras alternativas: desinfectantes químicos, solarización, biofumigación e incluso desinfección con vapor de agua. Los ensayos fueron realizados de forma secuencial entre el año 1999 y el 2011, comprobándose en primer lugar que el empleo de portainjertos híbridos de calabaza era posible con los cultivares de pepino corto tipo español, mayoritariamente empleados en los últimos años del siglo XX y primeros del XXI, fundamentalmente: Serena. Tras los primeros ensayos, Shintoza parecía el portainjerto híbrido de calabaza (Cucurbita maxima x C. moschata) con mejores perspectivas de empleo, pues presentaba la ventaja adicional de ser bien conocido por los semilleros que producen planta injertada al ser, en esos momentos, el portainjerto más empleado en sandía, lo que garantizaba por su lado, su empleo en pepino, y que los horticultores pudiesen disponer de planta injertada. Más adelante los trabajos se encaminaron hacia la determinación de la densidad y tipo de poda más adecuado para la planta injertada, realizándose múltiples ensayos en esta dirección, que culminaron con la conclusión de que el extravigor que los portainjertos conferían a las plantas permitía conducir estas a dos o más brazos (se suelen emplear dos, por mejor adaptación a los trabajos de manejo de la planta por parte de los agricultores), con lo que se podría disminuir la densidad de planta y por tanto ahorrar en este capítulo, cosa que preocupaba y preocupa a los agricultores. Se llegó a determinar que es posible reducir la densidad de plantación en alrededor de un 25%, estando la densidad de brazos más adecuada entre 3 y 3.5 br•m-2. Tras las primeras decisiones tomadas sobre el portainjerto y la densidad más adecuada, se continuó con el estudio de adaptación de estas propuestas a los nuevos cultivares que las empresas de semillas iban proponiendo y los agricultores adoptando. Estas acciones se complementaron con la introducción de nuevos portainjertos susceptibles de sustituir a Shintoza o rotar con él para cambiar de sistema radicular, lo que es conveniente cuando se emplean, como es el caso, portainjertos que no son resistentes a nematodos, principalmente de la especie Meloidogyne incognita, el mayor problema en la zona, debido al suelo. Cultivares como Trópico, en un primer momento, y Urano y Motril más recientemente, se adaptaron muy bien a esta técnica. Entre los portainjertos que mostraron buena adaptación a la técnica de injerto y suficientemente buena compatibilidad con la mayoría de los cultivares ensayados destacan: RS-841, Strongtosa y Camel. Azman también mostró un comportamiento relevante, pero este portainjerto no podrá ser empleado, al ser recientemente retirado del mercado por la empresa que lo obtuvo y comercializó Aunque no era el objetivo principal de esta Tesis Doctoral, se ha comprobado que puede ser interesante combinar el empleo del injerto con otras técnicas alternativas al bromuro de metilo para superar los problemas debidos a enfermedades del suelo o nematodos, pero debe seguirse trabajando pues este es un tema en continua evolución, tanto si se trata de desinfectantes, a la mayoría de los cuales les está siendo retirado el permiso para su comercialización, como de otros métodos como la biofumigación o el empleo de vapor de agua. Queda muy claro que el injerto puede considerarse entre los métodos respetuosos con el medio ambiente, si no el que más, en lo que alternativas al bromuro de metilo se refiere. También en otro momento, se comprobó que con plantas injertadas es posible reducir el aporte de nutrientes, sobre todo nitrógeno, lo que además de un ahorro supone una mejora más desde el punto de vista medioambiental. En definitiva, queda demostrado que es factible el empleo del injerto en pepino corto tipo español, que las selecciones de los híbridos entre Cucurbita maxima y C. moschata que habitualmente se están empleando en sandía son también de aplicación en estos pepinos y que su empleo puede llevarnos a producciones suficientemente remuneradoras, alargándose en muchos casos el ciclo y no modificando, de forma apreciable, la calidad. Queda también demostrado que aunque los portainjertos no sean resistentes a nematodos, su extravigor les hace desarrollarse, desde el punto de vista productivo, suficientemente, llegando por tanto, a “convivir” con ese problema. Al no ser resistentes los portainjertos, y permanecer e incluso agravarse el problema de nematodos es conveniente poder contar con diferentes portainjertos que nos permitan rotar entre ellos y utilizar diferentes sistemas radiculares que harán menos fácil el parasitismo de los nematodos, como recomiendan los nematólogos que se haga. ABSTRACT Vegetable grafting is one of the most current practices in horticulture, not only in Spain, but also in other Western and Asian countries, such as Japan, South Korea, China, the Philippines, etc. This is a decades-old, widespread technique: In fact, most cucurbit and solanaceous crops in Japan and Korea are grafted. At the end of the 1990s, it was clear that methyl bromide had an expiry date. Consequently, the areas strongly dependant on this soil disinfectant had to look for alternatives as quickly as possible. Besides, these had to be as environmentally friendly as possible and should not increase production costs significantly. The cucumber has been and still is the most important crop in greenhouses of the Comunidad de Madrid and in areas near Toledo and Guadalajara. Cucumber growers used methyl bromide systematically to disinfect the soil. The banning of this chemical product brought about uncertainty, which encouraged the search for different alternatives. After analyzing the different possibilities and taking into account the good results of watermelon grafting in Almería, it was decided to carry out the works that make up this doctoral thesis. Different trials were made in order to know if the cultivars used in cucumber grafting might be a real alternative for farmers, not only in Madrid, but also in the areas near Toledo and Guadalajara. The main aim was to assess the agronomic repercussions and whether that technique could be used alone, or if other complementary alternatives, such as chemical disinfectants, solarisation, biofumigation, or even steam disinfection, were necessary. Trials were carried out sequentially from 1999 to 2011. It was observed that the use of pumpkin hybrid rootstocks could be applied to cultivars of Spanish short cucumbers, mainly grown in the late 20th and early 21st centuries eg Serena. After the early trials, Shintoza (Cucurbita maxima x C. moschata), a pumpkin hybrid rootstock, seemed to be the best option, as it had the additional advantage of being well known by nurseries growing grafting plants. Bearing this in mind, Shintoza was then the hybrid rootstock to be used in cucumbers and consequently growers could have grafted plants at their disposal. Later on, research was focused on density and the most adequate type of pruning, by carrying out several trials. These experiments showed that, the extra vigour the rootstocks gave to the plants, allowed them to have two or three stems, (normally nurserymen use two, as it is easier for them to manage the plants). These findings would lead to the lessening the density of the plant and thus reduce costs, something which worried and still worries farmers. It was stated that it would be possible to reduce the density of the plants by about 25%, the optimum density of the stems ranging from 3 to 3.5 stem-m-2. Once decisions were taken both on the rootstock and the appropriate density, we went on to study how to apply these proposals to the new cultivars which the seed companies were proposing and the farmers were applying. These measures were complemented with the introduction of new rootstocks capable of replacing Shintoza, or rotating with it in order to change the root system. This is particularly necessary when rootstocks, non-resistant to nematodes, mainly of the species Meloidogyne incognita, are used. This is the main problem due to the soil of that area. Cultivars such as Trópico, at first, and Urano and Motril, more recently, adapted quite well to this technique. Among the rootstocks which adapted well to grafting and which were compatible with most tested cultivars, were, in particular, RS-841 Strongtosa and Camel. The behaviour of Azman was worth studying, but this rootstock was removed from the market by the company which had bought and commercialized it. Although not the main purpose of the research, it was observed that combining grafting with other alternatives to methyl bromide in order to overcome problems due to soil diseases or nematodes may be worthwhile. However, further research is needed, as this topic is in constant evolution, not only when we come to disinfectants, most of which are being affected by the removal of the permit for commercialization, but also when we refer to other techniques such as biofumigation or the use of steam. Results also showed that grafted plants may reduce the amount of fertilizers, particularly nitrogen, used: This means both saving money and the protection of the environment. We may conclude by saying that grafting Spanish short cucumbers is feasible, that the selections of the hybrids between Cucurbita maxima and C. moschata, habitually used in watermelon grafting, can also be applied to these cucumbers. It can also be concluded that the use of these grafting techniques may lead to profitable yields, by lengthening the growing cycle in many cases and by maintaining the quality to a large extent. Although these rootstocks are not resistant to nematodes, the results showed that their extra vigour enables them to develop in terms of production, and thus they cope with this problem. Since these rootstocks are not resistant to nematodes and the problem with these nematodes may even worsen, it is recommended that different types of rootstocks should be available to enable both the rotation and the use of different root systems, which will encourage the parasitism of nematodes.

Impact testing in melon (Cucumis melo L.)

The application of Rheology to study biological systems is a new and very extensive matter, in which melon is absolutely unknown. The goal of this work is to determine some physical characteristics of this fruit, immediately after harvest and during its conservation in cold storage. Portugal and Spain are the most interested countries in these studies, as they are important producers of melon. The varieties Branco da Leziria and Piel de sapo were chosen because they are the most popular in both countries. The fruit were studied on the day they were harvested, and then were conserved in cold storage in the "Instituto del Frio" in Madrid, and they were periodically tested again. Thus during seven days the same fruits, and new fruits, were picked up and tested. On the first day of testing we had 20 fruits to study and at the end of the testing period we had used 80 fruits. The results from the non-destructive impact test were very significant and they may contribute to standardise methods to measure fruit maturity. These results were confirmed by those obtained from compression tests. The results obtained during the Impact tests with melon were similar to those obtained previously with other fruits. There is a close relationship between the results of the Impact tests and Compression tests. Tests like Impact and Compression can be adapted to melon, varieties 'Piel de Sapo" and 'Branco de Leziria', allowing us to continue further work with this species. The great number of data obtained during performance of the tests allowed us to go on with this work and to contribute to standardise methods of measurement and expression of characteristics of a new biological product. During the "Impact damage in fruits and vegetables" workshop, held in Zaragoza in 1990, these matters were included in the priority list.

Insect growth regulators as potential insecticides to control olive fruit fly (Bactrocera oleae Rossi): insect toxicity bioassays and molecular docking approach

Olive fruit fly, Bactrocera oleae (Rossi), is a key pest in olive orchards, causing serious economic damage. To date, the pest has already developed resistance to the insecticides commonly applied to control it. Thus, in searching for new products for an accurate resistance management programme, targeting the ecdysone receptor (EcR)might provide alternative compounds for use in such programmes. RESULTS: Residual contact and oral exposure in the laboratory of B. oleae adults to the dibenzoylhydrazine-based compounds methoxyfenozide, tebufenozide and RH-5849 showed different results. Methoxyfenozide and tebufenozide did not provoke anynegative effectsontheadults,but RH-5849 killed98-100%of the treated insects15 days after treatment. Theligand-binding domain (LBD) of the EcR of B. oleae (BoEcR-LBD) was sequenced, and a homology protein model was constructed. Owing to a restricted extent of the ligand-binding cavity of the BoEcR-LBD, docking experiments with the three tested insecticides showed a severe steric clash in the case of methoxyfenozide and tebufenozide, while this was not the case with RH-5849. CONCLUSION: IGR molecules similar to the RH-5849 molecule, and different from methoxyfenozide and tebufenozide, might have potential in controlling this pest.

Determinaçao das características físicas de duas variedades de melâo (Cucumis melo L.).

O conhecimento preciso das características físicas dos frutos reveste-se do maior interesse pois permite minimizar as perdas por danos mecânicos, fornece dados para o desenho de novas maquinas e facilita a determinação das condições ideais de conservação. À determinação das características físicas de melão, a sua resistência aos danos físicos o seu comportamento quando sujeito a forças de deforma çao, sao estudadas mediante a utilização de diversos métodos. Foram seleccionadas duas variedades de melão ( Cucumis melo L.), as mais significativas em Espanha e Portugal respectivamente " Piei de Sapo " e " Melão Branco da Lezíria ". Ambos foram cultivados nas mesmas condições edafo-climáticas e sujeitos ã iguais práticas agronómicas, tendo sido os seus frutos sujeitos a diversos testes no " Laboratorio de Propiedades Físicas " da " Escola Técnica Superior de Ingenieros Agrónomos de Madrid ". Foram estudados diversos parâmetros como: " impact loading ", penetração, deformação e ruptura de frutos e ainda deformação e ruptura de amostras cilíndricas. O efeito do armazenamento em cámaras de frio sobre as propiedades físicas dos frutos foi também objecto de estudo.

Mapping and Introgression of QTL Involved in Fruit Shape Transgressive Segregation into 'Piel de Sapo' Melon (Cucumis melo L.)

A mapping F2 population from the cross ‘Piel de Sapo’ × PI124112 was selectively genotyped to study the genetic control of morphological fruit traits by QTL (Quantitative Trait Loci) analysis. Ten QTL were identified, five for FL (Fruit Length), two for FD (Fruit Diameter) and three for FS (Fruit Shape). At least one robust QTL per character was found, flqs8.1 (LOD = 16.85, R2 = 34%), fdqs12.1 (LOD = 3.47, R2 = 11%) and fsqs8.1 (LOD = 14.85, R2 = 41%). flqs2.1 and fsqs2.1 cosegregate with gene a (andromonoecious), responsible for flower sex determination and with pleiotropic effects on FS. They display a positive additive effect (a) value, so the PI124112 allele causes an increase in FL and FS, producing more elongated fruits. Conversely, the negative a value for flqs8.1 and fsqs8.1 indicates a decrease in FL and FS, what results in rounder fruits, even if PI124112 produces very elongated melons. This is explained by a significant epistatic interaction between fsqs2.1 and fsqs8.1, where the effects of the alleles at locus a are attenuated by the additive PI124112 allele at fsqs8.1. Roundest fruits are produced by homozygous for PI124112 at fsqs8.1 that do not carry any dominant A allele at locus a (PiPiaa). A significant interaction between fsqs8.1 and fsqs12.1 was also detected, with the alleles at fsqs12.1 producing more elongated fruits. fsqs8.1 seems to be allelic to QTL discovered in other populations where the exotic alleles produce elongated fruits. This model has been validated in assays with backcross lines along 3 years and ultimately obtaining a fsqs8.1-NIL (Near Isogenic Line) in ‘Piel de Sapo’ background which yields round melons.

Fungal microbiota from rain water and pathogenicity of Fusarium species isolated from atmospheric dust and rainfall dust

In order to determine the presence of Fusarium spp. in atmospheric dust and rainfall dust, samples were collected during September 2007, and July, August, and October 2008. The results reveal the prevalence of airborne Fusarium species coming from the atmosphere of the South East coast of Spain. Five different Fusarium species were isolated from the settling dust: Fusarium oxysporum, F. solani, F. equiseti, F. dimerum, and F. proliferatum. Moreover, rainwater samples were obtained during significant rainfall events in January and February 2009. Using the dilution-plate method, 12 fungal genera were identified from these rainwater samples. Specific analyses of the rainwater revealed the presence of three species of Fusarium: F. oxysporum, F. proliferatum and F. equiseti. A total of 57 isolates of Fusarium spp. obtained from both rainwater and atmospheric rainfall dust sampling were inoculated onto melon (Cucumis melo L.) cv. Piñonet and tomato (Lycopersicon esculentum Mill.) cv. San Pedro. These species were chosen because they are the main herbaceous crops in Almeria province. The results presented in this work indicate strongly that spores or propagules of Fusarium are able to cross the continental barrier carried by winds from the Sahara (Africa) to crop or coastal lands in Europe. Results show differences in the pathogenicity of the isolates tested. Both hosts showed root rot when inoculated with different species of Fusarium, although fresh weight measurements did not bring any information about the pathogenicity. The findings presented above are strong indications that long-distance transmission of Fusarium propagules may occur. Diseases caused by species of Fusarium are common in these areas. They were in the past, and are still today, a problem for greenhouses crops in Almería, and many species have been listed as pathogens on agricultural crops in this region. Saharan air masses dominate the Mediterranean regions. The evidence of long distance dispersal of Fusarium spp. by atmospheric dust and rainwater together with their proved pathogenicity must be taken into account in epidemiological studies.

Growth dynamics and yield of melon as influenced by nitrogen fertilizer

Nitrogen (N) is an important nutrient for melon (Cucumis melo L.) production. However there is scanty information about the amount necessary to maintain an appropriate balance between growth and yield. Melon vegetative organs must develop sufficiently to intercept light and accumulate water and nutrients but it is also important to obtain a large reproductive-vegetative dry weight ratio to maximize the fruit yield. We evaluated the influence of different N amounts on the growth, production of dry matter and fruit yield of a melon ‘Piel de sapo’ type. A three-year field experiment was carried out from May to September. Melons were subjected to an irrigation depth of 100% crop evapotranspiration and to 11 N fertilization rates, ranging 11 to 393 kg ha –1 in the three years. The dry matter production of leaves and stems increased as the N amount increased. The dry matter of the whole plant was affected similarly, while the fruit dry matter decreased as the N amount was increased above 112, 93 and 95 kg ha –1 , in 2005, 2006 and 2007, respectively. The maximum Leaf Area Index (LAI), 3.1, was obtained at 393 kg ha –1 of N. The lowest N supply reduced the fruit yield by 21%, while the highest increased the vegetative growth, LAI and Leaf Area Duration (LAD), but reduced yield by 24% relative to the N93 treatment. Excessive applications of N increase vegetative growth at the expense of reproductive growth. For this melon type, rates about 90-100 kg ha –1 of N are sufficient for adequate plant growth, development and maximum production. To obtain fruit yield close to the maximum, the leaf N concentration at the end of the crop cycle should be higher than 19.5 g kg –1

Nitrogen uptake dynamics, yield and quality as influenced by nitrogen fertilization in "Piel de sapo" melon.

The need to reduce nitrogen (N) fertilizer pollution strengthens the importance of improving the utilization efficiency of applied N to crops. This requires knowledge of crop N uptake characteristics and how fertilization management affects it. A three-year field experiment was conducted from May to September in central Spain to investigate the influence of different N rates, which ranged from 11 to 393 kg ha-1, applied through drip irrigation, on the dynamics of N uptake, nitrogen use efficiency (NUE), fruit yield and quality of a ?Piel de sapo? melon crop (Cucumis melo L. cv. Sancho). Both N concentration and N content increased in different plant parts with the N rate. Leaves had the highest N concentration, which declined by 40-50% from 34-41 days after transplanting (DAT), while the highest N uptake rate was observed from 30-35 to 70-80 DAT, coinciding with fruit development. In each year, NUE declined with increasing N rate. With N fertilizer applications close to the optimum N rate of 90-100 kg ha-1, the fruits removed approximately 60 kg N ha-1, and the amount of N in the crop residue was about 80 kg N ha-1; this serves to replenish the organic nutrient pool in the soil and may be used by subsequent crops following mineralization.

Storabelity of melon for different ripeness stages at harvest. Selection of instrumental procedures for quality assessment.

The consumption of melon (Cucumis melo L.) has been, until several years ago, regional, seasonal and without commercial interest. Recent commercial changes and world wide transportation have changed this situation. Melons from 3 different ripeness stages at harvest and 7 cold storage periods have been analysed by destructive and non destructive tests. Chemical, physical, mechanical (non destructive impact, compression, skin puncture and Magness- Taylor) and sensory tests were carried out in order to select the best test to assess quality and to determine the optimal ripeness stage at harvest. Analysis of variance and Principal Component Analysis were performed to study the data. The mechanical properties based on non-destructive Impact and Compression can be used to monitor cold storage evolution. They can also be used at harvest to segregate the highest ripeness stage (41 days after anthesis DAA) in relation to less ripe stages (34 and 28 DAA).Only 34 and 41 DAA reach a sensory evaluation above 50 in a scale from 0-100.

Fertilization Management: Assessing New Techniques and Key Interactions to Increase Crop Yields With Less N2O Emissions

El óxido nitroso (N2O) es un potente gas de efecto invernadero (GHG) proveniente mayoritariamente de la fertilización nitrogenada de los suelos agrícolas. Identificar estrategias de manejo de la fertilización que reduzcan estas emisiones sin suponer un descenso de los rendimientos es vital tanto a nivel económico como medioambiental. Con ese propósito, en esta Tesis se han evaluado: (i) estrategias de manejo directo de la fertilización (inhibidores de la nitrificación/ureasa); y (ii) interacciones de los fertilizantes con (1) el manejo del agua, (2) residuos de cosecha y (3) diferentes especies de plantas. Para conseguirlo se llevaron a cabo meta-análisis, incubaciones de laboratorio, ensayos en invernadero y experimentos de campo. Los inhibidores de la nitrificación y de la actividad ureasa se proponen habitualmente como medidas para reducir las pérdidas de nitrógeno (N), por lo que su aplicación estaría asociada al uso eficiente del N por parte de los cultivos (NUE). Sin embargo, su efecto sobre los rendimientos es variable. Con el objetivo de evaluar en una primera fase su efectividad para incrementar el NUE y la productividad de los cultivos, se llevó a cabo un meta-análisis. Los inhibidores de la nitrificación dicyandiamide (DCD) y 3,4-dimetilepyrazol phosphate (DMPP) y el inhibidor de la ureasa N-(n-butyl) thiophosphoric triamide (NBPT) fueron seleccionados para el análisis ya que generalmente son considerados las mejores opciones disponibles comercialmente. Nuestros resultados mostraron que su uso puede ser recomendado con el fin de incrementar tanto el rendimiento del cultivo como el NUE (incremento medio del 7.5% y 12.9%, respectivamente). Sin embargo, se observó que su efectividad depende en gran medida de los factores medioambientales y de manejo de los estudios evaluados. Una mayor respuesta fue encontrada en suelos de textura gruesa, sistemas irrigados y/o en cultivos que reciben altas tasas de fertilizante nitrogenado. En suelos alcalinos (pH ≥ 8), el inhibidor de la ureasa NBPT produjo el mayor efecto. Dado que su uso representa un coste adicional para los agricultores, entender las mejores prácticas que permitan maximizar su efectividad es necesario para posteriormente realizar comparaciones efectivas con otras prácticas que incrementen la productividad de los cultivos y el NUE. En base a los resultados del meta-análisis, se seleccionó el NBPT como un inhibidor con gran potencial. Inicialmente desarrollado para reducir la volatilización de amoniaco (NH3), en los últimos años algunos investigadores han demostrado en estudios de campo un efecto mitigador de este inhibidor sobre las pérdidas de N2O provenientes de suelos fertilizados bajo condiciones de baja humedad del suelo. Dada la alta variabilidad de los experimentos de campo, donde la humedad del suelo cambia rápidamente, ha sido imposible entender mecanísticamente el potencial de los inhibidores de la ureasa (UIs) para reducir emisiones de N2O y su dependencia con respecto al porcentaje de poros llenos de agua del suelo (WFPS). Por lo tanto se realizó una incubación en laboratorio con el propósito de evaluar cuál es el principal mecanismo biótico tras las emisiones de N2O cuando se aplican UIs bajo diferentes condiciones de humedad del suelo (40, 60 y 80% WFPS), y para analizar hasta qué punto el WFPS regula el efecto del inhibidor sobre las emisiones de N2O. Un segundo UI (i.e. PPDA) fue utilizado para comparar el efecto del NBPT con el de otro inhibidor de la ureasa disponible comercialmente; esto nos permitió comprobar si el efecto de NBPT es específico de ese inhibidor o no. Las emisiones de N2O al 40% WFPS fueron despreciables, siendo significativamente más bajas que las de todos los tratamientos fertilizantes al 60 y 80% WFPS. Comparado con la urea sin inhibidor, NBPT+U redujo las emisiones de N2O al 60% WFPS pero no tuvo efecto al 80% WFPS. La aplicación de PPDA incrementó significativamente las emisiones con respecto a la urea al 80% WFPS mientras que no se encontró un efecto significativo al 60% WFPS. Al 80% WFPS la desnitrificación fue la principal fuente de las emisiones de N2O en todos los tratamientos mientras que al 60% tanto la nitrificación como la desnitrificación tuvieron un papel relevante. Estos resultados muestran que un correcto manejo del NBPT puede suponer una estrategia efectiva para mitigar las emisiones de N2O. Con el objetivo de trasladar nuestros resultados de los estudios previos a condiciones de campo reales, se desarrolló un experimento en el que se evaluó la efectividad del NBPT para reducir pérdidas de N y aumentar la productividad durante un cultivo de cebada (Hordeum vulgare L.) en secano Mediterráneo. Se determinó el rendimiento del cultivo, las concentraciones de N mineral del suelo, el carbono orgánico disuelto (DOC), el potencial de desnitrificación, y los flujos de NH3, N2O y óxido nítrico (NO). La adición del inhibidor redujo las emisiones de NH3 durante los 30 días posteriores a la aplicación de urea en un 58% y las emisiones netas de N2O y NO durante los 95 días posteriores a la aplicación de urea en un 86 y 88%, respectivamente. El uso de NBPT también incrementó el rendimiento en grano en un 5% y el consumo de N en un 6%, aunque ninguno de estos incrementos fue estadísticamente significativo. Bajo las condiciones experimentales dadas, estos resultados demuestran el potencial del inhibidor de la ureasa NBPT para mitigar las emisiones de NH3, N2O y NO provenientes de suelos arables fertilizados con urea, mediante la ralentización de la hidrólisis de la urea y posterior liberación de menores concentraciones de NH4 + a la capa superior del suelo. El riego por goteo combinado con la aplicación dividida de fertilizante nitrogenado disuelto en el agua de riego (i.e. fertirriego por goteo) se considera normalmente una práctica eficiente para el uso del agua y de los nutrientes. Algunos de los principales factores (WFPS, NH4 + y NO3 -) que regulan las emisiones de GHGs (i.e. N2O, CO2 y CH4) y NO pueden ser fácilmente manipulados por medio del fertirriego por goteo sin que se generen disminuciones del rendimiento. Con ese propósito se evaluaron opciones de manejo para reducir estas emisiones en un experimento de campo durante un cultivo de melón (Cucumis melo L.). Los tratamientos incluyeron distintas frecuencias de riego (semanal/diario) y tipos de fertilizantes nitrogenados (urea/nitrato cálcico) aplicados por fertirriego. Fertirrigar con urea en lugar de nitrato cálcico aumentó las emisiones de N2O y NO por un factor de 2.4 y 2.9, respectivamente (P < 0.005). El riego diario redujo las emisiones de NO un 42% (P < 0.005) pero aumentó las emisiones de CO2 un 21% (P < 0.05) comparado con el riego semanal. Analizando el Poder de Calentamiento global en base al rendimiento así como los factores de emisión del NO, concluimos que el fertirriego semanal con un fertilizante de tipo nítrico es la mejor opción para combinar productividad agronómica con sostenibilidad medioambiental en este tipo de agroecosistemas. Los suelos agrícolas en las áreas semiáridas Mediterráneas se caracterizan por su bajo contenido en materia orgánica y bajos niveles de fertilidad. La aplicación de residuos de cosecha y/o abonos es una alternativa sostenible y eficiente desde el punto de vista económico para superar este problema. Sin embargo, estas prácticas podrían inducir cambios importantes en las emisiones de N2O de estos agroecosistemas, con impactos adicionales en las emisiones de CO2. En este contexto se llevó a cabo un experimento de campo durante un cultivo de cebada (Hordeum vulgare L.) bajo condiciones Mediterráneas para evaluar el efecto de combinar residuos de cosecha de maíz con distintos inputs de fertilizantes nitrogenados (purín de cerdo y/o urea) en estas emisiones. La incorporación de rastrojo de maíz incrementó las emisiones de N2O durante el periodo experimental un 105%. Sin embargo, las emisiones de NO se redujeron significativamente en las parcelas enmendadas con rastrojo. La sustitución parcial de urea por purín de cerdo redujo las emisiones netas de N2O un 46 y 39%, con y sin incorporación de residuo de cosecha respectivamente. Las emisiones netas de NO se redujeron un 38 y un 17% para estos mismos tratamientos. El ratio molar DOC:NO3 - demostró predecir consistentemente las emisiones de N2O y NO. El efecto principal de la interacción entre el fertilizante nitrogenado y el rastrojo de maíz se dio a los 4-6 meses de su aplicación, generando un aumento del N2O y una disminución del NO. La sustitución de urea por purín de cerdo puede considerarse una buena estrategia de manejo dado que el uso de este residuo orgánico redujo las emisiones de óxidos de N. Los pastos de todo el mundo proveen numerosos servicios ecosistémicos pero también suponen una importante fuente de emisión de N2O, especialmente en respuesta a la deposición de N proveniente del ganado mientras pasta. Para explorar el papel de las plantas como mediadoras de estas emisiones, se analizó si las emisiones de N2O dependen de la riqueza en especies herbáceas y/o de la composición específica de especies, en ausencia y presencia de una deposición de orina. Las hipótesis fueron: 1) las emisiones de N2O tienen una relación negativa con la productividad de las plantas; 2) mezclas de cuatro especies generan menores emisiones que monocultivos (dado que su productividad será mayor); 3) las emisiones son menores en combinaciones de especies con distinta morfología radicular y alta biomasa de raíz; y 4) la identidad de las especies clave para reducir el N2O depende de si hay orina o no. Se establecieron monocultivos y mezclas de dos y cuatro especies comunes en pastos con rasgos funcionales divergentes: Lolium perenne L. (Lp), Festuca arundinacea Schreb. (Fa), Phleum pratense L. (Php) y Poa trivialis L. (Pt), y se cuantificaron las emisiones de N2O durante 42 días. No se encontró relación entre la riqueza en especies y las emisiones de N2O. Sin embargo, estas emisiones fueron significativamente menores en ciertas combinaciones de especies. En ausencia de orina, las comunidades de plantas Fa+Php actuaron como un sumidero de N2O, mientras que los monocultivos de estas especies constituyeron una fuente de N2O. Con aplicación de orina la comunidad Lp+Pt redujo (P < 0.001) las emisiones de N2O un 44% comparado con los monocultivos de Lp. Las reducciones de N2O encontradas en ciertas combinaciones de especies pudieron explicarse por una productividad total mayor y por una complementariedad en la morfología radicular. Este estudio muestra que la composición de especies herbáceas es un componente clave que define las emisiones de N2O de los ecosistemas de pasto. La selección de combinaciones de plantas específicas en base a la deposición de N esperada puede, por lo tanto, ser clave para la mitigación de las emisiones de N2O. ABSTRACT Nitrous oxide (N2O) is a potent greenhouse gas (GHG) directly linked to applications of nitrogen (N) fertilizers to agricultural soils. Identifying mitigation strategies for these emissions based on fertilizer management without incurring in yield penalties is of economic and environmental concern. With that aim, this Thesis evaluated: (i) the use of nitrification and urease inhibitors; and (ii) interactions of N fertilizers with (1) water management, (2) crop residues and (3) plant species richness/identity. Meta-analysis, laboratory incubations, greenhouse mesocosm and field experiments were carried out in order to understand and develop effective mitigation strategies. Nitrification and urease inhibitors are proposed as means to reduce N losses, thereby increasing crop nitrogen use efficiency (NUE). However, their effect on crop yield is variable. A meta-analysis was initially conducted to evaluate their effectiveness at increasing NUE and crop productivity. Commonly used nitrification inhibitors (dicyandiamide (DCD) and 3,4-dimethylepyrazole phosphate (DMPP)) and the urease inhibitor N-(n-butyl) thiophosphoric triamide (NBPT) were selected for analysis as they are generally considered the best available options. Our results show that their use can be recommended in order to increase both crop yields and NUE (grand mean increase of 7.5% and 12.9%, respectively). However, their effectiveness was dependent on the environmental and management factors of the studies evaluated. Larger responses were found in coarse-textured soils, irrigated systems and/or crops receiving high nitrogen fertilizer rates. In alkaline soils (pH ≥ 8), the urease inhibitor NBPT produced the largest effect size. Given that their use represents an additional cost for farmers, understanding the best management practices to maximize their effectiveness is paramount to allow effective comparison with other practices that increase crop productivity and NUE. Based on the meta-analysis results, NBPT was identified as a mitigation option with large potential. Urease inhibitors (UIs) have shown to promote high N use efficiency by reducing ammonia (NH3) volatilization. In the last few years, however, some field researches have shown an effective mitigation of UIs over N2O losses from fertilized soils under conditions of low soil moisture. Given the inherent high variability of field experiments where soil moisture content changes rapidly, it has been impossible to mechanistically understand the potential of UIs to reduce N2O emissions and its dependency on the soil water-filled pore space (WFPS). An incubation experiment was carried out aiming to assess what is the main biotic mechanism behind N2O emission when UIs are applied under different soil moisture conditions (40, 60 and 80% WFPS), and to analyze to what extent the soil WFPS regulates the effect of the inhibitor over N2O emissions. A second UI (i.e. PPDA) was also used aiming to compare the effect of NBPT with that of another commercially available urease inhibitor; this allowed us to see if the effect of NBPT was inhibitor-specific or not. The N2O emissions at 40% WFPS were almost negligible, being significantly lower from all fertilized treatments than that produced at 60 and 80% WFPS. Compared to urea alone, NBPT+U reduced the N2O emissions at 60% WFPS but had no effect at 80% WFPS. The application of PPDA significantly increased the emissions with respect to U at 80% WFPS whereas no significant effect was found at 60% WFPS. At 80% WFPS denitrification was the main source of N2O emissions for all treatments. Both nitrification and denitrification had a determinant role on these emissions at 60% WFPS. These results suggest that adequate management of the UI NBPT can provide, under certain soil conditions, an opportunity for N2O mitigation. We translated our previous results to realistic field conditions by means of a field experiment with a barley crop (Hordeum vulgare L.) under rainfed Mediterranean conditions in which we evaluated the effectiveness of NBPT to reduce N losses and increase crop yields. Crop yield, soil mineral N concentrations, dissolved organic carbon (DOC), denitrification potential, NH3, N2O and nitric oxide (NO) fluxes were measured during the growing season. The inclusion of the inhibitor reduced NH3 emissions in the 30 d following urea application by 58% and net N2O and NO emissions in the 95 d following urea application by 86 and 88%, respectively. NBPT addition also increased grain yield by 5% and N uptake by 6%, although neither increase was statistically significant. Under the experimental conditions presented here, these results demonstrate the potential of the urease inhibitor NBPT in abating NH3, N2O and NO emissions from arable soils fertilized with urea, slowing urea hydrolysis and releasing lower concentrations of NH4 + to the upper soil layer. Drip irrigation combined with split application of N fertilizer dissolved in the irrigation water (i.e. drip fertigation) is commonly considered best management practice for water and nutrient efficiency. Some of the main factors (WFPS, NH4 + and NO3 -) regulating the emissions of GHGs (i.e. N2O, carbon dioxide (CO2) and methane (CH4)) and NO can easily be manipulated by drip fertigation without yield penalties. In this study, we tested management options to reduce these emissions in a field experiment with a melon (Cucumis melo L.) crop. Treatments included drip irrigation frequency (weekly/daily) and type of N fertilizer (urea/calcium nitrate) applied by fertigation. Crop yield, environmental parameters, soil mineral N concentrations, N2O, NO, CH4, and CO2 fluxes were measured during the growing season. Fertigation with urea instead of calcium nitrate increased N2O and NO emissions by a factor of 2.4 and 2.9, respectively (P < 0.005). Daily irrigation reduced NO emissions by 42% (P < 0.005) but increased CO2 emissions by 21% (P < 0.05) compared with weekly irrigation. Based on yield-scaled Global Warming Potential as well as NO emission factors, we conclude that weekly fertigation with a NO3 --based fertilizer is the best option to combine agronomic productivity with environmental sustainability. Agricultural soils in semiarid Mediterranean areas are characterized by low organic matter contents and low fertility levels. Application of crop residues and/or manures as amendments is a cost-effective and sustainable alternative to overcome this problem. However, these management practices may induce important changes in the nitrogen oxide emissions from these agroecosystems, with additional impacts on CO2 emissions. In this context, a field experiment was carried out with a barley (Hordeum vulgare L.) crop under Mediterranean conditions to evaluate the effect of combining maize (Zea mays L.) residues and N fertilizer inputs (organic and/or mineral) on these emissions. Crop yield and N uptake, soil mineral N concentrations, dissolved organic carbon (DOC), denitrification capacity, N2O, NO and CO2 fluxes were measured during the growing season. The incorporation of maize stover increased N2O emissions during the experimental period by c. 105 %. Conversely, NO emissions were significantly reduced in the plots amended with crop residues. The partial substitution of urea by pig slurry reduced net N2O emissions by 46 and 39 %, with and without the incorporation of crop residues respectively. Net emissions of NO were reduced 38 and 17 % for the same treatments. Molar DOC:NO3 - ratio was found to be a robust predictor of N2O and NO fluxes. The main effect of the interaction between crop residue and N fertilizer application occurred in the medium term (4-6 month after application), enhancing N2O emissions and decreasing NO emissions as consequence of residue incorporation. The substitution of urea by pig slurry can be considered a good management strategy since N2O and NO emissions were reduced by the use of the organic residue. Grassland ecosystems worldwide provide many important ecosystem services but they also function as a major source of N2O, especially in response to N deposition by grazing animals. In order to explore the role of plants as mediators of these emissions, we tested whether and how N2O emissions are dependent on grass species richness and/or specific grass species composition in the absence and presence of urine deposition. We hypothesized that: 1) N2O emissions relate negatively to plant productivity; 2) four-species mixtures have lower emissions than monocultures (as they are expected to be more productive); 3) emissions are lowest in combinations of species with diverging root morphology and high root biomass; and 4) the identity of the key species that reduce N2O emissions is dependent on urine deposition. We established monocultures and two- and four-species mixtures of common grass species with diverging functional traits: Lolium perenne L. (Lp), Festuca arundinacea Schreb. (Fa), Phleum pratense L. (Php) and Poa trivialis L. (Pt), and quantified N2O emissions for 42 days. We found no relation between plant species richness and N2O emissions. However, N2O emissions were significantly reduced in specific plant species combinations. In the absence of urine, plant communities of Fa+Php acted as a sink for N2O, whereas the monocultures of these species constituted a N2O source. With urine application Lp+Pt plant communities reduced (P < 0.001) N2O emissions by 44% compared to monocultures of Lp. Reductions in N2O emissions by species mixtures could be explained by total biomass productivity and by complementarity in root morphology. Our study shows that plant species composition is a key component underlying N2O emissions from grassland ecosystems. Selection of specific grass species combinations in the context of the expected nitrogen deposition regimes may therefore provide a key management practice for mitigation of N2O emissions.

A non-persistently transmitted-virus induces a pull?push strategy inits aphid vector to optimize transmission and spread

Plant viruses are known to modify the behaviour of their insect vectors, both directly and indirectly,generally adapting to each type of virus?vector relationship in a way that enhances transmissionefficiency. Here, we report results of three different studies showing how a virus transmitted in a non-persistent (NP) manner (Cucumber mosaic virus; CMV, Cucumovirus) can induce changes in its host plant,cucumber (Cucumis sativus cv. Marumba) that modifies the behaviour of its aphid vector (Aphis gossypiiGlover; Hemiptera: Aphididae) in a way that enhances virus transmission and spread non-viruliferousaphids changed their alighting, settling and probing behaviour activities over time when exposed toCMV-infected and mock-inoculated cucumber plants. Aphids exhibited no preference to migrate fromCMV-infected to mock-inoculated plants at short time intervals (1, 10 and 30 min after release), butshowed a clear shift in preference to migrate from CMV-infected to mock-inoculated plants 60 min afterrelease. Our free-choice preference assays showed that A. gossypii alates preferred CMV-infected overmock-inoculated plants at an early stage (30 min), but this behaviour was reverted at a later stage andaphids preferred to settle and reproduce on mock-inoculated plants. The electrical penetration graph(EPG) technique revealed a sharp change in aphid probing behaviour over time when exposed to CMV-infected plants. At the beginning (first 15 min) aphid vectors dramatically increased the number of shortsuperficial probes and intracellular punctures when exposed to CMV-infected plants. At a later stage (sec-ond hour of recording) aphids diminished their feeding on CMV-infected plants as indicated by much lesstime spent in phloem salivation and ingestion (E1 and E2). This particular probing behaviour includingan early increase in the number of short superficial probes and intracellular punctures followed by aphloem feeding deterrence is known to enhance the transmission efficiency of viruses transmitted in aNP manner. We conclude that CMV induces specific changes in a plant host that modify the alighting,settling and probing behaviour of its main vector A. gossypii, leading to optimum transmission and spreadof the virus. Our findings should be considered when modelling the spread of viruses transmitted in a NPmanner.

Integration of physical, chemical and biological tactics against insect pests and virus diseases in horticultural crops

Actualmente, la gestión de sistemas de Manejo Integrado de Plagas (MIP) en cultivos hortícolas tiene por objetivo priorizar los métodos de control no químicos en detrimento del consumo de plaguicidas, según recoge la directiva europea 2009/128/CE ‘Uso Sostenible de Plaguicidas’ (OJEC, 2009). El uso de agentes de biocontrol como alternativa a la aplicación de insecticidas es un elemento clave de los sistemas MIP por sus innegables ventajas ambientales que se utiliza ampliamente en nuestro país (Jacas y Urbaneja, 2008). En la región de Almería, donde se concentra el 65% de cultivo en invernadero de nuestro país (47.367 ha), MIP es la principal estrategia en pimiento (MAGRAMA, 2014), y comienza a serlo en otros cultivos como tomate o pepino. El cultivo de pepino, con 8.902 ha (MAGRAMA, 2013), tiene un protocolo semejante al pimiento (Robledo et al., 2009), donde la única especie de pulgón importante es Aphis gossypii Glover. Sin embargo, pese al continuo incremento de la superficie de cultivo agrícola bajo sistemas MIP, los daños originados por virosis siguen siendo notables. Algunos de los insectos presentes en los cultivos de hortícolas son importantes vectores de virus, como los pulgones, las moscas blancas o los trips, cuyo control resulta problemático debido a su elevada capacidad para transmitir virus vegetales incluso a una baja densidad de plaga (Holt et al., 2008; Jacas y Urbaneja, 2008). Las relaciones que se establecen entre los distintos agentes de un ecosistema son complejas y muy específicas. Se ha comprobado que, pese a que los enemigos naturales reducen de manera beneficiosa los niveles de plaga, su incorporación en los sistemas planta-insecto-virus puede desencadenar complicadas interacciones con efectos no deseables (Dicke y van Loon, 2000; Jeger et al., 2011). Así, los agentes de biocontrol también pueden inducir a que los insectos vectores modifiquen su comportamiento como respuesta al ataque y, con ello, el grado de dispersión y los patrones de distribución de las virosis que transmiten (Bailey et al., 1995; Weber et al., 1996; Hodge y Powell, 2008a; Hodge et al., 2011). Además, en ocasiones el control biológico por sí solo no es suficiente para controlar determinadas plagas (Medina et al., 2008). Entre los métodos que se pueden aplicar bajo sistemas MIP están las barreras físicas que limitan la entrada de plagas al interior de los invernaderos o interfieren con su movimiento, como pueden ser las mallas anti-insecto (Álvarez et al., 2014), las mallas fotoselectivas (Raviv y Antignus, 2004; Weintraub y Berlinger, 2004; Díaz y Fereres, 2007) y las mallas impregnadas en insecticida (Licciardi et al., 2008; Martin et al., 2014). Las mallas fotoselectivas reducen o bloquean casi por completo la transmisión de radiación UV, lo que interfiere con la visión de los insectos y dificulta o impide la localización del cultivo y su establecimiento en el mismo (Raviv y Antignus, 2004; Weintraub, 2009). Se ha comprobado cómo su uso puede controlar los pulgones y las virosis en cultivo de lechuga (Díaz et al., 2006; Legarrea et al., 2012a), así como la mosca blanca, los trips y los ácaros, y los virus que estos transmiten en otros cultivos (Costa y Robb, 1999; Antignus et al., 2001; Kumar y Poehling, 2006; Doukas y Payne, 2007a; Legarrea et al., 2010). Sin embargo, no se conoce perfectamente el modo de acción de estas barreras, puesto que existe un efecto directo sobre la plaga y otro indirecto mediado por la planta, cuya fisiología cambia al desarrollarse en ambientes con falta de radiación UV, y que podría afectar al ciclo biológico de los insectos fitófagos (Vänninen et al., 2010; Johansen et al., 2011). Del mismo modo, es necesario estudiar la compatibilidad de esta estrategia con los enemigos naturales de las plagas. Hasta la fecha, los estudios han evidenciado que los agentes de biocontrol pueden realizar su actividad bajo ambientes pobres en radiación UV (Chyzik et al., 2003; Chiel et al., 2006; Doukas y Payne, 2007b; Legarrea et al., 2012c). Otro método basado en barreras físicas son las mallas impregnadas con insecticidas, que se han usado tradicionalmente en la prevención de enfermedades humanas transmitidas por mosquitos (Martin et al., 2006). Su aplicación se ha ensayado en agricultura en ciertos cultivos al aire libre (Martin et al., 2010; Díaz et al., 2004), pero su utilidad en cultivos protegidos para prevenir la entrada de insectos vectores en invernadero todavía no ha sido investigada. Los aditivos se incorporan al tejido durante el proceso de extrusión de la fibra y se liberan lentamente actuando por contacto en el momento en que el insecto aterriza sobre la malla, con lo cual el riesgo medioambiental y para la salud humana es muy limitado. Los plaguicidas que se emplean habitualmente suelen ser piretroides (deltametrina o bifentrín), aunque también se ha ensayado dicofol (Martin et al., 2010) y alfa-cipermetrina (Martin et al., 2014). Un factor que resulta de vital importancia en este tipo de mallas es el tamaño del poro para facilitar una buena ventilación del cultivo, al tiempo que se evita la entrada de insectos de pequeño tamaño como las moscas blancas (Bethke y Paine, 1991; Muñoz et al., 1999). Asimismo, se plantea la necesidad de estudiar la compatibilidad de estas mallas con los enemigos naturales. Es por ello que en esta Tesis Doctoral se plantea la necesidad de evaluar nuevas mallas impregnadas que impidan el paso de insectos de pequeño tamaño al interior de los invernaderos, pero que a su vez mantengan un buen intercambio y circulación de aire a través del poro de la malla. Así, en la presente Tesis Doctoral, se han planteado los siguientes objetivos generales a desarrollar: 1. Estudiar el impacto de la presencia de parasitoides sobre el grado de dispersión y los patrones de distribución de pulgones y las virosis que éstos transmiten. 2. Conocer el efecto directo de ambientes pobres en radiación UV sobre el comportamiento de vuelo de plagas clave de hortícolas y sus enemigos naturales. 3. Evaluar el efecto directo de la radiación UV-A sobre el crecimiento poblacional de pulgones y mosca blanca, y sobre la fisiología de sus plantas hospederas, así como el efecto indirecto de la radiación UV-A en ambas plagas mediado por el crecimiento de dichas planta hospederas. 4. Caracterización de diversas mallas impregnadas en deltametrina y bifentrín con diferentes propiedades y selección de las óptimas para el control de pulgones, mosca blanca y sus virosis asociadas en condiciones de campo. Estudio de su compatibilidad con parasitoides. ABSTRACT Insect vectors of plant viruses are the main agents causing major economic losses in vegetable crops grown under protected environments. This Thesis focuses on the implementation of new alternatives to chemical control of insect vectors under Integrated Pest Management programs. In Spain, biological control is the main pest control strategy used in a large part of greenhouses where horticultural crops are grown. The first study aimed to increase our knowledge on how the presence of natural enemies such as Aphidius colemani Viereck may alter the dispersal of the aphid vector Aphis gossypii Glover (Chapter 4). In addition, it was investigated if the presence of this parasitoid affected the spread of aphid-transmitted viruses Cucumber mosaic virus (CMV, Cucumovirus) and Cucurbit aphid-borne yellows virus (CABYV, Polerovirus) infecting cucumber (Cucumis sativus L). SADIE methodology was used to study the distribution patterns of both the virus and its vector, and their degree of association. Results suggested that parasitoids promoted aphid dispersal in the short term, which enhanced CMV spread, though consequences of parasitism suggested potential benefits for disease control in the long term. Furthermore, A. colemani significantly limited the spread and incidence of the persistent virus CABYV in the long term. The flight activity of pests Myzus persicae (Sulzer), Bemisia tabaci (Gennadius) and Tuta absoluta (Meyrick), and natural enemies A. colemani and Sphaerophoria rueppellii (Weidemann) under UV-deficient environments was studied under field conditions (Chapter 5). One-chamber tunnels were covered with cladding materials with different UV transmittance properties. Inside each tunnel, insects were released from tubes placed in a platform suspended from the ceiling. Specific targets were located at different distances from the platform. The ability of aphids and whiteflies to reach their targets was diminished under UV-absorbing barriers, suggesting a reduction of vector activity under this type of nets. Fewer aphids reached distant traps under UV-absorbing nets, and significantly more aphids could fly to the end of the tunnels covered with non-UV blocking materials. Unlike aphids, differences in B. tabaci captures were mainly found in the closest targets. The oviposition of lepidopteran T. absoluta was also negatively affected by a UV-absorbing cover. The photoselective barriers were compatible with parasitism and oviposition of biocontrol agents. Apart from the direct response of insects to UV radiation, plant-mediated effects influencing insect performance were investigated (Chapter 6). The impact of UV-A radiation on the performance of aphid M. persicae and whitefly B. tabaci, and growth and leaf physiology of host plants pepper and eggplant was studied under glasshouse conditions. Plants were grown inside cages covered by transparent and UV-A-opaque plastic films. Plant growth and insect fitness were monitored. Leaves were harvested for chemical analysis. Pepper plants responded directly to UV-A by producing shorter stems whilst UV-A did not affect the leaf area of either species. UV-A-treated peppers had higher content of secondary metabolites, soluble carbohydrates, free amino acids and proteins. Such changes in tissue chemistry indirectly promoted aphid performance. For eggplants, chlorophyll and carotenoid levels decreased with supplemental UVA but phenolics were not affected. Exposure to supplemental UV-A had a detrimental effect on whitefly development, fecundity and fertility presumably not mediated by plant cues, as compounds implied in pest nutrition were unaltered. Lastly, the efficacy of a wide range of Long Lasting Insecticide Treated Nets (LLITNs) was studied under laboratory and field conditions. This strategy aimed to prevent aphids and whiteflies to enter the greenhouse by determining the optimum mesh size (Chapter 7). This new approach is based on slow release deltamethrin- and bifenthrin-treated nets with large hole sizes that allow improved ventilation of greenhouses. All LLITNs produced high mortality of M. persicae and A. gossypii although their efficacy decreased over time with sun exposure. It was necessary a net with hole size of 0.29 mm2 to exclude B. tabaci under laboratory conditions. The feasibility of two selected nets was studied in the field under a high insect infestation pressure in the presence of CMV- and CABYV-infected cucumber plants. Besides, the compatibility of parasitoid A. colemani with bifenthrin-treated nets was studied in parallel field experiments. Both nets effectively blocked the invasion of aphids and reduced the incidence of both viruses, however they failed to exclude whiteflies. We found that our LLITNs were compatible with parasitoid A. colemani. As shown, the role of natural enemies has to be taken into account regarding the dispersal of insect vectors and subsequent spread of plant viruses. The additional benefits of novel physicochemical barriers, such as photoselective and insecticide-impregnated nets, need to be considered in Integrated Pest Management programs of vegetable crops grown under protected environments.