Tesi sul tema "Crop protection"

Chemotaxis in Agrobacierium tumefaciens was studied. Several plant derived monocyclic phenolic compounds were analysed for their ability to act as chemoattractants for A. tumefaciens C58C (^1) and as inducers of the Ti-plasmid virulence operons. The results divided the phenolics into 4 groups. A strong correlation between vir- inducing ability and Ti-plasmid requirement for chemo taxis was established and chemical structure rules for vir induction and chemo taxis are outlined. Furthermore, virA and virG were found to be the Ti-plasmid virulence genes required for chemo taxis towards the monocyclic phenolic compound acetosyringone. Chemotaxis towards both monocotyledonous and dicotyledonous plant extracts was analysed. Undiluted shoot and root extracts from both sources elicited a response from both Ti-plasmid harbouring and cured A. tumefaciens C58C(^1) However, when diluted extracts of Wheat and Kalanchoe shoot homogenate were analysed, a distinct enhancement of chemotaxis was conferred by the Ti-plasmid, suggesting that recognition of, and attraction towards, susceptible plants is not the step blocked in monocot transformation. Analysis of cell wall material revealed that native cell wall components are not required for chemotaxis of A. tumefaciens C58C (^1) towards plant extracts. Results obtained on chemotaxis along with current knowledge of vir- induction allowed the development of a novel idea involving Agrobacterium as a biocontrol agent. A chitinase gene from Serratia marcescens was manipulated such that its promotor was removed. The promotorless cassette was linked to the virB pro-motor from an octopine Ti-plasmid and the construct introduced into Agrobacterium harbouring virA and virG. The potential benefit of this biocontrol system with respect to other existing biocontrol systems is that expression of the pesticidal gene is regulated by components of wound exudate and therefore is a conservative process, pesticide being produced only when a plant is wounded, at a time when it is most susceptible to attack by plant pathogens, and then exclusively in the microrhizosphere around the wound site.

Els fongs patògens de plantes causen importants pèrdues en les collites, posant en perill la seguretat i qualitat alimentària. Els pèptids antimicrobians (AMPs) mostren una activitat lítica potent i duradora específicament enfront de microorganismes, de manera que tenen un gran potencial com a nous fungicides naturals per al control dels fongs patògens. La seva explotació requereix de sistemes de producció ràpids, eficaços, econòmics i segurs. El principal objectiu d’aquest treball era desenvolupar sistemes de producció sostenibles de AMPs, i la seva caracterització en el control d’infeccions fúngiques per avançar en la seva aplicació en l’agricultura. Les proteïnes antifúngiques (AFPs) secretades per fongs filamentosos són un grup de AMPs rics en cisteïnes, molt estables, actius específicament enfront de fongs. En aquest estudi vam demostrar que les plantes de Nicotiana bentamiana són una excel·lent biofactoria de AFPs mitjançant expressió transitòria usant un nou vector derivat del virus de mosaic del tabac. Utilitzant aquest sistema de producció en plantes, hem produït eficientment dues AFPs molt actives enfront de fongs fitopatògens, la AfpA de Penicillium expansum i la AfpB de Penicillium digitatum. Hem descobert que el compartiment subcel·lular on s’acumulen les AFPs té un impacte important en la producció obtinguda, probablement perquè la seva compartimentació evita la toxicitat cap a les cèl·lules vegetals. Els valors més alts es van obtenir quan les proteïnes es van acumular en els vacúols, aconseguint fins 0,170 mg / g de fulla en el cas de la proteïna més activa AfpA i fins a vuit vegades més per a la AfpB (1,2 mg / g de fulla). També vam demostrar que els extractes crus de plantes que contenen AFP són actius enfront a fongs, sense necessitat de purificar les proteïnes reduint considerablement el processament del material vegetal i els costos de producció. Per tant, el sistema desenvolupat és eficient per a la producció de AFPs, i també és econòmic i segur ja que es basa en plantes. A més, hem desenvolupat un sistema alternatiu per a la producció del pèptid antifúngic PAF102 que prèviament no s’havia pogut produir biotecnològicament. Aquest sistema es basa en acumular el pèptid en les gotes lipídiques (LDs) mitjançant la fusió a una proteïna oleosina de plantes. Mitjançant aquesta estratègia, hem produït PAF102 en llavors d’arròs en quantitats de 20 mg per gram de llavor. No obstant això, la producció en llavors és lenta i per accelerar el procés hem transferit la tecnologia de la fusió a oleosinas de plantes al sistema de Pichia pastoris. Usant aquest nou sistema hem obtingut rendiments comercialment rellevants amb produccions de 180 mg / l de cultiu en només 4 dies. L’acumulació de PAF102 a les LDs de les llavors d’arròs i del llevat facilita enormement la seva extracció per simple flotació en solucions denses, permetent la recuperació de pèptid biològicament actiu. Finalment, hem demostrat que tant AfpA i AfpB produïdes en plantes, com els extractes de plantes enriquits en aquestes proteines, són eficaços en la prevenció d’infeccions fúngiques en cultius econòmicament rellevants, com ara la podridura grisa causada per Botrytis cinerea en fulles i fruits de tomàquet, la piriculariosis causada per Magnaporthe oryzae, o les infeccions de les llavors d’arròs per Fusarium proliferatum. Els nostres resultats proporcionen un sistema de producció sostenible de AFPs i demostren la seva eficàcia en la protecció de les plantes contra les infeccions fúngiques, donant ferm suport per al seu ús com a “fungicides verds” eficaços i respectuosos amb el medi ambient en la protecció de cultius, mentres són al canmp o durant el periode postcollita.
Los hongos patógenos de plantas causan importantes pérdidas en las cosechas, poniendo en peligro la seguridad y calidad alimentaria. Los péptidos antimicrobianos (AMPs) muestran una actividad lítica potente y duradera específicamente frente a microorganismos, por lo que tienen un gran potencial como nuevos fungicidas naturales para el control de los hongos patógenos. Su explotación requiere de sistemas de producción rápidos, eficaces, económicos y seguros. El principal objetivo de este trabajo era desarrollar sistemas de producción sostenibles de AMPs, y su caracterización en el control de infecciones fúngicas para avanzar en su aplicación en la agricultura. Las proteínas antifúngicas (AFPs) secretadas por hongos filamentosos son un grupo de AMPs ricos en cisteínas, muy estables, activos específicamente frente a hongos. En este estudio demostramos que las plantas de Nicotiana bentamiana son una excelente biofactoría de AFPs mediante expresión transitoria usando un nuevo vector derivado del virus de mosaico del tabaco. Utilizando este sistema de producción en plantas, hemos producido eficientemente dos AFPs muy activas frente a hongos fitopatógenos, la AfpA de Penicillium expansum y la AfpB de Penicillium digitatum. Hemos descubierto que el compartimento subcelular donde se acumulan las AFPs tiene un impacto importante en la producción obtenida, probablemente porque su compartimentalización evita la toxicidad hacia las células vegetales. Los valores más altos se obtuvieron cuando las proteínas se acumularon en las vacuolas, alcanzando hasta 0,170 mg/g de hoja en el caso de la proteína más activa AfpA y hasta ocho veces más para la AfpB (1,2 mg/g de hoja). También demostramos que los extractos crudos de plantas que contienen AFP son activos frente a hongos, sin necesidad de purificar las proteínas reduciendo considerablemente el procesamiento del material vegetal y los costes de producción. Por lo tanto, el sistema desarrollado es eficiente para la producción de AFPs, y también es económico y seguro ya que se basa en plantas. Además, hemos desarrollado un sistema alternativo para la producción del péptido antifúngico PAF102 que previament no había podido producirse biotecnológicamente. Este sistema se basa en acumular el péptido en las gotas lipídicas (LDs) mediante la fusión a una proteína oleosina de plantas. Mediante esta estrategia, hemos producido PAF102 en semillas de arroz en cantidades de 20 mg por gramo de semilla. Sin embargo, la producción en semillas es lenta y para acelerar el proceso hemos transferido la tecnología de la fusión a oleosinas de plantas al sistema de Pichia pastoris. Usando este nuevo sistema hemos obtenido rendimientos comercialmente relevantes con producciones de 180 mg/l de cultivo en sólo 4 días. La acumulación de PAF102 en las LDs de las semillas de arroz y de la levadura facilita enormemente su extracción por simple flotación en soluciones densas, permitiendo la recuperación de péptido activo frente a hongos patógenos. Finalmente, hemos demostrado que tanto AfpA y AfpB producidas en plantas, como los extractos de plantas enriquecidos estas proteinas, son eficaces en la prevención de infecciones fúngicas en cultivos económicamente relevantes, tales como la podredumbre gris causada por Botrytis cinerea en hojas y frutos de tomate, el quemado del arroz causado por Magnaporthe oryzae, o las infecciones de las semillas de arroz por Fusarium proliferatum. Nuestros resultados proporcionan un sistema de producción sostenible de AFPs y demuestran su eficacia en la protección de las plantas contra las infecciones fúngicas, apoyando firmemente su uso como "fungicidas verdes" eficaces y respetuosos con el medio ambiente en la protección de cultivos y postcosecha.
Plant diseases caused by pathogenic fungi are responsible of important crop losses endangering food security and safety. Antimicrobial peptides (AMPs), exhibiting potent and durable lytic activity specifically against microorganisms, have a great potential as novel natural fungicides for the control of pathogenic fungi. However, viable exploitation of AMPs requires fast, cost-efficient, and safe production systems. The main goal of this work was to develop a sustainable platform for the production of bioactive AMPs, and to characterize them in the control of fungal infections in plants to advance in their application in agriculture. Antifungal proteins (AFPs) secreted by filamentous fungi are a group of highly stable cysteine-rich AMPs that specifically target fungal cells. In this study, we demonstrate that Nicotiana benthamiana plants are an excellent biofactory for producing AFPs through transient expression using a new vector derived from the tobacco mosaic virus. Using this plant-based production system we efficiently produced two different bioactive AFPs, the Penicillium expansum AfpA and Penicillium digitatum AfpB. We found that the subcellular compartment where AFPs are accumulated has an important impact on protein yield, probably avoiding toxicity towards plant cells. The highest yields were achieved when targeting AFPs to vacuoles, reaching up to 0.170 mg/g of fresh leaves of the highly active AfpA and eight times more of AfpB (1.2 mg/g of leaf). We also show that plant crude extracts containing AFPs are fully active against plant pathogens without requiring further protein purification, thus reducing significantly downstream processing. Therefore, the developed system is efficient for the production of AFPs, and also it is economic and safe since it is based on plants. We also developed an alternative system for the production of the linear PAF102 antifungal peptide that was recalcitrant to be produced in biological systems. This system is based on targeting the peptide to lipid droplets (LDs) through the fusion to a plant oleosin protein. Using this oleosin fusion technology, we produced PAF102 in rice seed LDs, reaching moderate yields of about 20 mg of peptide per gram of grain. Production on rice seeds is long process in order to speed the process, we successfully transferred the plant oleosin fusion technology to the Pichia pastoris system. We produced commercially relevant yields of PAF102 in these yeast LDs, reaching values of 180 mg/l of culture in only 4 days. The accumulation of PAF102 in the LDs of rice seeds and yeast facilitated its downstream extraction and recovery by simple flotation on dense solutions, with the recovered PAF102 being biologically active against pathogenic fungi. Finally, we demonstrate that in planta produced AfpA and AfpB, either purified protein or protein extracts enriched with these two proteins, are efficient in controlling important fungal diseases on economically relevant crops, including Botrytis gray mold disease in tomato leaves and fruits, blast disease in rice plants and Fusarium proliferatum infection in rice seeds. Our results provide a sustainable production system of AFPs, and evidence their efficacy on protecting plants from fungal infection, strongly supporting the use of AFPs as environmental friendly and effective “green fungicides” in crop and postharvest protection.
Universitat Autònoma de Barcelona. Programa de Doctorat en Biologia i Biotecnologia Vegetal

This study presents the theoretical and experimental results of natural ventilation rates induced by stack, wind and the combination of both stack and wind effects for a typical crop protection structure suitable for the tropics. The structure consists of simple structural frame, transparent roofing and insect screen side walls. It was found the relative importance of the stack and wind effects is dependent on the ratio between wind speed and the square root of the inside-outside temperature difference (u/AT°.5). In this study, the wind effect dominates over the stack effect when the ratio u/AT" becomes greater than 0.5. Ventilation rate induced by the stack effect was found to increase with increasing temperature difference between inside and outside of the crop protection structure according to a power law, with an index of 0.5. The wind effect ventilation rate was found to increase linearly with increasing outside wind speed measured at eaves level. In addition, the combination of the stack and wind effects could be represented as the vectorial sum of two the independent effects (40sw = (43)k2 + (1)„,nd21 0.5). However, the result of the wind effect in the combined effects was insignificant when the ratio of ventilator opening to the total wall area is higher than 20 %. Different methods have been used to determine the natural ventilation rates. The dynamic tracer gas was used as the control; direct airspeed measurement, energy balance and neutral plane methods were used to quantify ventilation induced by the stack effect. Pressure field measurements were used to quantify ventilation by wind effect. In addition, the dynamic tracer gas, energy balance, and stack and wind methods were used to quantify ventilation induced by the combined effects. However, these methods have their constraints and limitations because of statistically significant differences in the comparison between the methods. The tracer gas method was found very difficult to use in the highly porous structure. In addition, the ventilation rate measured by this method was 30-40 % less than the other methods. The energy balance method has the advantage that it estimates many important climatic and crop parameters, however, the errors were found to be the highest. The neutral plane method was suitable for measuring ventilation induced by stack effect, the simplest method, requiring only the measurement of the inside and outside temperatures. The direct airspeed measurement method was much easier to handle and the result was comparable to other methods suitable for determining the ventilation induced by the wind effect. The physical properties of the covering materials, namely light transmission, coefficient of discharge and airflow characteristics were also determined in this study. It was found that the light transmissions of transparent polythene film and insect screens were close to each other. The coefficient of discharge and light transmission were dominant parameters in the ventilation rate calculation. It was found that when air flows through a screen, the pressure drop increases linearly with the square of approach airspeed. Airflow distributions inside the crop protection structure induced by the stack and wind effects are also presented in this study. Finally, this study presents information on natural ventilation for tropical greenhouses that was not previously available.

In agricultural research, plant genetic resources (PGR) are “non-traditional infrastructural resources”, which may generate higher social value and positive externalities if they are managed in an openly accessible manner. The privatisation of crop biodiversity is based on the assumption that the internalisation of these externalities is the panacea to fostering private research investment. However, if the domestic plant breeding and biotechnology capacity is limited, the above normative approach may fall short of expectations because the social costs of establishing or strengthening exclusion rights are higher than their social benefits. The International Treaty on Plant Genetic Resources for Food and Agriculture (ITPGRFA) is the only international agreement whose normative approach reflects in part this economic reality. However, its constructively ambiguous intellectual property rights-related provisions do not effectively fence off crop biodiversity from private appropriation. Besides, the desire of most countries not to prejudice the negotiation of an international access and benefit sharing regime under the UN Convention of Biological Diversity may prevent the extension of the ITPGRFA’s “commons” management principles to a larger number of essential food crops. The scope of this study, which focuses on PGR and agricultural innovation, derives from the paramount importance that both the design and allocation of rights in these areas might have for global food security. The innovation system perspective shows that social and economic development depends on the institutional context in which technological change occurs. Finally, the study of the transition between property regimes shows that the global reform of the institutional arrangements, which govern the present and future allocation of wealth from agriculture, is insufficient to achieve international equity so as to meet the target of reducing the proportion of people who suffer from hunger in accordance with goal 1 of the Millennium Development Goals.

An Enzyme-Linked Immunosorbent Assay (ELISA) was developed which detected slug antigens in postmortem gut analysis of carabid beetles. The ELISA was used to identify beetles which fed on slugs in three fields of oilseed rape and winter wheat in the Tyne valley, Northumberland. Generalist species such as Harpalus rufipes, Pterostichus melanarius, Pterostichus nladidus, Anlara silnilata and Nebria brevicollis fed on slugs in the field. Molluscan specialists such as Carabus violaceus and Cychrus caraboides also fed on slugs in the field. Laboratory studies indicated that many large and medium sized carabids were able to predate small slugs. Some beetle species did not eat slugs but exposure to the beetles increased slug mortality. Therefore, postmortem investigations may underestimate the impact that carabids exert on slugs as they do not measure the number of slugs killed. Slug mucus affected the locomotory activity of generalist and specialist beetle species. Beetles foraged longer, covered greater distances, made more turns, walked slower and spent more time stationary on soil covered in slug mucus compared to control areas. Abax parallelepipedus, P.melanarius, Pterostichus niger and H.rufipes all reduced slug damage to a chinese cabbage crop in a miniplot experiment compared with unprotected plots. However, these differences were not significant. A.parallelepipedus was most effective at reducing slug damage to the chinese cabbage but was rare in arable land. H.rufipes was least effective at reducing slug damage but was abundant in arable land in both years of the study. A high proportion of H.rufipes beetles fed on slugs in the field. None of these four species occurred at densities in the field which reduced slug damage in the miniplot experiment.

Ce doctorat a permis la conception de nouvelles molécules destinées aux développements de futurs produits phytosanitaires. Tout d’abord, la synthèse d’alpha,alpha-difluoro-beta-hydroxy cétones a été réalisée. Motif déjà reconnu dans le domaine pharmaceutique, nous voulions étendre son champ d’application à l’agrochimie. Une série de composés possédant une activité biologique en tant qu’agonistes des récepteurs GABA a été synthétisée. Ils ont été obtenus à l’issu d’une synthèse convergente nécessitant une réaction de couplage entre un aldéhyde aromatique et un intermédiaire alpha,alpha-difluoro-beta-trifluoromethyldihydroxy cétone. L’analyse biologique de nos produits a fait ressortir un type de famille spécifique. Une approche prodrug a débuté afin d’en affiner la structure et d’en faire ressortir un hit. Dans un second temps, le développement d’une série de quinoléines substituées par des groupements fluorés en position 2 et 4 a été conduit. Ces molécules peu décrites dans la littérature fûrent synthétisées dans des conditions douces avec de bons rendements et une complète régiosélectivité, inspirée par les réactions de Combes et de Meth-Cohn utilisant un Réactif Fluoroalkyl Amine (FARs). La post-fonctionnalisation en position 3 et 8 a permis l’exemplification de ces composés. Une étude physico-chimique réalisée sur une série homogène a apporté des informations complémentaires sur leurs propriétés électroniques. Bien qu’aucune molécule n’ait montré d’activité biologique, nous avons pu lors de ce projet réaliser la synthèse de nouvelles quinoléines et évaluer des FARs dans la synthèse de molécules inconnues de la littérature jusqu’à ce jour
This PhD thesis allowed the conception of new molecules for the development of novel phytosanitary ingredients. First, the synthesis of alpha,alpha-difluoro-betahydroxy ketones was performed. Since this motif is already known in the pharmaceutical field, we decided to extend their application to the agrochemical field. A series of compounds with biological activities as GABA agonist receptors was synthesized. They were obtained by a convergent method after a coupling reaction between benzaldehydes and alpha,alpha-difluoro-beta-trifluoromethyldihydroxy ketone intermediates. Biological analysis highlighted a specific family of compounds. A prodrug approach was applied to tune the structure and allowed the discovery of a hit. Second, the development of a series of 2,4-(fluoroalkyl)-substituted quinoline derivatives was conducted. Scarcely described in literature, these molecules were obtained under smooth conditions, with good yields and a complete regioselectivity, inspired by Combes and Meth- Cohn reactions using Fluoroalkyl Amino Reagents (FARs). Post-functionalization in position 3 and 8 allowed us to increase the scope of the reaction. A physico-chemical study gave complementary informations on their electronical properties. Although none of these molecules have shown biological activity, we have during this project realized the synthesis of new quinolines and evaluated the use of FARs in the synthesis of unknown fluorinated molecules

Artificial diet bioassays were carried out to investigate the impact of GNA and Con A upon the development of L. oleracea larvae. GNA, at 2 % of total dietary protein, exerted a significantly detrimental effect upon larval development, growth and consumption, with little effect upon survival. Con A was shown to be the more toxic of the 2 lectins. When tested at concentrations of 2.0 %, 0.2 % and 0.02 %, Con A caused a significant decrease in survival and larval development, and caused greater reductions in larval growth and consumption compared to GNA. The potential for GNA and Con A to exert insecticidal effects via binding to the brush border membrane (BBM) and peritrophic membrane (PM) of L. oleracea larvae was investigated. Con A, which specifically binds a-D-mannopyranoside and a-D- glucopyranoside residues, was shown to bind in vitro to the majority of BBM and PM proteins. In contrast GNA, which exhibits strict specificity for a(l,3) and a(l,6)-linked D-mannose residues, bound to only 5 BBMV and 2 PM proteins. In agreement, higher levels of Con A, compared to GNA, were shown to accumulate in larval gut tissue after feeding the proteins in vivo. Despite this both lectins were shown to have a similar ability to disrupt the digestive capacity of the larval midgut. GNA and Con A stimulated similar short term elevations in BBM enzyme and soluble trypsin activities and a long-term reduction in a-glucosidase activity. Increases in levels of trypsin activity in faecal material collected from lectin-fed larvae suggested that the proteins may act by disrupting mechanisms of enzyme recycling. Aminopeptidase, an abundant and avidly binding BBM protein (120 kDa), was identified as a major Con A binding species in L. oleracea. A 98 kDa GNA-binding BBM protein was purified and amino acid sequence data was obtained from digest polypeptides allowing oligonucleotide primers to be designed. Subsequent attempts to amplify (by PCR and RT-PCR) fragments containing coding sequence corresponding to the 98 kDa protein were unsuccessful. This was attributed to oligonucleotide degeneracy together with the low abundance and relatively large size of the protein. The potential for GNA and Con A to exert systemic effects upon I. oleracea was demonstrated by the detection of both lectins in the haemolymph of larvae exposed to experimental diets. GNA was detected in haemolymph of larvae exposed to experimental diet for just 2 hours. In contrast, no Con A was detectable in haemolymph extracted from larvae fed for 24 hours, although it was present in the haemolymph after 5 days of exposure to the diet. That GNA and Con A may act directly upon organs other than the insect gut was indicated by the detection of lectins in vivo in malpighian tubule and fat body tissue extracts. A significant reduction in haemocyte abundance in haemolymph samples extracted from lectin-fed larvae suggested that both GNA and Con A may also act by disrupting the immune system of L. oleracea.

There is increasing pressure from government legislation and supermarket quality assurance schemes to reduce chemosynthetic pesticide-inputs in conventional production systems. This has resulted in the need to study potential management based approaches to develop alternative management (e.g. rotational design or crop nutrition) strategies and/or crop breeding/variety selection based approaches to reduce disease pressure or crop resistance to biotic stress. Two long-term, factorial field trials (the NSCS and the NUE-crops trials) were used to assess effects of fertiliser input types (composted manure vs mineral fertilisers), fertiliser input levels, pre-crop/rotational position, crop protection (with and without the use of chemosynthetic pesticides) and wheat genetics/variety choice on phenolic profiles in flag leaves, foliar disease severity and grain yield in winter wheat. Interactions between crop management practices and growing season were investigated using univariate and redundancy analysis approaches. Flag leaf concentrations of phenolic compounds differed between years and plant growth stages and were higher (approx 40%) in crops fertilised with organic compared to mineral fertilisers, but there were virtually no effects of crop protection on phenolic profiles. In contrast, both disease severity and crop yield were significantly affected by crop protection and fertilisation practices. The use of conventional, pesticide based crop protection resulted in lower disease severity and higher yields and mineral NPK fertilisers resulted in lower Septoria and higher mildew and stripe rust severity. Effects of precrop/rotational position and wheat genetics/variety choice, and interactions between precrop, fertilisation, crop protection and/or variety choice were also detected for phenolic profiles, disease severity and grain yield. RDA identified (a) positive associations between organic fertilisation and radiation, and concentrations of phenolic compounds in leaves, (b) positive associations between organic crop protection and relative humidity, and disease severity and (c) negative associations between concentrations of most phenolic compounds and disease severity. Results indicate that it is possible to increaseing concentrations of phenolic compounds in cereals via changes to fertilisation practices, and that this may (a) reduce disease severity and/or increase crop yield in cropping systems which omit chemosynthetic pesticides, but (b) may have no effect on disease and reduced crop yields in cereal cropping systems which use pesticides. However, varietal differences in leaf phenolic expression under organic fertilisation could not be linked to differences in disease severity and yields.

The RNAi response suppresses gene expression at the post-transcriptional level and the potential of this technique to give control of insect pests in crops has been recognised for a decade. This project focuses on a comparison of RNAi responses in insects of different orders by injecting and feeding dsRNA directed against thread (APIN) and V-Type- ATPase E homologues in the target species. The results showed systemic RNAi responses, and mortality, occurred in larvae of the Tribolium castaneum (T. castaneum), Coleopteran, but not a Hemipteran, the Acyrthosiphon pisum (A. pisum), where comparatively low levels of gene down-regulation were only achieved by injection of dsRNA. DsRNA injection produced both a lethal phenotype and gene down- regulation in larvae of the dipteran species Musca domestica (M. domestica), and Delia radicum (D. radicum), although the effects were found to be stage dependent. Rapid dsRNA degradation in the extracellular environment could lead to a limitation of RNAi reponses. In vitro experiments show that dsRNA was degraded rapidly by A. pisum haemolymph and gut extracts, and less rapidly by D. radicum larval extracts. However, T. castaneum larval extracts differ in both the amount and qualitative nature of their RNase activity; dsRNA was degraded at a slow rate, predominantly by exonuclease activity rather than endonuclease activity. A strategy using recombinant proteins was used to address limitations of RNAi effects after feeding dsRNA in insects. A recombinant protein containing an RNA binding domain (RBD) was selected to conjugate dsRNA forming a protein-RNA complex. The complexed protein enhanced the stability of dsRNA and protected it from degradation from insect extracts. A fusion protein containing snowdrop lectin (GNA) linked to RBD was also developed to produce a "systemic" RNAi effect, by transporting the protein-RNA complex to the insect haemolymph using the lectin as a "carrier".

Quizalofop-p-ethyl is a herbicide introduced in the mid 1980's with apparent low toxicity and is readily degradable.Quizalofop-p-ethyl is a member of the aryloxyphenoxypropionate group of herbicides and is a postemergence herbicide used for pulses (including soybean) and vegetables. The aim of the project discussed in this study is to develop an analytical method for the sensitive and reliable determination of quizalofop-p-ethyl and quizalofop acid in soybean using HPLC. Soybean is chosen as a typical agricultural crop. It has 15 to 20 percent oil content and is a crop which has been successfully used with theis herbicide.Two different methods of extraction, i.e. the solvent-solvent extraction method and solid phase extraction method, and clean up are discussed in some detail.
Doctor of Philosophy (PhD)

The major digestive enzymes of larval Diabrotica undecimpunctata howardi, the southern corn rcotworm (SCR), have been investigated. A number of proteases have been identified, the majority cysteine proteases, some aspartic acid proteases are also present. In vitro assays of cysteine proteases showed that almost all activity could be arrested by E64 or chicken egg-white cystatin. This activity was also affected by TLCK, CpTI and thaumatin. Two inhibitory activities were demonstrated in protein extracts from rice seed. The first, oryzacystatin-I, caused marked inhibition of both the insect cysteine proteases and papain. The second produced strong inhibition of insect cysteine proteases but left papain virtually unaffected. Amino acid sequence data for this novel inhibitor was obtained, and significant homology demonstrated to the rice allergenic proteins (Izumi et a].,1992; Adachi et al., 1993). Oryzacystatin-I was expressed as a fully functional fusion protein (Rozc) in Escherchia coli, isolated, characterised and employed in feeding trials with larval SCR, a significant rise in mortality was demonstrated. Other protease inhibitors were also assayed in vivo, but none exhibited the efficacy of Rozc. A single iso-form of a-araylase was identified and characterised. In vitro assays with amylase inhibitors demonstrated the effectiveness of WAAI and the weak effect of BAAI. WAAI, employed in bioassay, produced a significant decrease in survival. Five lectins were tested by bioassay. WGA and GNA caused marked alterations in larval development, GNA was most effective. Saporin caused little effect when incorporated into artificial diet. WAAI, CpTI and GNA were employed in combination bioassays. An enhanced level of effectiveness was demonstrated with the double and triple combinations assayed. While further work is necessary, especially assaying protein efficacy in planta and establishing mechanisms of action, this project has successfully demonstrated the clear potential of plant antimetabolic proteins for use in the enhancement of inherent resistance of crop plants to insects, and of employing a number of proteins in a multi-mechanistic defence.

The main focus of this research was to study in detail the selected plant growth promoting bacteria (PGPR) from Bacillus group. PGPR have been described as possible solution to the negative effects of chemicals fertilizers experienced by human beings, other living organisms and the environment through reducing or replacing the use of chemical fertilizers and pesticides. The work aimed at finding plant promotion and plant protection activity of selected PGPR species. Different bioassays were performed in the laboratory and greenhouse to find if the selected bacteria promote plant growth and protect plants through inhibiting pathogens, or protect them during drought conditions. RNA sequencing technology was carried out to study gene expression regulation of a Bacillus atrophaeus UCMB-5137. The selected bacteria showed some plant growth promotion and protection abilities. Bacillus atrophaeus UCMB-5137 and Bacillus amyloliquefaciens ssp. plantarum UCMB-5007 actively promoted wheat growth. All strains showed some pathogen inhibition, while some strains induced drought tolerance abilities. Genes responsible for plant colonization of the Bacillus atrophaeus UCMB-5137 and transcription factors regulating gene expression were identified. This work will create a basis for the possible development of industrially potential biofertilizers and biopesticides for agricultural practices.
Thesis (PhD)--University of Pretoria, 2017.
Biochemistry
PhD
Unrestricted

Crop insurance is an essential risk management tool for America's agricultural producers because a single crop failure or disastrous year can eliminate the results of multiple years of profitability. Crop insurance is designed to provide financial protection to farmers; however, insurance managers who lack managerial underwriting strategies disrupt companies to the point of financial distress. Self-insurance and self-protection theory were the conceptual frameworks for this single-case study to explore successful strategies that insurance managers used to mitigate net underwriting losses. Four insurance and senior strategic managers from an insurance company in the midwestern United States were recruited through a purposeful sampling method to participate in semistructured interviews. Data gathered from these interviews and from the company's website and its public financial reports were analyzed through a reflective interpretation process, which was guided by the Van Kaam method. Five themes that emerged from this study, including disruptive technology, traditional underwriter vs. integrated profit-and-loss expert, streamlined applications by in-house technology or strategic alliances, opportunity assessment, and underwriting discipline. By implementing executive support for strategies to mitigate net underwriting losses, managers of crop insurance companies can overcome the challenges of net underwriting losses. The findings from this study may promote positive social change by lowering insurance premiums to the farm community and enabling managers to reduce risk to companies and farmers by distributing financial risk across a pool of participants thus enriching the stakeholders' investments.

The use of crop protection products (CPPs) is essential to improve crop production levels and feed the rising global population. Safety testing of CPPs is also essential to ensure that their use does not adversely affect human health or the environment. Currently, laboratory-based studies typically over-estimate the environmental persistence of CPPs in the environment. The inclusion of environmental variables that are currently omitted from laboratory studies, such as non-UV light, could reduce the disparity between laboratory and field degradation studies. The inclusion of light resulted in a significant reduction in extractable parent compound for benzovindiflupyr, chlorotoluron, prometryn, imidacloprid, and fludioxonil compared to dark conditions. In contrast, a significantly slower rate of cinosulfuron transformation was observed under light compared to dark conditions. In a separate experiment, terminal restriction fragment length polymorphism showed that under light conditions, the soil surface (0-3 mm) harboured distinct phototroph, bacterial and fungal communities compared to the underlying bulk soil (3-12 mm), or dark incubated soil. 454 pyrosequencing revealed that light selected for diazotrophs at the soil surface, including Nostoc punctiforme, in addition to heterotrophic bacteria, particularly within the phylum Firmicutes. Metatranscriptomic analysis showed that light also selected for actively transcribing photosynthetic eukaryotes at the soil surface, such as Chlorophyceae and Saccharomycetes, in addition to heterotrophs, such as Nostocaceae. Finally, light selected for major soil functions such as photosynthesis, and reduced the alpha and beta diversity of predicted protein coding regions at the soil surface. This work has important implications for CPP regulatory studies and soil surface management practices.

Background: Workers in outdoor occupations are expected to be at high risk of increased morbidity and mortality, and diminished productivity, as a result of global warming in the 21st century. A previous modelling study of geographic variations in heat-related mortality risk in projected U.S. populations mid-century showed the states of Tennessee, Kentucky and North Carolina to be highly vulnerable. Methods: Under both the RCP4.5 and RCP8.5 emissions scenarios of IPCC AR5, we evaluate the effect of future warming on estimated Wet Bulb Globe Thermometer (WBGT) temperatures using model-simulated future climate variables that were dynamically downscaled by a regional meteorology model for years 2049-2052. Select Appalachian counties in the three states that are presently dependent upon agricultural crop production are the focus of this analysis. Results: Using predicted WBGT temperatures, together with estimated work loads for hand harvesting of crops, alterations in work-rest routines under heat stress prevention guidelines are presented. Conclusions: Assumptions needed to translate these altered work-rest routines into measures of productivity and economic loss are discussed. Issues in extending the modelling to heat-related morbidity and mortality in outdoor worker populations are considered in terms of data gaps and major sources of uncertainty.

The environmental conditions created in the Southwestern deserts of the United States are conducive to the production of green microalgae biomass, for use as a feedstock intended for conversion to carbon neutral liquid biodiesel. One promising heat-tolerant, rapidly-growing, high lipid content species is the chlorophyte, Chlorella sorokiniana (Shihira and Krauss, 1965) (isolate DOE 1412), which has been selected for pilot-scale production as part of a larger algal biofuels project to assess its potential for long-term productivity in open, outdoor monoculture production systems. Molecular analysis exposed the presence of the pathogenic bacterium, Vampirovibrio chlorellavorus (Gromov & Mamkaeva, 1972) causing infection and death of DOE 1412, which occurred most rapidly at air temperatures exceeding 34 °C. This Gram-negative bacterium has been reported to attach to and utilize the cellular contents of several Chlorella species, leading to yellowing and flocculation of algal cells, and death of the host. A quantitative PCR assay was developed to monitor pathogen accumulation using the 16S ribosomal RNA gene, in addition to the algal 18S ribosomal RNA gene for normalization. The assay is highly sensitive, with limits of quantification for the 16S and 18S gene targets calculated to be 19 and 131 copies, respectively. The qPCR assay was used to monitor several outdoor reactors inoculated with the DOE1412, throughout the warm season growth-to-harvest cycle to understand the disease cycle and inform disease management decisions. Further, the bacterium was monitored in paddlewheel DOE 1412 cultures treated with benzalkonium chloride (BAC), a biocide tested for the ability V. chlorellavorus attack of DOE 1412. The treatment resulted in a reduced growth rate for DOE 1412, but prolonged the duration of the production cycle resulting in increased total harvestable yield, compared to untreated control cultures.

Crop protection products (CPPs) are an essential component of modern agriculture, necessary to improve crop yield to feed the ever-increasing world population. Regulation and safety testing of CPPs entering the environment is mandatory to ensure that their use is not at the detriment of environmental or human health. Regulatory laboratory studies typically over-estimate the persistence of CPPs within the environment as they are not representative of environmental conditions. This study investigated the role of non-UV light on CPP degradation and the development of soil surface communities. The inclusion of non-UV light in laboratory studies impacted the degradation of fludioxonil and cinosulfuron, increasing and decreasing the rate of transformation relative to dark conditions, respectively. Further, the inclusion of light increased non-extractable residues (NER) formation in fludioxonil, paclobutrazol and benzovindiflupyr. In a field based degradation experiment, the availability of photosynthetically active radiation (PAR) increased the transformation of benzovindiflupyr relative to when PAR was restricted. Further, the formation of paclobutrazol and benzovindiflupyr NERs was increased when PAR was not restricted, and the proportion of CPP remaining at the soil surface (0-5 mm) was higher when PAR was restricted. Targeted amplicon sequencing (Illumina MiSeq) revealed that bacterial and phototrophic communities at the soil surface changed with time, and that communities formed when PAR was available were structurally distinct relative to communities when PAR was restricted. In a further experiment, analysis of bacterial and phototrophic communities under crops with differing canopy characteristics showed that distinct communities formed at the soil surface relative to bulk soil, and that phototrophic communities of bare soil and under low-density canopies were structurally distinct to those that formed under high-density canopies. This work has potential implications for regulatory CPP degradation studies, and furthers the understanding of soil surface community development in temperate environments.

Potatoes (Solanum tuberosum L.) are the most important food crop in the world after rice and wheat. Potato tubers contain many types of essential nutrients. They contain high concentrations of carbohydrates and also vitamins, minerals and protein. They also contain toxic compounds called glycoalkaloids. Potato is classed as a protective vegetable because of its high vitamin C content. The objectives of this study were to determine the effects of different fertiliser types (composted cattle manure, mineral NPK fertiliser), pre-crop (beans, barley) crop protection protocols (organic, conventional) and growing season on the nutritional quality of vitamin C, glycoalkaloids and minerals and yield of different varieties of potato (Sante, Sarpo Mira, Nicola, Fontane, Agria and Cara). Field experiments were carried out during the 2010 and 2011 growing seasons at Nafferton Farm (Newcastle University). The experiment was a split split split plot-design. The main plot was crop rotation, the sub-plot was crop protection, the sub-sub plot was fertility management and sub-sub-sub plot was variety. The vitamin C content was determined by two different methods of analyses; colorimetric titration and high- performance liquid chromatography (HPLC). Mineral content was assessed by the Dumas combustion method at Sabanci University, Turkey. Total glycoalkaloids were determined according to the AOAC method 997. 13. A significant effect of fertilisation was detected for potato yield for both of years. In plots treated with NPK fertilisers, higher yield, compared to compost was obtained for both seasons. In addition conventional crop protection increased yield in both years. Pre-crop significantly affected yield in both years, with higher yields being recorded after beans than barley in 2010 while, a pre-crop of barley resulted in the significantly higher yield than beans in 2011. However, no significant difference between varieties was detected. Also, no significant interactions were detected for tuber yield in 2010. However a significant 2-way interaction between crop protection and pre-crop was observed in 2011, with a pre-crop of beans resulting in the highest yields under conventional crop protection, while a pre-crop of barley resulted in the significantly higher yield under organic crop protection systems. Results of the second growing season (2011) were based on the NUE potato experiment and showed that there was significant difference (P < 0.05) in yield between varieties, fertilisation iii and crop protection. A significant interaction between fertilisation and variety was detected for the yield. There was a significant effect of variety on tuber vitamin C content (P < 0.001), in 2010 and 2011. The concentrations were higher in Sante than Sarpo Mira. There were no significant influences of other treatments (fertility, rotation, crop protection). However, a 2-way interaction between fertilisation and variety was detected in 2011. In 2011 there was a significant effect of varieties on vitamin C content, but no significant effect of fertility and crop protection treatments. However, a significant interactions between fertility, variety and crop protection treatments was detected but only for the titration analysis for vitamin C content. There were no significant effects (P > 0.05) of fertilisation, pre-crop and crop protection on glycoalkaloid concentrations. However, a significant 3-way interaction between fertilisation, variety and rotation was detected for alpha solanine in 2011. A significant 3-way interaction between crop protection, fertilisation and variety was detected for alpha chaconine in 2010. In 2010 the mineral content (N, P, K, Na and Ca) of potatoes was significantly affected by fertilisation treatments. N, P and Ca contents were significantly increased by NPK application while the K and Na contents were significantly increased by compost application. In 2011 Ca contents were affected by fertilisation treatments only. Effects of variety, rotation and crop protection and interactions between factors were also detected for macro mineral contents. However, in 2011 no effect of pre-crop on tuber macronutrient concentration was detected. There was a significant difference in micronutrient concentration between variety, fertilisation and crop protection. No significant effect of rotations was detected for micronutrient concentrations in both of seasons. In conclusion, in both years, 2010 and 2011, variety significantly affected vitamin C content of potato tubers, but there were no significant effects of fertility, pre-crop and crop protection treatments irrespective of the analytical method (titration and HPLC) analysis. Sante tubers contained about 20% more vitamin C than Sarpo Mira. Therefore, it appears that variety choice is a reliable means of manipulating vitamin C as a component of tuber quality compared with other agronomic treatments. In addition, mineral content was also affected by treatments (fertility, variety, rotation and crop protection) and was highly variable. iv For the two years, similar results were obtained in alpha solanine and alpha chaconine content. Significant interactions between rotation, fertilisation and variety were only detected on alpha solanine content during 2011. Also, significant interactions between variety, fertility and crop protection were observed on alpha chaconine content during 2010. The 2010 and 2011 growing seasons appeared to have a significant effect on alpha solanine, macro minerals and micro minerals but did not appear to affect yield, vitamin C and alpha chaconine contents.

The field slug Deroceras reticulatum is a major pest in UK agriculture and amidst growing concern and regulatory pressures surrounding chemical molluscicides, innovation is required to advance the current repertoire of slug controls. This study set out to investigate the bacteria associated with D. reticulatum to assess their importance to the slug and potential as a target for biological control. Slug gut bacterial isolates identified using the phenotypical API system (BioMérieux) and 16S rRNA gene sequencing, were mainly soil-dwelling organisms of the phyla Proteobacteria and Bacteriodetes some of which may be important in human or plant disease. A ribosomal intergenic spacer analysis (RISA) was developed to study microbial communities in the slug gut. Slugs had an average species richness of 12 and comparing the bacterial communities in slugs from different locations yielded a mean similarity of 0.159 (Jaccard index) which was significantly lower than similarity indices of slugs collected within a single location (Mean Jaccard index 0.205, p<0.001, ANOVA). Cloning and sequencing of RISA bands common to slugs and slug eggs, but absent from the surrounding soil and plants identified bacteria for future investigation as potential beneficial symbionts. Bacteria extracted from the slug gut were tested for sensitivity to 16 antibiotics and greatest inhibition of growth was observed for chloramphenicol, gentamicin and tetracycline. These antibiotics administered to slugs by feeding and injection caused a reduction in gut-associated bacteria in plate counts, and in bacterial 16S rDNA quantities estimated by real-time quantitative PCR. Field collected D. reticulatum has a large transient gut bacterial population which is reduced upon starvation to a low background level. No significant detrimental effect of antibiotic treatment on the fitness and survival of the slugs was seen, in some instances control slugs suffered greater mortality than slugs that had been injected with antibiotic. Slugs that died during bioassays had a significantly greater amount of bacterial 16S rDNA in their gut than slugs that were sacrificed as healthy individuals suggesting the presence of a bacterial pathogen. This study has found little evidence that a bacterial symbiont may exist and be important for optimal fitness and survival of D. reticulatum, but insight into slug associated bacteria will be valuable in the direction of future studies in this field.

Chemical inhibition of the activity of poly (ADP-ribose) polymerases (PARPs) is associated with enhanced stress tolerance and growth in response to a broad range of abiotic plant stressors. This led to the suggestion that PARP inhibitors might have application in future crop protection strategies. However, the vast majority of studies to date have involved short-term, in vitro assays which are not representative of the conditions crop plants experience in the field. This work aimed to quantify the impact of chemical PARP inhibitor application on photosynthesis, growth and yield in planta, under well-watered and droughted conditions. In Chapter 2, a protocol for the quantification of the impact of drought stress on photosynthesis was developed, mainly using chlorophyll fluorescence imaging. In Chapter 3, the impacts of PARP inhibitors on photosynthesis, growth and survival in response to drought were measured. PARP inhibitors enhanced survival to severe stress but there was a cost associated with application under well-watered and moderate drought conditions. Chlorophyll fluorescence and growth measurements indicated that PARP inhibitors also had a damaging effect. Results from Chapter 4 suggested that PARP and PSII inhibitors had broadly negative impacts on yield. There was a strong relationship between growth (maximum plant object sum area) and yield under stress, which enabled the effects that compounds had on yield to be predicted approximately 40 days earlier than measuring at harvest. By fitting a model to the growth data it was possible to predict the impacts even earlier still. In Chapter 5, application of a PARP inhibitor reduced stomatal conductance but did not alter opening/closing kinetics, indicating the compound had an anti-transpirant effect. The enhanced stress tolerance of PARP-deficient plants likely protected against severe drought. However, a trade-off arises because of the costs associated with application under more moderate conditions. If PARP inhibitors are to be used in agriculture the cost/benefit balance will have to be carefully considered.

Purple nutsedge (Cyperus rotundus) is a troublesome weed that outcompetes crops and contributes to poor yields. In the past, agriculturalists controlled purple nutsedge by fumigating soil with methyl bromide but the fumigant has since been classified as a controlled substance under the Montreal Protocol. This study evaluated the effectiveness of several alternative purple nutsedge control techniques and compared them with results obtained from the application of Roundup. Concentration treatment effects for the allelopathic seed powders of watercress and turnip were tested in a field trial while seed powders of yellow mustard and sunflower were tested in a potted trial. The allelopathic amendments significantly delayed weed emergence but several factors interfered with long-term effectiveness. Roundup was determined to be the most effective season-long weed control among the treatments consistently leaving the least amount of surviving weeds and underground organs.

L'utilisation de pesticides conventionnels, en particulier les pesticides chimiques de synthèse, a considérablement réduit les pertes de récoltes et a connu un succès commercial. Cependant, l'utilisation excessive de pesticides chimiques qui manquent de toxicité spécifique a provoqué une série de problèmes environnementaux et de santé publique. L'intérêt de la recherche vers de nouveaux biopesticides naturels avec de nouveaux modes d'actions vise à un meilleur équilibre entre l'efficacité des pesticides et la réduction des méfaits possibles pour l'environnement et les humains. Les plantes sont une source importante de biopesticides. Les acides chlorogéniques (CQA), isolés à partir de diverses plantes et présentent in vivo et in vitro un large spectre d'activités biologiques, ont attiré l'attention avec un potentiel comme biopesticides basé sur la toxicité brevetée de l'acide 3,5-di-O-caféoylquinique contre les larves de Myzus persicae. L'étude des propriété insecticides notamment la mode d'action et l'étude de certaines relations structure-activité pourrait bénéficier de la synthèse de différents dérivés et analogues de CQA. Des analogues des acides 4-désoxy-3,5-dicinnamoy quiniques et 3,4- et 4,5-dicinnamoylquiniques naturels ont été synthétisés. Sept analogues dans la série 4-désoxy ont été soumis à des essais insecticides et deux composés présentent une activité insecticide plus élevée que l'acide 3,5-dicaféoylquinique naturel. Comme perspectives à ce travail, confirmer l'activité des composés synthétisés sur d'autres espèces de pucerons d'importance agronomique pourrait être réalisé. De plus, d’autres collaborations avec des biologistes pourraient être établies afin d’évaluer d'autres activités des composés synthétisés ou les utiliser comme outils pour étudier des mécanismes de biosynthése
The use of conventional pesticides, especially the synthetic chemical pesticides, has greatly reduced the crop losses and gained a commercial success. However, the excessive use of pesticides lacking toxic specificity has caused a series of environmental and public health problems. The research interest toward new naturally-occurring biopesticides with novel modes of actions aims at a better balance between the efficiency of pesticide and reducing possible harms to environment and humans. Botanicals are an important source of biopesticides. Cinnamoyl quinic acids (CQA), isolated from various plants and shown to exhibit in vivo and in vitro a wide spectrum of biological activities, have attracted the attention with potential as biopesticides based on the patented toxicity of 3,5-di-O-caffeoylquinic acid against the larvae of Myzus persicae. The investigation of the insecticidal activity of CQA including their mode of action and the study of some structure-activity relationships could benefit from the synthesis of different CQA derivatives and analogs. A series of natural CQA derivatives natural 3,4- and 4,5-dicinnmamoylquinic acid derivatives but also analogs like 4-deoxy-3,5-dicinnamoylquinic acids were so synthesized. Seven targeted 4-deoxy CQA analogs were subjected to insecticidal assays, and two compounds were found to exhibit higher insecticidal activities than natural 3,5-dicaffeoylquinic acid. As perspectives to this work, confirming the activity of the synthesized compounds on other aphid species of agronomic importance could be performed. Furthermore, other collaborations could be established with biologists dedicated to measure other bioactivities of the synthesized compounds or use them as tools to investigate various biological pathways

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The earthworm breeding corresponds to the creation of earthworm for different purposes like producing arrays and cocoons and worms for the production of vermicompost. The vermicompost, widely used as a fertilizer in agriculture and family farming systems ecologically based, also has important role as fitoprotetor. The study aimed to produce vermicompost of Eisenia andrei Bouché from cattle manure (CM) and combinations of this with peanut husk (PH), as structural materials, and spent coffee grounds (SCG), nutritional qualities of the source, and its evaluation in liquid and aerated form in suppressiveness Sclerotium rolfsii Sacc. The work was divided into two stages. At first, vermicompost produced up to four treatments and six replications: H1 CM 100%); H2 CM 75% + PH 25%; H3 CM 75% + SCG 25%; H4 CM 50% + SCG 25% + PH 25%. We evaluated the biomass of earthworms, cocoon production, yield and vermicompost microbiological and chemical variables. Treatments H3 and H4 had higher biomass and cocoon production. When assessed the density of bacterial colonies and fungal treatments H2 and H4 registered the highest rates. In the second step, vermicompost tea aerated were prepared from different combinations of vermicompost, in order to test their antagonistic effect against S. rolfsii. The vermicompost were packed in sachets and immersed in water and aerated for a period of 24 hours. Were quantified bacterial and fungal colonies present in vermicompost tea (VT), in selective culture media. Representative samples of VT obtained from each of the four treatments were evaluated on mycelial growth of S. rolfsii from mycelium and sclerotic under two conditions: unfiltered and filtered. The VT filtered did not inhibit mycelial growth of S. rolfsii, as opposed to VT unfiltered. Colonies of microorganisms coming from the unfiltered treatments, once isolated and purified, were paired with discs of mycelium of S. rolfsii, to evaluate the effect of antagonistic fungi and bacteria present in vermicompost on the sclerotic. The F3 isolated, on VT on treatment H2, identified as belonging to the genus Trichoderma, was the one who showed the greatest inhibition to S. rolfsii, among other microorganisms matched. The production and use of vermicompost in family farming should be encouraged, because besides the already established benefits of fertilization on cropping systems can also aid in the control of plant diseases.
A minhocultura corresponde à criação de minhocas destinadas a diferentes fins, como produção de matrizes e casulos, e minhocas destinadas a produção de húmus. O húmus de minhoca, amplamente utilizado como fertilizante na agricultura familiar e sistemas agrícolas de base ecológica, também tem relevante papel como fitoprotetor. O estudo teve por objetivo produzir húmus de minhoca da espécie Eisenia andrei Bouché a partir de esterco bovino (EB) e combinações deste com cascas de amendoim (CA), como material estruturante, e borra de café (BC), na qualidade de fonte nutricional, e sua avaliação na forma líquida e aerada na supressividade de Sclerotium rolfsii Sacc. O trabalho foi dividido em duas etapas. Na primeira, produziu-se húmus em quatro tratamentos e seis repetições, sendo: H1 EB 100%; H2 EB 75% + CA 25%; H3 EB 75% + BC 25%; H4 EB 50% + BC 25% + CA 25%. Avaliou-se a biomassa de minhocas, produção de casulos, rendimento de húmus e variáveis microbiológicas e químicas. Os tratamentos H3 e H4 apresentaram maiores índices de biomassa e produção de casulos. Quando avaliadas as densidades de colônias bacterianas e fúngicas, os tratamentos H2 e H4 registraram os maiores índices. Na segunda etapa, foram preparados húmus líquidos aerados a partir das diferentes combinações de húmus, com o objetivo de testar seus efeitos sobre escleródios de S. rolfsii. Os húmus foram acondicionados em sachês e mergulhados em água, sendo aerados pelo período de 24 horas. Foram quantificadas as colônias bacterianas e fúngicas presentes nos húmus líquidos (HL), em meios de cultura seletivos. Amostras representativas de HL obtidos de cada um dos quatro tratamentos foram avaliadas no crescimento micelial de S. rolfsii, a partir de micélio e escleródio, em duas condições: filtradas e não filtradas. Os HL filtrados não inibiram o crescimento micelial de S. rolfsii, em oposição aos HL não filtrados. As colônias de microrganismos oriundas dos tratamentos não filtrados, uma vez isoladas e purificadas, foram pareadas com discos de micélio de S. rolfsii, objetivando avaliar o efeito antagônico de fungos e bactérias presentes nos húmus sobre o escleródio. O isolado F3, do HL oriundo do tratamento H2, identificado como pertencente ao gênero Trichoderma, foi quem apresentou a maior inibição ao S. rolfsii, dentre os demais microrganismos pareados. A produção e o uso de húmus de minhoca na agricultura familiar devem ser estimulados, pois além dos benefícios já consagrados acerca da fertilização em sistemas de cultivo também podem auxiliar no controle de doenças de plantas.

Many economists and policy analysts have conducted studies on crop insurance. Three research gaps are identified: i) moral hazard in prevented planting (PP), ii) choice of PP and planting a second crop, and iii) selecting margin protection in the Dairy Margin Protection Program (MPP-Dairy). The first essay analyzes the existence of moral hazard in PP. The PP provision is defined as the “failure to plant an insured crop by the final planting date due to adverse events”. If the farmer decides not to plant a crop, the farmer receives a PP indemnity. Late planting (LP) is an option for the farmer to plant a crop while maintaining crop insurance after the final planting date. Crop insurance may alter farmers’ behavior in selecting PP or LP and could increase the likelihood of PP claims even though farmers can choose LP. This study finds evidence that a farmer with higher insurance coverage tends to choose PP more often (moral hazard). Spatial panel models attest to the existence of moral hazard in PP empirically. If a farmer chooses PP, s/he receives the PP indemnity and may either leave the acreage unplanted or plant a second crop, e.g., soybean for corn. If the farmer plants a second crop after the PP claim, the farmer receives a 35% of PP payment. The current PP provision fails to provide farmers with an incentive to plant a second crop; 99.9% of PP claiming farmers do not plant a second crop. Adjusting PP indemnity payment may encourage farmers to plant a second crop. The second essay explores this question using a stochastic simulation and suggests to increase the PP payment by 10%-15%. The third essay investigates why Wisconsin dairy farmers purchase more supplementary protection than California farmers in a MPP-Dairy introduced in the 2014 Farm Bill. MPP-Dairy provides dairy producers with margin protection when the national dairy margin is below a farmer selected threshold. This study determines whether conditional probabilities regarding regional and national margins have a role in farmer’s decision-making to purchase supplementary coverages using Copula models. Results indicate that Wisconsin farmers have higher conditional probabilities and purchase more buy-up coverages.

There is a remarkable growth in the field of Information Communication Technology (ICT) in Developing Countries (DCs). Telecommunication is one of the areas where ICT is recording an ongoing rapid change. Mobile phones are becoming pervasive in daily scenario; and among the beneficiaries of this are farmers. Farmers are using mobile phones in executing their farming business and daily life. At the same time, Wireless Sensor Networks (WSNs) are also showing a result in developed part of our world. WSNs potential in sensing various environmental condition, their affordability and applicability motivated conducting of this master thesis. Therefore, the objective of conducting this master thesis is to investigate and identify how the use of mobile phones in conjunction with WSN enable farmers in Ethiopia monitor and control their farm field. We use firsthand qualitative data we gathered during our field work in Ethiopia to design our proposed prototype. Functional requirements and system design guideless are obtained from observation we make and interviews we carry out on irrigation based farmers around town of Meki in region of Oromia. We use our prototype to demonstrate and evaluate how irrigation based farmers benefit from existence of such system.

Proportional loss models commonly used in disease surveys are based on the assumption that per cent yield loss is the same in all crops, regardless of their yield potential. Estimates of regional crop loss may be inaccurate if the relationship between disease and yield loss is affected by crop yield potential. The importance of crop yield potential in disease: yield loss modelling was investigated and models for more accurate regional crop loss estimates were developed, taking crop yield potential into account. Two spring sown barley (cv. Triumph) experiments were conducted in 1987/88 and 1988/89 in Canterbury, New Zealand, to study the effect of crop yield potential on the relationship between disease and yield loss. Crop yield potentials of 323 to 806gDM/m² were generated in seven crops by varying nitrogen and water inputs, sowing date (mid-spring and early-summer) and season. Leaf rust (Puccinia hordei Otth) epidemics of different severity were generated by applying fungicides at different times, frequencies and rates to control the natural epidemics. Disease was measured as per cent disease severity (%DS), green leaf area, radiation interception and near-infrared radiation (NIR) reflectance from crop canopies. Yield was measured as total and grain dry weight. Epidemics were severe in the fully diseased plots from GS 34 and 46 to maturity in the late and early sown crops respectively. Disease reduced grain yield by 50 to 63% in 1987/88 and 24 to 38% in 1988/89 in the fully diseased plots. Disease: yield loss models were derived by regression analysis for each crop in 1987/88. Single point, multiple point and area under curve models were derived from %DS and GLAI variables, and proportional (%) and actual (gDM/m²) grain yield. The effect of yield potential was determined by comparing regression equation coefficients for each crop with crop yield potential. An area under green leaf area index curve (AUGLAIC): actual yield model was best suited to determining the effect of yield potential on yield loss. This model was selected because AUGLAIC summarised the effect of disease on plant growth over the season and actual yield represented the crop yield potential in the absence of disease and the response of actual yield to disease. Crop yield potential did not affect actual yield loss caused by leaf rust. Disease measured as AUGLAIC explained most of the variation in yield (R²adj=0.93) for all crops in both years. Assessment of GLAI is not suitable for estimation of regional crop loss because of the requirement for a rapid and low cost method. Reflectance of NIR from the crop canopy was investigated as an alternative to GLAI measurements. Reflectance was correlated significantly (P<0.001) with GLAI (r=0.66 to 0.89) and green area index (r=0.76 to 0.92). Reflectance measured at grain-filling (GS 85-87) explained most (R²adj=0.94) of the variation in yield for all crops in both years. The relationship between AUGLAIC and yield was validated with data from independent diseased and healthy barley crops. The AUGLAIC: yield model described the effects of disease on yield accurately but overestimated yield by 49 to 108% in the healthy crops. Models based on accumulated PAR (photosynthetically active radiation) intercepted by green leaves explained the observed deviations in yield of these crops from the AUGLAIC: yield model. Accumulated PAR models accounted for differences in incident radiation, canopy structure, radiation interception by green leaves, radiation use efficiency and harvest index which are important in determining dry matter production and grain yield. Accumulated PAR models described the effects of disease on crop growth which were not represented by GLAI alone. Variation in crop yield potential at the regional scale is important in disease: yield loss modelling and can be accounted for by using either separate equations for each yield potential crop or crop category, robust models, inclusion of a form function for yield potential or choice of disease and yield variables which integrate yield potential.

Dans un objectif de réduction de l'utilisation des pesticides, des méthodes alternatives doivent assurer la protection des cultures. Les dégâts causés par les insectes ravageurs sont les manifestations de dynamiques de population dont certains déterminants paysagers sont des éléments structuraux potentiellement relocalisables. Nous explorons ici, par la simulation numérique, la modification du paysage comme stratégie de protection des cultures.Les éléments paysagers peuvent impacter les populations d'insectes par leur influence sur les comportements individuels. Nous avons développé un modèle de population tenant compte de l'approvisionnement individuel. Ce processus résulte (1) de la perception des ressources distantes par l'individu, (2) de la localisation de la population sur une dimension additionnelle quantifiant les réserves énergétiques, et (3) d'une procédure d'optimisation qui définit des mouvements dirigés adaptés.Une heuristique évolutionnaire de modification de paysages a été développée. Elle produit des réarrangements sous contraintes de composition et de structure. Les modèles de paysages et de dynamique de population ont été appliqués à un problème théorique d'aménagement du paysage. La description mécaniste de l'approvisionnement optimal offre des leviers d'action efficaces, basés sur la perturbation des comportements d'approvisionnement. Nos travaux montrent la réactivité de la population aux modifications du paysage, ainsi que la capacité des algorithmes évolutionnaires à proposer des paysages résistants, sous contraintes agronomiques
Environmental and health issues call for a switch in crop protection towards less chemically driven strategies. Pest damages on crops result of population dynamics that are influenced by landscape features. Those features may be relocated or dismissed to improve landscapes resistance to pest damages. Here we explore numerically the landscape modification as a crop protection strategy.Landscape features can influence population dynamics through their impact on individuals. Therefore, we developed a multiscale population dynamics model accounting for individual foraging. The foraging process results of (1) the perception of distant features used as resources by the individuals, (2) the localisation of the population along an additional dimension quantifying individual energy supply, (3) an optimisation procedure defining adapted directed motionsWe developed an evolutionary heuristic for landscape modification. It is able to rearrange landscapes with respect to compositional and structural constraints. Population and landscape models were applied to a theoretical landscape planning problem. The mechanistic description of the optimal foraging process enables new and efficient levers for crop protection, building on the disruption of the foraging behaviours. This application enlightens the responsiveness of the simulated population to landscape modifications, as well as the ability of evolutionary algorithms to produce resilient landscapes under agronomic productive constraints

Thesis (PhD)--Stellenbosch University, 2002.
ENGLISH ABSTRACT: The traditional ways in which manufacturers of crop protection products exercised control over distribution systems have diminished in their impact. Control was based on quality products, the power of the brand, an installed customer base and a broad portfolio of mostly patented products. Manufacturers of crop protection products need to review the manner in which these products are marketed, which distribution system management models are to be used, and the importance that is placed on distribution system management in the marketing mix. The primary aim of this study was defined as: "The development of appropriate distribution system management models for application in South Africa and Australia~. The secondary aim was defined as: "Establishing the key factors which determine the relationship between manufacturers and distributorsn . The dominant economic characteristics of the global crop protection industry are deemed to consist of a global market valued at US $28,090 mio, a mature market in a decline phase, the rapid consolidation of industry players, increased generic product manufacturer activity and an overall decline in manufacturer profitability. In South Africa crop protection product manufacturers use approximately 46 independent distributors to market products on farms through 600 affiliated commissioned sales agents. In Australia the distribution of crop protection products is much more concentrated. Distribution is essentially controlled by five national distributors and their coupled salaried representatives. Manufacturers therefore rely on third parties for the marketing of their products to farmers in both countries. The applicable problem statement has been formulated to select a distribution system management model that will: (i) optimally balance direct distribution related cost and subsequent levels of control over distributors; (ii) maximise the probability that a distributor will buy and actively promote the complete product portfolio of a for high levels of interpersonal relationship maintenance; and (vi) manufacturers have to instill the philosophy that distribution system management is part of a manufacturer's strategic business and marketing focus and not simply a task to be performed by a third party.
AFRIKAANSE OPSOMMING: Die tradisionele maniare waarop die vervaardigers van oesbeskermingsprodukte beheer kan uitoefen oor verspreidingsisteme is nie meer so suksesvol nie. Beheer was gebaseer op kwaliteil produkte, die mag van die handelsmerk:n gevestigde klientebasis en 'n wye produktereeks,bestaande uit gepatenteerde produkte, Vervaardigers van oesbeskermingsprodulcte moet die wyse waarop hierdie produkte bemark word, watter verspreidingsisteem bestuursmodelle gebruik word en die belangrikheid wat geplaas word op die bestuur van die verspreidingsisteme in die totala bemarkingspoging in heroorweging neern. Die primere doelstelling van hierdie studie is: "Die ontwikkeling van toepaslike verspreidingsisteem bestuursmodelle vir toe passing in Suid-Afrika en Australia. "Die sekondere doelstelling is: "Die vas stelling van die sleutelfaktore wat die verhouding tussen velVaardigers (verskaffers) en verspreiders bepaar. Die dominante ekonomiese kenmerke van die globale oesbeskermingsindustrie is 'n markwaarde in VSA $ 28,090 miljoen, 'n stagnante mark in 'n agteruitgang lase, die drastiese konsolidasie van die industrie spelers, 'n toename in die aktiwiteite van die generiese produkvervaardigers en 'n algemene daling in die winsgewindheid van die vervaardigers. In Suid-Afrika gebruik die vervaardigers van die oesbeskermingsprodukte ongeveer 46 onafhanklike verspreiders om die produkte op plaasvlak te bemark met behulp van 600 geaffilieerde kommissie agente. Die verspreiding van oesbeskermingsprodukte is baie meer gekonsentreerd in Australie. Verspreiding word in wese beheer deur vyf nasionale verspreiders en hulle spanne van verkoopsteenwoordigers wat 'n salaris verdien. In beide lande moet velVaardigers van oesbeskermingsprodukte staatmaak op onafhanklike instansies vir die bemarking van hul produkte op plaasvlak. Die probleemsteiling is dus die uitdaging om 'n verspreidingsisteem bestuursmodel t9 ontwikkel wat: (i) . n optima Ie balans sal gee tussen direkte verspreidingskoste en gekoppelde vlakke van beheer oor verspreiders; (ii) die waarskynlikheid dat . n verspreider die betrokke produkreeks van die vervaardiger sal aankoop en aktief sal promoveer; (iii) dit kan bereik in . n mark waar verspreiders talle bronne het van soortgelyke produkte teen kompeterende pryse. Gebaseer op die sekondere navorsing wat gedoen is wit dit blyk dat daar 'n algemene konsensus is dat daar n nei9in9 weg is van transaksie spesifieke besigheidsverhoudings na besigheidsverhoudings gebaseer op vennootskap gebaseerde benaderings in die interaksie tussen vervaardigers (verskaffers) en verspreiders. Hierdie tipe besigheidsverhouding word gekenmerk deur hoe vlakke van onderlinge vertroue, wedersydse verbintenis, konflik hanteringsmeganismes en goeie wedersydse kommunikasie. Primere navorsing is in Suid-Afrika (253 respondente) en Australia (180 respondente) gedoen. Implikasies vir die konstruksie van verspreidingsisteem bestuursmodelle wat voortvloei uit hierdie navorsing sluit in die belangrikheid dat on vervaardiger on wye en gesogte produkreeks het, die vereiste dat produkte beproefde effektiwiteit het, produkte kompeterend geprys is, die vereiste vir hoe vlakke van verskaffer opvolgdiens en die vereiste vir goeie interpersoonlike verhouding tussen werknemers van beide die vervaardigers en die verspreiders. Hierdie elemente moet ondermeer gerugsteun word deur goeie kommunikasiesisteme. Die "idea Ie" verspreidingsisteem bestuursmodelle is vervolgens voorgestel vir beide Suid-Afrika en Australie, gebaseer op die bevindinge van ondermeer die sekondere- en primere navorsing. Hierdie mode lie het ten doel om toepaslike strukture en besigheidsbenaderings vir vervaardigers daar te stel om hulle behulpsaam te wees om die geidentifiseerde behoeftes van die verspreiders aan te spreek.

Los péptidos antimicrobianos (AMP) son una alternativa prometedora para el desarrollo de nuevos antifúngicos que puedan sustituir a los fungicidas usados en agricultura. Sin embargo, el alto coste de la síntesis química y la dificultad para su producción a gran escala han limitado su aplicación. Las proteínas antifúngicas (AFP) son AMP naturales, pequeñas, catiónicas, secretadas y ricas en cisteína con gran potencial para el control de hongos fitopatógenos. Las AFPs se encuentran en hongos filamentosos, son estables y pueden producirse en grandes cantidades. Sin embargo, el papel biológico en su hongo productor no se conoce en profundidad. En esta tesis, se estudió la diversidad de AFPs en genomas de hongos ascomicetos y se propuso una nueva clasificación en tres clases (A, B y C). Penicillium digitatum es el principal patógeno postcosecha de cítricos y codifica solo una AFP en su genoma de clase B (AfpB), mientras que Penicillium expansum, el principal patógeno postcosecha de manzana, codifica una AFP de cada clase (AfpA, AfpB y AfpC). En este trabajo describimos la producción biotecnológica y la caracterización de estas cuatro AFPs. Se ha caracterizado el papel biológico del gen afpB en P. digitatum mediante estudios de expresión génica y generación de mutantes nulos y de expresión constitutiva. Los resultados indicaron que afpB es prescindible para la biología y el ciclo vital del hongo, aunque la expresión del gen afpB bajo el promotor constitutivo gpdA de Aspergillus nidulans es perjudicial para su crecimiento y virulencia. Sorprendentemente, ni la cepa parental ni las cepas constitutivas produjeron cantidades detectables de AfpB a pesar de la alta expresión del gen codificante. El modelado molecular y el diseño racional permitieron predecir la estructura terciaria de AfpB y diseñar péptidos sintéticos para mapear motivos antifúngicos en su secuencia primaria. Confirmamos que los bucles catiónicos L2 y L3 mostraron actividad antifúngica moderada y que pueden actuar sinergísticamente. Con el objetivo de producir AfpB mediante biotecnología, usamos un casete de expresión de AFPs basado en las regiones promotora y terminadora del gen paf de Penicillium chrysogenum, hongo que produce naturalmente grandes cantidades de su propia proteína PAF. Este casete funcionó en P. digitatum y permitió la producción homóloga de AfpB. Los datos también mostraron que las secuencias del péptido señal (SP) y el pro-péptido de la SP-Pro-AfpB no determinan la producción de proteína. También demostramos la estabilidad térmica y la resistencia a la proteólisis de AfpB, y aportamos datos que sugieren que la estructura terciaria no es necesaria para la actividad antifúngica. Similar a lo descrito en P. digitatum, ninguna de las tres AFPs se detectó en los sobrenadantes de cultivo en medio rico de P. expansum. Sin embargo, AfpA se produjo en grandes cantidades en cultivos de medio mínimo de P. expansum. Para completar el repertorio de AFPs, produjimos las tres AFPs de P. expansum (AfpA, AfpB y AfpC) en P. chrysogenum con el casete paf. Las tres proteínas de P. expansum se produjeron, purificaron y caracterizaron con éxito. Ninguna de las AFPs producidas en este trabajo fue citotóxica frente a eritrocitos de mamíferos. AfpA de P. expansum seguida de AfpB de P. digitatum fueron las AFPs más activas contra hongos filamentosos, incluyendo patógenos de plantas y humanos, productores de micotoxinas y sus propios hongos productores, una característica previamente no descrita en las AFPs. Además, la AfpA de P. expansum y la AfpB de P. digitatum protegieron frente a la infección causada por el hongo Botrytis cinerea en plantas de tomate, y AfpA de P. expansum protegió frente a P. digitatum en frutos de naranja. Estos resultados confirman nuestra hipótesis de que las AFPs son buenas candidatas para el desarrollo de nuevos antifúngicos en protección vegetal y conservación postcosecha, pero ta
Els pèptids antimicrobians (AMP) són una alternativa prometedora per al desenvolupament de nous antifúngics que puguen substituir als fungicides utilitzats en agricultura. No obstant això, l'alt cost de la síntesi química i la dificultat per a la producció biotecnològica a gran escala han limitat la seua aplicació. Les proteïnes antifúngiques (AFP) són AMPs naturals, xicotetes, catiòniques, secretades i riques en cisteína que oferixen un gran potencial per al control de fongs fitopatogens. Les AFPs estan presents de en fongs filamentosos, són molt estables i poden produir-se en grans quantitats. No obstant això, el paper biològic d'estes AFPs en el seu fong productor encara no està clar. En esta tesi es va estudiar la diversitat d'AFPs en genomes de fongs ascomicets i es va proposar una nova classificació en tres clases (A, B i C). Penicillium digitatum, el principal patogen postcollita de cítrics, codifica només una AFP en el seu genoma de classe B (AfpB). Penicillium expansum, el principal patogen postcollita de poma, codifica una AFP de cada classe (AfpA, AfpB i AfpC). En este treball presentem la producció biotecnològica i la caracterització d'estes quatre AFPs. Hem caracteritzat el paper biològic del gen afpB en P. digitatum mitjançant estudis d'expressió gènica i la generació de mutants nuls i d'expressió constitutiva. Els resultats van indicar que afpB és prescindible per a la biologia i el cicle de vida d'este fong, encara que l'expressió del gen afpB davall el promotor constitutiu gpdA d'Aspergillus nidulans és perjudicial per al seu creixement i virulència sobre fruits cítrics. Sorprenentment, ni el cep parental ni els ceps constitutius van produir quantitats detectables d'AfpB malgrat l'alta expressió del gen afpB. El modelatge molecular i el disseny racional van permetre predir l'estructura terciària d'AfpB i dissenyar pèptids sintètics per a identificar motius antifúngics dins de la seqüència primària. Confirmarem que les estructures catiòniques L2 i L3 mostren activitat antifúngica i que poden actuar de forma sinèrgica. Amb l'objectiu de la producció biotecnològica d'AfpB, utilitzarem un casset d'expressió d'AFPs basat en les regions promotora i terminadora del gen paf de Penicillium chrysogenum, el qual produïx naturalment grans quantitats de la seua pròpia proteïna PAF. Este casset va funcionar en P. digitatum i va permetre la producció homòloga d'AfpB. Les dades també van mostrar que les seqüències del pèptid señal (SP) i el propèptid de la SP-Pro-AfpB no determinaren la producció de proteïna. També demostrarem l'extrema estabilitat tèrmica i la resistència proteolítica d'AfpB, i proporcionem dades que suggerixen que l'estructura terciària no és necessària per a l'activitat antifúngica. Semblant a P. digitatum, cap de les tres AFPs es van detectar en els sobrenadants de medi de cultiu ric de P. expansum. Al contrari, AfpA es va produir en grans quantitats en cultius de P. expansum en medi mínim. Per a completar el repertori d'AFPs, vam produir les tres AFPs de P. expansum (AfpA, AfpB i AfpC) en P. chrysogenum mitjançant l'ús del casset paf. Així, les tres proteïnes de P. expansum es van produir, purificar i caracteritzar amb èxit. Cap de les AFPs produïdes en este treball va ser citotóxica front eritròcits de mamífer. AfpA de P. expansum seguida d'AfpB de P. digitatum van ser les AFPs més actives contra fongs filamentosos, incloent patògens de plantes i humans, productors de micotoxines i els seus propis productors, una característica prèviament no descrita per a les AFPs. A més, AfpA de P. expansum i AfpB de P. digitatum van protegir front la infecció causada pel fong Botrytis cinerea en plantes de tomaca, i l'AfpA de P. expansum va protegir front P. digitatum en fruits de taronja. Estos resultats confirmen la nostra hipòtesi anterior de que les AFPs són bones candidates per al desenvolupament d'antifúngics en protecció
Antimicrobial peptides (AMPs) are promising antifungal alternatives to the fungicides used in agriculture. However, the high cost of chemical synthesis and the difficulties of large-scale production have limited their application. Antifungal proteins (AFPs) are a group of natural, small, cationic, secreted, cysteine-rich AMPs that offer a great potential to develop new biomolecules for the control of phytopathogenic fungi. AFPs are naturally present in filamentous fungi, are very stable, and can be produced in large amounts. However, the biological role of these AFPs in their producer fungus is still unclear. In this thesis, we first studied the diversity of AFPs in ascomycetous genomes and proposed a new classification in three different classes (A, B and C). Penicillium digitatum is the main citrus postharvest pathogen and encodes only one AFP from class B in its genome (AfpB), while Penicillium expansum is the main pome postharvest pathogen and encodes one AFP from each class (AfpA, AfpB and AfpC). In this work, we report the identification, efficient biotechnological production and characterization of these four AFPs. We characterized the biological role of the afpB gene in P. digitatum by the study of its gene expression pattern and the generation of null and constitutive expression mutants. Results indicated that afpB is dispensable for the biology and life cycle of this fungus, although expression of the afpB gene under the constitutive Aspergillus nidulans gpdA promoter is detrimental to growth and virulence to citrus. Surprisingly, neither the wild type nor the constitutive strains produced detectable amounts of AfpB in spite of the high afpB gene expression. Molecular modeling and rational design allowed us to predict the AfpB tertiary structure and design synthetic peptides to map antifungal motifs within the AfpB primary sequence. We confirmed that the cationic exposed loops L2 and L3 showed moderate antifungal activity and that they can act synergistically. With the objective of the biotechnological production of AfpB, we used an AFP expression cassette based on the promoter and terminator regions of the well-studied paf gene from Penicillium chrysogenum, which naturally produces high amounts of its own protein PAF. This paf cassette worked efficiently in P. digitatum and allowed the homologous production of AfpB. Data also showed that the signal peptide (SP) and pro-peptide sequences of the translated SP-Pro-AfpB do not determine protein production. We also demonstrated the thermal stability and resistance to proteolytic cleavage of the P. digitatum AfpB, and provided data that suggest that tertiary structure is not required for antifungal activity. Similar to P. digitatum, none of the three AFPs were detected in supernatants of cultures of P. expansum in rich medium. By contrast, AfpA was produced with very high yields in P. expansum cultures in minimal medium. To complete the repertoire of AFPs from P. expansum we produced the three AFPs from P. expansum (AfpA, AfpB and AfpC) in P. chrysogenum with the use of the paf cassette. With this combined approach, the three P. expansum proteins were successfully produced, purified and characterized. None of the four AFPs produced in this work were cytotoxic against mammal erythrocytes. The P. expansum AfpA followed by the P. digitatum AfpB were the most active AFPs against filamentous fungi, including plant and human pathogens, mycotoxin-producer fungi, and their own producers, a feature that had not been previously described for AFPs. Moreover, AfpA from P. expansum and AfpB from P. digitatum protected against fungal infection caused by Botrytis cinerea in tomato plants, and additionally the P. expansum AfpA protected against P. digitatum in orange fruits. These results confirm our previous hypothesis that AFPs are good candidates for the development of antifungals in plant protection and postharvest conservation, but also in clinic or food preservation.
Garrigues Cubells, SM. (2018). Producción y utilización Biotecnológica de nuevas proteínas antifúngicas de hongos filamentosos [Tesis doctoral no publicada]. Universitat Politècnica de València. https://doi.org/10.4995/Thesis/10251/113162
TESIS

Crop rotation may be used to prevent the continuous selection of particular weed species adapted to one crop type. This might be useful for weed management, economy in herbicide applications and promoting biodiversity. Common simple crop sequences might be diversified by introducing perennial forage crops. Impacts of such perennial crops on weeds were studied with four approaches : 1) Large-scale weed surveys in 632 fields in western France showed that weed species composition differed most strongly between perennial alfalfa crops and annual crops. Comparisons of fields before, during and after perennial alfalfa suggested that community composition varies in a cyclic way during such crop rotations. Several weed species problematic in annual crops were suppressed during and after perennial crops, but the appearance of other species led to equal or even higher plant diversities. 2) A 3-year field experiment with contrasting crop management options allowed an investigation of the underlying mechanisms for this: The absence of soil tillage reduced weed emergence but increased the survival of established plants. The permanent vegetation cover and frequent hay cuttings reduced weed growth, plant survival and seed production. 3) Greenhouse experiments testing the regrowth ability of individual plants after cutting showed strong differences between species and functional groups. An two-factorial experiment suggested that the negative impacts of cutting and competition on weed growth were mainly additive. 4) Special measurements of weed seed predation in the field experiment showed positive correlations with vegetation cover, indicating that this ecosystem service may be particularly fostered by perennial crops. Consistent preferences of seed predators for certain weed species indicates that seed predation may be another cause of the observed weed community shifts.

En l’actual situació de creixement continuat de la població mundial, la producció suficient d’aliments no pot ser garantida sense l’ús de productes fitosanitaris (PF). No obstant, el seu ús no resta exempt de notables dificultats com l’elevat cost dels programes de lluita química pel control de les plagues i malalties que, en el cas de la fructicultura i la viticultura, representen fins al 30% dels costos totals de producció. A més a més, l’ús de PF comporta riscs importants per la salut humana i el medi ambient. A tot això, s’hi ha de sumar la creixent desconfiança, quan no el rebuig, d’amplis sectors de la societat. Per tal de reduir el seu impacte, la Comissió Europea mitjançant el pacte verd i, en concret, a través de l’estratègia de la granja a la taula, pretén reduir el consum i el risc associat als PF en un 50% en l’any 2030. En aquest context, són necessàries eines cientificotècniques validades per reduir de les dosis a les mínimes imprescindibles sense comprometre la collita. En aquesta Tesi es presenta i valida el sistema d'ajuda a la presa de decisió DOSA3D, el qual permet ajustar el volum de caldo i la dosi de PF en cultius arboris (cultius 3D) a partir de les característiques del cultiu (àrea foliar objectiu i geometria ), el polvoritzador i la plaga o malaltia a controlar. La validació del sistema DOSA3D s’ha dut a terme en tractaments uniformes i en tractaments zonals en base a mapes de vigor atenent a dos criteris, les deposicions sobre l’objectiu tractat i l’eficàcia en el control de plagues i malalties. En base a això, les hipòtesis contrastades han estat: i) el “Leaf Area Index” (LAI) o índex d’àrea foliar (paràmetre més rellevant per l’ajust de la dosis) és estimat correctament a partir de la geometria de la vegetació i de l’estat fenològic de la vinya; ii) l’eficiència del tractament calculada mitjançant el sistema DOSA3D s’ajusta a la realitat operativa dels tractaments; iii) la deposició a les fulles és un paràmetre que es relaciona bé amb l’eficàcia biològica del tractament; iv) el volum de caldo establert pel sistema DOSA3D permet obtenir deposicions similars a l’objectiu llindar (1.2 µl cm-2) i, conseqüentment, obtenir l’eficàcia adequada; i, v) el tractament zonal de la vinya a partir de mapes de vigor, ajustant la dosi mitjançant el sistema DOSA3D, permet assolir deposicions optimes i l’eficàcia biològica esperada en els diferents vigors. El cos de la Tesi Doctoral es divideix en tres blocs. El primer està dedicat a la revisió de l’estat de l’art en matèria d’expressió i l’ajustament de la dosi i a la comparació entre els diferents sistemes existents per a la presa de decisió en l’ajustament de la dosi. El segon bloc de la Tesi inclou dos capítols dedicats als fonaments del sistema DOSA3D i a la seva validació en el cultiu de la de vinya. això Amb aquesta finalitat, s’han practicat defoliacions de trams de vinyes en espatllera, donant-se per vàlid el model d’estimació del LAI del sistema DOSA3D vinyes de fins a 1.25 m d’amplada. També, mitjançant la metodologia descrita a la norma ISO 22522:2007, s’ha determinat la deposició foliar en tractaments ajustats, posant-se de manifest la marcada tendència a la uniformització de les deposicions en la mesura que incrementa el LAI. També, el sistema DOSA3D ha estat validat al llarg de tota la campanya per l’eficàcia en el control de l’oïdi (Erysiphe necator Schw.) en quatre assajos independents en els que les dosis de PF s’han reduït fins al 60% respecte a l’estratègia realitzada per l’agricultor. En el tercer bloc s’han validat els tractaments zonals a dosis ajustades mitjançant el sistema DOSA3D en vinyes espacialment variables. Mitjançant l’índex de vigor “Plant Cell Density” (PCD), establert a partir d’imatges multiespectrals adquirides des d’un avió, s’han confeccionat mapes de dos vigors i els consegüents mapes de prescripció. En tots els casos, la deposició foliar no ha presentat diferencies significatives entre classes de vigor i l’eficàcia dels tractaments en el control del àcar groc (Eotetranychus carpini Oud.) i el mosquit verd (Empoasca vitis G. y Jacobiasca lybica Berg. & Zanon) ha permès situar la densitat de la plaga per sota dels llindars econòmics de danys en ambdues classes de vigor, comportant estalvis de PF entre el 16,6 i el 24,8% en les zones de baix vigor. Les conclusions obtingudes en la present Tesi Doctoral proporcionen les evidències cientificotècniques necessàries per utilitzar el sistema DOSA3D de manera sanitàriament segura (garantint l’eficàcia biològica). El sistema es mostra com una eina a ser considerada pels seus efectes beneficiosos per part dels agents interessats (agricultors, assessors en sanitat vegetal, indústria química, constructors d’equips de tractaments i autoritats reguladores) així com per la seva contribució a la consecució dels objectius de la Comissió Europea en matèria de reducció de l'ús dels PF.
En la situación de crecimiento continuado de la población mundial, la producción suficiente de alimentos no puede ser garantizada sin los productos fitosanitarios (PF). No obstante, su empleo no está exento de graves dificultades como el elevado coste de los programas de lucha química contra las plagas y enfermedades que, para el caso de la producción frutal y el viñedo, pueden representar hasta el 30% del total de los costes de producción. Además, el uso de PF conlleva riesgos importantes para la salud humana y el medio ambiente. A todo ello hay que añadir la desconfianza creciente, cuando no el rechazo, de amplias capas de la sociedad en relación a los PF. Para reducir su impacto, la Comisión Europea mediante el pacto verde y, en concreto, a través de la estrategia de la granja a la mesa, pretende reducir el consumo y el riesgo asociado a los PF en un 50% para el año 2030. En este contexto, se hacen necesarias herramientas científico-técnicas validadas para el ajuste de las dosis a las mínimas imprescindibles sin comprometer la cosecha. En esta Tesis se presenta y valida el sistema de ayuda a la toma de decisión DOSA3D, el cual permite ajustar el volumen de caldo y la dosis de PF en cultivos arbóreos (o cultivos 3D) a partir de las características del cultivo (área foliar objetivo y geometría), el pulverizador y la plaga o enfermedad a controlar. La validación del sistema DOSA3D se ha llevado a cabo en tratamientos uniformes y en tratamientos zonales en base a mapas de vigor atendiendo a dos criterios, las deposiciones sobre el objetivo tratado y la eficacia en el control de plagas y enfermedades. A tal fin, las hipótesis contrastadas han sido: i) el Leaf Area Index (LAI) o índice de área foliar (parámetro más relevante para el ajuste de la dosis) es estimado correctamente a partir de la geometría de la vegetación y del estado fenológico del viñedo; ii) la eficiencia del pulverizador calculada mediante el sistema DOSA3D se ajusta a la realidad operativa de los tratamientos; iii) la deposición en hojas es un parámetro que se relaciona bien con la eficacia biológica del tratamiento; iv) el volumen de caldo establecido por el sistema DOSA3D permite obtener deposiciones similares al objetivo umbral (1.2 µl cm-2) y, consecuentemente, conseguir la eficacia adecuada; y, v) el tratamiento zonal en viñedo a partir de mapas de vigor, ajustando la dosis mediante el sistema DOSA3D, permite alcanzar deposiciones óptimas y la eficacia biológica esperada en los distintos vigores. El cuerpo de la Tesis se divide en tres bloques. El primero está dedicado a la revisión del estado del arte en materia de la expresión y ajuste de la dosis y a la comparación entre los diferentes sistemas al uso para el ajuste de la dosis. El segundo bloque de la tesis incluye dos capítulos dedicados a los fundamentos del sistema DOSA3D y a su validación en viñedo. Para ello se han practicado defoliaciones de tramos de vides en espaldera, dándose por válido el modelo de estimación del LAI del sistema DOSA3D para espalderas de hasta 1.25 m de anchura. Asimismo, mediante la norma ISO 22522:2007, se ha determinado la deposición foliar, lo que ha puesto de manifiesto la tendencia marcada a la homogenización de las deposiciones en la medida que incrementa el LAI. También, el sistema DOSA3D ha sido validado a lo largo de toda la campaña por eficacia en el control de oídio (Erysiphe necator Schw.) en cuatro ensayos independientes en los que se ha reducido la dosis entre 0% y 60% respecto a la aplicada por el agricultor. En el tercer bloque se han validado los tratamientos zonales a dosis ajustadas mediante el sistema DOSA3D en viñedos espacialmente variables. Mediante el índice de vigor Plant Cell Density (PCD), establecido a partir de imágenes multiespectales adquiridas desde avión, se han confeccionado mapas de clasificación zonal en dos vigores y el consiguiente mapa de prescripción. La deposición foliar no ha presentado diferencias significativas entre clases de vigor y la eficacia de los tratamientos en el control de ácaro amarillo (Eotetranychus carpini Oud.) y mosquito verde (Empoasca vitis G. y Jacobiasca lybica Berg. & Zanon) han permitido situar la densidad de las plagas por debajo de los umbrales económicos de daño en ambas clases de vigor, comportando ahorros de PF entre el 16.6 y el 24.8% en las zonas de bajo vigor.
In the current situation of a growing world population, it is not possible to ensure the production of sufficient food without phytosanitary products (PPs). However, their use has considerable drawbacks such as high costs of chemical control programs against pests and diseases. For instance, in the integrated pest control strategies implemented in orchards and vineyards they may represent up to 30% of the total production costs. Furthermore, the use of PPs can pose important risks to human health and to the environment. In addition, broad sectors of society increasingly distrust and reject the use of PPs. In order to reduce the negative impacts of PPs, the European Union developed the green deal and, through the implementation of the “from farm to fork” strategy, aims to reach a 50% reduction of the consumption and risks associated to PPs for the year 2030. In this context, reliable scientific and technical tools are needed to allow adjustment of application doses to the minimum doses required for effective control, that is without reducing the harvest. Throughout this thesis, the DOSA3D decision support system is presented and validated, this system allows to adjust the volume and dose rates of the PPs for fruit crops (3D crops) according to the characteristics of the crop (leaf area and geometry), the sprayer and the target pest or disease. The validation of the DOSA3D system has been carried out for both uniform applications and zonal applications based on vigour maps, according to the following two criteria: on-target product deposition and efficacy of pest and disease control. In order to do so, the following hypothesis were tested: i) the LAI or leaf area index (the most relevant parameter for adjusting the dose) is estimated correctly from the geometry of the vegetation and the phenological stages of the vineyard; ii) the efficiency of the sprayer, calculated by means of the DOSA3D system is adjusted to the operational reality of the applications; iii) deposition on leaves is a parameter that correlates well to the biological efficacy of the application; iv) the volume rate established by the DOSA3D system allows to obtain leaf depositions close to the objective threshold (1.2 µl cm-2) and, consequently, achieve the appropriate efficacy; and v) the zonal application in vineyards based on vigour maps, after adjusting the dose by means of the DOSA3D system, allows to achieve optimal depositions and therefore the expected biological efficacy for the different vigour zones. The body of this thesis is divided into three parts. The first part consists of a revision of the state of the art regarding dose expression and adjustment and different existing dose adjustment decision support systems. The second part of the thesis includes two chapters dedicated to the technical rationale of the DOSA3D system and its validation in vineyards. Sections along the vine trellis were defoliated in order to validate the LAI estimation model of the DOSA3D system for trellises up to 1.25 m wide. Likewise, the ISO standard 22522:2007 was used to determine the foliar deposition, this evidenced a marked tendency to the homogenisation of the depositions as LAI increases. In addition, the DOSA3D system has been validated throughout the entire season for the control of powdery mildew (Erysiphe necator Schw.), in four independent trials in which the dose was reduced up to 60%, compared to the doses used by the farmer. In the third part of the thesis, the DOSA3D system is validated for zonal applications of adjusted doses in spatially variable vineyards. Zonal classification maps, and the corresponding prescription maps, were built based on the Plant Cell Density (PCD) vigour index, established from aerial multispectral images. Leaf deposition did not present significant differences between vigour classes and the reduced-dose applications for control of yellow spider-mite (Eotetranychus carpini Oud.) and leafhoppers (Empoasca vitis G. and Jacobiasca lybica Berg. & Zanon) allowed to reduce pest density bellow the economic damage thresholds in both vigour classes, resulting in PPs reductions between 16.6 and 24.8% in low vigour zones. The conclusions reached in this thesis provide the scientific and technical evidences required for the safe use of the DOSA3D system for delivery of reliable control of pests and diseases. The system has proved to be a useful tool due to its benefits for stakeholders (farmers, advisors in plant protection, the chemical industry, constructors of application equipment and regulatory authorities) and its contribution to the achievement of European Commission objectives regarding reduction in the use of PPs.