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Thesis (MSc)--Stellenbosch University, 2012. / ENGLISH ABSTRACT: The latest strategies in the protection of crops against microbial pathogens are rooted in harnessing the natural, highly complex defense mechanisms of plants through genetic engineering to ultimately reduce the application of chemical pesticides. This approach relies on an in-depth understanding of plant-pathogen interactions to develop reasonable strategies for plant improvement. Among the highly specialized defense mechanisms in the plant’s arsenal against pathogen attack, is the de novo production of proteinaceous antimicrobial peptides (AMPs) as part of the plant’s innate immunity. These AMPs are small, cysteine-rich peptides such as plant defensins that are known for their broad-spectrum of antifungal activity. These plant defensin peptides have been found to be present in most, if not all plant species and the defensin encoding genes are over-represented in plant genomes. Most of these defensins are generally the products of single genes, allowing the plant to deliver these molecules relatively rapidly and with minimal energetic expense to the plant. These factors contribute to establishing AMPs as excellent candidates for genetic engineering strategies in the pursuit of alternative crop protection mechanisms. The first antimicrobial peptide identified and isolated from grapevine, Vv-AMP1, was found to be developmentally regulated and exclusively expressed in berries from the onset of ripening. Recombinantly produced Vv-AMP1 showed strong antifungal activity against a wide range of plant pathogenic fungi at remarkably low peptide concentrations in vitro, however, no in planta defense phenotype could thus far be linked to this peptide. In this study, the antifungal activity of Vv-AMP1 constitutively overexpressed in its native host (Vitis vinifera) was evaluated against grapevine-specific necrotrophic and biotrophic fungi. Firstly, a hardened-off genetically characterised transgenic V. vinifera (cv. Sultana) population overexpressing Vv-AMP1 was generated and morphologically characterized. In order to evaluate the in planta functionality of Vv-AMP1 overexpressed in grapevine, this confirmed transgenic population was subjected to antifungal assays with the necrotrophic fungus, B. cinerea and the biotrophic powdery mildew fungus, Erysiphe necator. For the purpose of infection assays with a biotrophic fungus, a method for the cultivation and infection with E. necator was optimized to generate a reproducible pathosystem for this fungus on grapevine. Detached leaf assays according to the optimized method with E. necator revealed programmed cell death (PCD) associated resistance linked to overexpression of Vv-AMP1 that can be compared to that of the highly resistant grapevine species, Muscadinia rotundifolia. Contrastingly, whole-plant infection assays with B. cinerea revealed that Vv-AMP1 overexpression does not confer V. vinifera with elevated resistance against this necrotrophic fungus. An in silico analysis of the transcription of defensin-like (DEFL) genes previously identified in grapevine was included in this study. This analysis revealed putative co-expression of these DEFL genes and other genes in the grapevine genome driven by either tissue- or cultivar specific regulation or the plant’s response to biotic and abiotic stress stimuli. In conclusion, this study contributed to our knowledge regarding Vv-AMP1 and revealed an in planta defense phenotype for this defensin in grapevine. In silico analysis of the DEFL genes in grapevine further revealed conditions driving expression of these genes allowing for inferences to be made regarding the possible biological functions of DEFL peptides in grapevine. / AFRIKAANSE OPSOMMING: Die nuutste strategieë wat deel vorm van die beskerming van plant gewasse teen mikrobiese patogene het hul oorsprong in die inspanning van die natuurlike, hoogs gekompliseerde verdedigingsmeganismes van die plant deur middel van genetiese enginieurswese ten einde die gebruik van chemiese plaagdoders te verlaag. Hierdie benadering maak staat op ‘n in-diepte begrip van plant-patogeen interaksies om verstandige strategieë vir plantverbetering te kan ontwikkel. Van hierdie hoogs-gespesialiseerde verdedigingsmeganismses in die plant se arsenaal teen patogeen aanvalle sluit die de novo produksie van proteinagtige antimikrobiese peptiede (AMPs) in as deel van die plant se ingebore immuunstelsel. Hierdie AMPs is klein, sisteïen-ryke peptiede soos die plant “defensins” en is bekend vir hul breë-spektrum antifungiese aktiwiteit. Hierdie plant defensinpeptiede word aangetref in meeste, indien nie alle plant spesies nie en die defensin koderende gene word oor-verteenwoordig in plant genome. Meeste van hierdie defensins is gewoonlik die produkte van enkele gene wat die plant in staat stel om hierdie molekules relatief spoedig en met minimale energie verbruik in die plant te vorm. Hierdie faktore dra by tot die vestiging van AMPs as uitstekende kandidate vir genetiese ingenieursstrategieë as deel van die strewe na alternatiewe gewasbeskermingsmeganismes. Die eerste antimikrobiese peptied wat geïdentifiseer en geïsoleer is uit wingerd, Vv-AMP1, word beheer deur die ontwikkelingsstadium en word eksklusief uitgedruk in korrels vanaf die aanvang van rypwording. Rekombinant-geproduseerde Vv-AMP1 het sterk antifungiese aktiwiteit getoon teen ‘n wye reeks plantpatogeniese swamme teen merkwaardige lae peptied konsentrasies in vitro, alhoewel geen in planta verdedigingsfenotipe tot dusver gekoppel kon word aan hierdie peptied nie. In hierdie studie was die antifungiese aktiwiteit van Vv-AMP1 wat ooruitgedruk is in sy natuurlike gasheerplant (Vitis vinifera) ge-evalueer teen wingerd-spesifieke nekrotrofiese- en biotrofiese swamme. Eerstens is ‘n afgeharde geneties-gekarakteriseerde transgeniese V. vinifera (cv. Sultana) populasie wat Vv-AMP1 ooruitdruk gegenereer en morfologies gekarakteriseer. Om die in planta funksionaliteit van Vv-AMP1 ooruitgedruk in wingerd te evalueer is hierdie bevestigde transgeniese populasie blootgestel aan antifungiese toetse met die nekrotrofiese swam, B. cinerea en die biotrofiese swam, Erysiphe necator. Vir die doel om infeksiestudies uit te voer met ‘n biotrofiese swam is ‘n metode geoptimiseer vir die kweek en infeksies met E. necator wat gelei het tot ‘n herhaalbare patosisteem vir hierdie swam op wingerd. Blaarstudies, volgens die pas-verbeterde metode vir E. necator infeksies het ‘n geprogrammeerde seldood-geassosieërde weerstand, gekoppel aan die ooruitdrukking van Vv-AMP1 onthul, wat vergelyk kan word met dié van die hoogs-weerstandige wingerdspesie, Muscadinia rotundifolia. Hierteenoor het heel-plant infeksie studies met B. cinerea onthul dat Vv-AMP1 ooruitdrukking geen verhoogde weerstand teen dié nekrotrofiese swam aan V. vinifera bied nie. ‘n In silico analise van die transkripsie van defensin-agtige (DEFL) gene wat vroeër in wingerd geïdentifiseer is, is by hierdie studie ingesluit. Hierdie analise het vermeende gesamentlike uitdrukking van hierdie DEFL gene en ander gene in die wingerd genoom onthul wat aangedryf word deur weefsel- of kultivar-spesifieke regulering of die plant se reaksie tot biotiese en abiotiese stress stimuli. Ten slotte, hierdie resultate het bygedra tot ons kennis in verband met Vv-AMP1 en het ‘n in planta verdedigingsfenotipe vir hierdie defensin in wingerd onthul. In silico analiese van die DEFL gene in wingerd het verder toestande onthul wat die uitdrukking van hierdie gene aandryf wat ons toelaat om aannames te maak ten opsigte van die moontlike biologiese funksies van DEFL peptiede in wingerd en ondersteun die opstel en toets van hipoteses vir die rol en megansimes van aksie van die wingerd defensin familie.

The optimisation of spore production by the potential fungal biological control agent for aphids, Erynia neoaphidis Remaudiere and Hennebert (Zygomycetes: Entomophthoraceae) was studied. The fungus was able to grow in semi-defined Frynia medium (SDEM) containing glucose, yeast extract, mycological peptone, and 0.02% oleic acid buffered to a pH 6. Oleic acid was fungicidal at 0.1 % (v/v) while 0.02% (v/v) oleic acid was the optimum for radial grovvth. Plugs cut 5-10 mm from the margin ofa colony produced more conidia than plugs cut 13-20 mm from the colony margin. Renewed grovvth continued through two subcultures on solid SDEM lacking yeast extract (SDEML YE), and SDEM lacking mycological peptone (SDEMLMP). The continued growth was attributed to the carry over of nutrient in the inoculum. Growth was supported on SDEMNH4S04 when ammonium sulphate was used as the nitrogen source instead of mycological peptone suggesting that the fungus could obtain the growth factors it required from yeast extract. When chitin was added to SDEM in insoluble powder form instead ofglucose (SDEMC 1 & SDEMC2), the absence of a clearing zone around the developing colony suggested that chitin was not metabolised by E. neoaphidis. Biomass grown on SEMA and on SDEMDG (containing double the original concentration ofglucose 3 2grl), resulted in production of fewer conidia oflarger volume compared to SDEMDMP containing double and half the original concentration of mycological peptone (SDEMHP), SDEM containing halfthe original concentration ofglucose (SDEMHG). Increasing the glucose to double the original concentration resulted to an increase in biomass. Erynia neoaphidis grown on aphid cadavers produced many, smaller conidia. Mycelial mats harvested from biomass grown in fed-batch liquid fermenter culture in SDEMDG at the end ofthe exponential phase and placed on water agar discharged conidia at a rate of 6,700 conidia mm -2 h-1which persisted for approximately 3 days. When E. neoaphidis was subcultured onto SDEM from SEMA medium, the colony growth rate increased on the second subculture on SDEM where more lipases and aminopeptidases were detected at higher concentrations using the API ZYM system. This shows that attenuation might have taken place by either a phenotypic or genotypic (eg mutation) change or both when E. neoaphidis was grown on SDEM from SEMA medium. Growth in GASP medium resulted in the production of more biomass and a delay in the onset of decline phase compared to cultures grown in SDEM. Fewer enzymes were detected at a lower concentration in cultures grown in GASP compared to cultures grown in SDEM, this difference might be more likely to relate to the balance of nutrients and the fact that GASP medium is more similar in composition to the nutrients found in the haemocoel of an aphid. Based on this research. It is recommend that E. neoaphidis be grown in SDEM liquid cultures containing 32 grl glucose instead of 16 grl glucose. Biomass for field applications should be harvested at the end ofthe exponential growth and mycelial mats made. The mycelial mats should be maintained at high relative humidity and can be expected to discharge conidia for 3 days.

Predation by thrips was referred to as early as 1882 when Pergande discussed their habits. Later, Plank (1936) found Leptothrips mali (Fitch) to be predaceous on lepidopterous eggs, and Sharp (1938) cited it as a being a predator of the pecan nut caseborer, Acrobasis caryae Grote. Bailey (1939) stated that the six-spotted thrip, Scolothrips sexmaculatus (Pergande), is predaceous on Tetranychus telarius (L.), Tetranychus pacificus McG., Bryobia praetiosa Koch, Paratetranychus citri McG., Paratetranychus pilosus (C & F.), Paratetranychus indicus Hirst, Paratetranychus yothersi (McG.), Paratetranychus ilicis (McG.), and Phyllocoptes oleivorus Aschm. and in 1943 Bailey and Keifer noted that L. mali fed on tomato russet mite, Phyllocoptes desctructor (Keifer). Muma (1955) discussed the predation of S. sexmaculatus, L. mali, and Aleurodothrips fasciapennis (Franklin) on citrus insects and mites in Florida.

Thesis (PhD(Agric))--Stellenbosch University, 2012. / ENGLISH ABSTRACT: Grapevines are one of the most important and diverse crops in the world, but tend to be susceptible for numerous pests and diseases. The dagger nematode, Xiphinema index (X. index) is a well-known soil-borne pest of grapevine and vector of grapevine fanleaf virus. Several Vitis species showed resistance to this pest. Breeding efforts have been underway for several decades to create resistant rootstocks. However, conventional breeding efforts are time consuming due to grapevines being a perennial crop, its heterozygosity, as well as its long growth cycle. Breeding new grapevine varieties are also expensive and work intensive. The development of marker-assisted selection introduced a way to overcome some of the abovementioned problems. The aim of this study was to broaden the genetic evaluation and breeding efforts for improved X. index resistance in grapevine rootstocks. In 2007 several crosses were made in the University of California, Davis vineyards. The background for all these crosses consisted of V. arizonica. These V. arizonica plants are part of a collection obtained by H.P. Olmo during the 1960’s. In recent studies it was established that X. index resistance is controlled by a single dominant gene. The 0701 (R8916-07 (Wichita Refuge x b40-14) x R8916-32), 0704 (161-49C x b40-14) and 0705 (161-49C x R8916-22) populations were created to confirm the homozygous nature of b40-14, a V. arizonica accession. In addition, several V. arizonica species were screened to confirm their resistance or susceptibility towards X. index feeding. The 0705 population was also used to create a genetic map for X. index resistance. In this study a new and improved screening method was developed to inoculate vines under greenhouse conditions. This screening method proved to be quicker and less damaging on the nematodes than traditional systems. Control varieties were used and O39-16, a commercial rootstock showed no damage, even with high nematode pressure, whereas V. rupestris Saint George had severe root damage and decline after eight weeks of exposure. A range of V. arizonica accessions was tested for their resistance to X. index feeding. Of the 18 genotypes tested, half showed resistance and the rest were susceptible. It is possible that these genotypes are not pure V. arizonica genotypes. Genotypes with V. arizonica in the background were also tested. Wichita Refuge was used as a susceptible female parent and the progeny were expected to be heterozygous resistant. Some of the progeny allowed low levels of feeding damage, which may have been the result of the more effective inoculation method described above. The 0701 population confirmed the hypothesized model of 3:1 (Resistance (R):Susceptible (S)) segregation although 13 of the genotypes showed significantly higher gall numbers than the susceptible female parent. The possibility of transgressive segregation exists, but needs to be confirmed. All progeny from the 0704 population should be heterozygous resistant, but a 1:1 (R:S) segregation pattern was observed. The 0705 population was created as a mapping population to study X. index resistance. This population was also tested in the greenhouse for its X. index resistance and was expected to segregate 1:1 (R:S). The X2 analysis did not fully support this model. A genetic map covering all 19 linkage groups, and positioning 175 polymorphic SSR markers was created for the 164 progeny in the 0705 population. MapQTL analysis revealed a major QTL on linkage group 9 and two minor QTL’s on groups 13 and 19. The major QTL placed between markers VMC1c10 and CTG1032918 with a LOD score of 33.4 explaining 70.5% of the phenotypic variance for X. index. This QTL is the second major QTL discovered for X. index resistance. With the discovery of a second major QTL, the two types of resistance can be pyramided. Work is underway to saturate the area around the major QTL on linkage group 9 and to move towards physical mapping of X. index resistance. The b40-14 V. arizonica accession is also known for its resistance to Pierce’s disease and the possibility of simultaneous expression of two types of resistance is created. The 0705 population can also be used to evaluate phenotypical characteristics in the field to determine if useful rootstocks can be selected. Taken together, the results obtained in this study provide improved methods and highly characterized plant populations to support the efforts in obtaining improved X. index resistance in grapevine rootstocks. / AFRIKAANSE OPSOMMING: Wingerde is van die belangrikste en mees diverse gewasse op aarde, maar hulle neig om vir ‘n verskeidenheid plae en siektes vatbaar te wees. Die dolk-aalwurm, Xiphinema index (X. index), is ‘n bekende grondgedraagde plaag van wingerd en ‘n vektor vir wingerd- netelblaarvirus. Verskeie Vitis-spesies toon weerstand teen hierdie plaag. Daar word reeds vir dekades pogings aangewend om weerstandbiedende onderstokke te kweek. Konvensionele kweekpogings is egter tydrowend omdat wingerd ‘n meerjarige gewas is, op grond van die heterosigositeit van die gewas, sowel as die lang groeisiklus. Dit is ook duur en arbeidsintensief om nuwe wingerdvariëteite te kweek. Die ontwikkeling van merkerondersteunde seleksie het dus ‘n metode verskaf om sommige van bogenoemde probleme te oorkom. Die doelwit van hierdie studie was om die genetiese evaluerings- en kweekpogings vir verbeterde X. index-weerstand in wingerd-onderstokke te verbreed. In 2007 is verskeie kruisings in die wingerde by die Universiteit van Kalifornië, Davis gemaak. Die agtergrond vir al hierdie kruisings het bestaan uit V. arizonica. Hierdie V. arizonica-plante vorm deel van ‘n versameling wat in die 1960’s deur H.P. Olmo verkry is. In onlangse studies is daar bepaal dat X. index-weerstand deur ‘n enkele dominante geen beheer word. Die 0701 (R8916-07 (Wichita Refuge x b40-14) x R8916-32), 0704 (161-49C x b40-14) en 0705 (161-49C x R8916-22) bevolkings is geskep om die homosigotiese geaardheid van b40-14, ’n V. arizonicaafstammeling, te bevestig. Daarbenewens is verskeie V. arizonica-spesies gesif om hulle weerstand teen of vatbaarheid vir X. index voeding te bevestig. Die 0705 bevolking is ook gebruik om ‘n genetiese kaart vir X. indexweerstand te skep. In hierdie studie is ‘n nuwe en verbeterde siftingsmetode ontwikkel om wingerdstokke onder glashuistoestande te inokuleer. Daar is gewys dat hierdie siftingsmetode vinniger en minder skadelik vir die aalwurms as tradisionele metodes is. Beheervariëteite is gebruik en O39-16, ‘n kommersiële onderstok, het geen skade getoon nie, selfs met hoë aalwurmdruk, terwyl V. rupestris Saint George ernstige wortelskade en agteruitgang na agt weke se blootstelling getoon het. ‘n Verskeidenheid V. arizonica-afstammelinge is vir hulle weerstand teen X. index-voeding getoets. Van die 18 genotipes wat getoets is, het die helfte weerstand getoon en die res was vatbaar. Dit is moontlik dat hierdie genotipes nie suiwer V. arizonica-genotipes was nie. Genotipes met V. arizonica in hulle agtergrond is ook getoets. Wichita Refuge is as ‘n vatbare vroulike ouer gebruik en die verwagting was dat die nageslag heterosigoties weerstandbiedend sou wees. Sommige van die nageslag het lae vlakke van voedingskade toegelaat, wat moontlik die gevolg was van die meer doeltreffende inokulasiemetode wat hierbo beskryf word. Die 0701 bevolking het die veronderstelde model van 3:1 (Weerstandbiedend (W):Vatbaar (V)) segregasie bevestig, hoewel 13 van die genotipe noemenswaardig hoër galgetalle as die vatbare vroulike ouer getoon het. Die moontlikheid van transgressiewe segregasie bestaan, maar dit moet nog bevestig word. Alle nageslag van die 0704 bevolking behoort heterosigoties weerstandbiedend te wees, maar ‘n 1:1 (W:V) segregasiepatroon is waargeneem. Die 0705 bevolking is as ‘n karteringsbevolking geskep om X. index-weerstand te bestudeer. Hierdie bevolking is ook in die glashuis vir sy X. index-weerstand getoets en daar is verwag dat dit 1:1 (W:V) sou segregeer. Die X2 analise het nie hierdie model ten volle ondersteun nie. ‘n Genetiese padkaart wat al 19 skakelingsgroepe en die posisies van 175 polimorfiese SSR merkers toon, is vir die 164 afstammelinge in die 0705 bevolking geskep. MapQTL analise het ‘n groot kwantitatiewe eienskap lokus (QTL) op skakelingsgroep 9 en twee kleiner QTL’e op groepe 13 en 19 onthul. Die groot QTL is tussen merkers VMC1c10 en CTG1032918 met ‘n LOD telling van 33.4 geplaas en het 70.5% van die fenotipiese variansie van X. index verklaar. Hierdie QTL is die tweede groot QTL wat vir X. index-weerstand ontdek is. Met die ontdekking van ‘n tweede groot QTL, kan die twee soorte weerstand gepiramideer word. Werk word reeds onderneem om die area rondom die groot QTL op skakelingsgroep 9 te versadig en om na die fisiese kartering van X. index-weerstand te beweeg. Die b40-14 V. arizonica-afstammeling is ook bekend vir sy weerstand teen Pierce se siekte en die moontlikheid word geskep vir die gelyktydige uitdrukking van twee soorte weerstand. Die 0705 bevolking kan ook gebruik word om die fenotipiese kenmerke in die veld te evalueer om te bepaal of bruikbare onderstokke geselekteer kan word. In kombinasie behoort die resultate wat in hierdie studie verkry is, verbeterde metodes en hoogs gekarakteriseerde plantbevolkings te lewer wat die pogings sal ondersteun om verbeterde X. index-weerstand in wingerdonderstokke te verkry.

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Made available in DSpace on 2015-03-26T13:37:39Z (GMT). No. of bitstreams: 1 texto completo.pdf: 2474594 bytes, checksum: 6cdab11f886d77e7d0c8a3d024dde318 (MD5) Previous issue date: 2008-07-30 / Conselho Nacional de Desenvolvimento Científico e Tecnológico / The genus Begomovirus (family Geminiviridae) includes viruses with a genome comprised of one or two molecules of circular, single-stranded DNA, transmitted to dicot species by the whitefly Bemisia tabaci. In Brazil, after the introduction of the B biotype of B. tabaci in the early 1990s, the incidence of begomoviruses in tomato has become frequent, with several reports of new viral species. Some of these species have become prevalent under field conditions, including Tomato severe rugose virus (ToSRV) and Tomato yellow vein streak virus (ToYVSV). The purpose of this study was to characterize two isolates of these species through the cloning of their whole genomes followed by molecular analysis, and the production of infectious clones for determination of their host ranges. Total DNA extracts of infected plants were used for whole genome amplification using the phi29 phage DNA polymerase. The amplification products were cloned into plasmids and completely sequenced. Sequence analysis indicated that the DNA-A and DNA-B of ToSRV-[BR:Pir1:05] and ToYVSV- [BR:Pda30:05] isolates had greater than 90% identities with other isolates of ToSRV and ToYVSV, respectively. The molecular analysis indicated that the DNA-A of the BR:Pir1:05 isolate may be the result of a recombination event, in which the virus acquired the rep ORF of Tomato rugose mosaic virus (ToRMV) and the cp ORF of an unidentified virus. Analysis of the DNA-B of the same isolate indicated the existence of a relationship with viruses that infect weed/wild hosts in Brazil, corroborating the hypothesis that viruses present in wild hosts led to the virus currently found in tomatoes. Additionally, because of the high identity observed between the DNA-B of ToSRV-[BR:Pir1:05] and ToRMV, it is possible that this genomic component is shared by the two viruses. In host range assays, plants showing latent infections were observed for both isolates. Such plants can act as natural reservoirs and serve as a primary source of inoculum for host plants of economic importance such as the tomato. The infectious clones of ToYVSV- [BR:Pda30:05] had low infectivity in the host range assays. It is possible that the inoculation method was not effective in the transmission of this isolate, or one or both clones could contain mutations that reduce the efficiency of their replication. / O gênero Begomovirus (família Geminiviridae) inclui vírus com genoma composto por uma ou duas moléculas de DNA fita simples, transmitidos a espécies de plantas dicotiledôneas pela mosca-branca Bemisia tabaci. No Brasil, após a introdução do biótipo B de B. tabaci no início da década de 1990, a incidência de begomovírus em tomateiro tornou-se freqüente, com vários relatos de novas espécies virais. Algumas dessas espécies tornaram-se prevalentes em condições de campo no Brasil, incluindo o Tomato severe rugose virus (ToSRV) e o Tomato yellow vein streak virus (ToYVSV). O objetivo deste trabalho foi caracterizar dois isolados dessas espécies, por meio da clonagem de seus genomas completos seguida de análise molecular, e da obtenção de clones infecciosos para determinação de suas gamas de hospedeiros. Extratos de DNA total de plantas infectadas foram utilizados para a amplificação do genoma viral completo utilizando-se a DNA polimerase do fago φ29. Os produtos da amplificação foram clonados em plasmídeos e completamente seqüenciados. A análise das seqüências do DNA-A e DNA-B dos isolados ToSRV- [BR:Pir1:05] e ToYVSV- [BR:Pda30:05] indicou valores de identidade acima de 90% com outros isolados de ToSRV e ToYVSV, respectivamente. A análise molecular indica que o DNA-A do isolado BR:Pir1:05 pode ser resultado de um evento de recombinação, no qual o vírus adquiriu a ORF rep do Tomato rugose mosaic virus (ToRMV) e a ORF cp de um vírus não identificado. A análise do DNA-B do mesmo isolado indica a existência de relacionamento com vírus que infectam plantas daninhas/silvestres no Brasil, reforçando a hipótese de que vírus presentes em hospedeiros silvestres deram origem aos vírus atualmente encontrados em tomateiro. Adicionalmente, devido à alta identidade observada entre os DNAs-B dos isolados BR:Pir1:05 e do ToRMV, é possível que este componente genômico seja compartilhado pelos dois vírus. Nos testes de gama de hospedeiros, plantas apresentando infecções latentes foram observadas para os dois isolados. Tais plantas podem atuar como reservatórios naturais e servir de fonte de inóculo primário para plantas hospedeiras de importância econômica, como o tomateiro. Os clones infecciosos do isolado BR:Pda30:05 apresentaram baixa infectividade nos testes de gama de hospedeiros. É possível que o método de inoculação não tenha sido eficiente na transmissão deste isolado, ou que os clones apresentem mutações que reduzam a eficiência de sua replicação.

Os modelos de crescimento econômico mostram que a única forma de promover o crescimento sustentado da economia é por meio do progresso tecnológico. Porém, a manutenção de um fluxo crescente de inovações tecnológicas requer uma forte estrutura de pesquisa e desenvolvimento (P&D), o que implica em uma série de gastos por parte de ambos os setores, público e privado. Conseqüentemente, as instituições de pesquisa são crescentemente cobradas quanto aos resultados dos recursos nelas investidos, o que torna interessante a realização de estudos que procurem avaliar os retornos de tais investimentos e determinar quão proveitosos estes vêm-se revelando para a sociedade. Encontram-se na literatura diversos estudos objetivando medir os retornos econômicos provenientes dos investimentos em P&D na agricultura em geral, porém, existem poucos trabalhos para setores específicos. Desta forma, o presente estudo teve como principal objetivo medir o retorno econômico dos investimentos em pesquisa na citricultura paulista. A justificativa para a escolha do setor analisado é que o Brasil, além de representar sozinho cerca de 80% do total de suco de laranja concentrado e congelado comercializado no mercado internacional, responde por 28% da produção mundial de laranja. O Estado de São Paulo, por sua vez, responde por cerca de 80% da produção nacional de laranja e 98% das exportações brasileiras de suco concentrado. A relevância econômica dessa atividade no Estado tem estimulado inúmeras pesquisas, especialmente em relação aos aspectos fitossanitários, uma vez que a citricultura paulista é fortemente atacada por uma série de pragas e doenças, desde o seu surgimento. A metodologia utilizada para a estimativa do retorno aos investimentos na pesquisa citrícola foi o cálculo da produtividade total dos fatores (PTF), através do Índice de Tornqüist, para posterior comparação com os gastos em pesquisa. Além disso, utilizou-se o método do valor presente líquido (VPL) para comparação entre diferentes cenários com relação à contaminação dos pomares, de modo a estimar o dano econômico evitado pela descoberta e pelo controle das doenças. Dentre os principais resultados encontrados verificou-se que para cada R$1,00 investido na pesquisa citrícola obtém-se um aumento de R$ 13,67 no valor da produção de laranja no Estado de São Paulo. Com relação às perdas de produtividade evitadas graças ao controle das principais doenças, verificou-se que se o cancro cítrico não fosse rigorosamente controlado através da erradicação, o prejuízo teria sido ao redor de R$ 2 bilhões nos últimos 35 anos. Finalmente, a partir da simulação de diferentes níveis de contaminação pelo greening os resultados sugeriram que o prejuízo causado pela possível disseminação desta doença nos pomares paulistas seria bastante significativo nos próximos 20 anos, especialmente se estiverem associados a um cenário de manejo integrado do cancro cítrico, ao invés da erradicação. Em outras palavras, os resultados do estudo corroboram a importância dos investimentos em P&D na citricultura paulista. / The economic growth models show that the only way to promote a sustained growth of the economy is through technological progress. However, keeping a growing technological innovation flow requires a strong research and development - R&D structure, demanding expenses from both public and private sectors. Consequently, the research institutions have been increasingly charged about the outcomes from applied funds, which turn necessary to conceptualize and evaluate such R&D investment returns, as well as to highlight what benefits they can reveal to society. There are several studies aiming to assess the whole agriculture R&D investments returns, whereas only few studies has been doing towards to specific sectors. Thus, the general objective of this study is to assess the economic return from R&D investments in São Paulo State's citrus sector over the last 35 years. Brazil represents 80% of the total frozen concentrated orange juice - FCOJ commercialized in the international market, and 28% of the world orange production. São Paulo State is responsible by 80% of national orange production and 98% of Brazilian FCOJ exportation. The economic importance of this industry has been stimulating several research activities, especially related to plant disease aspects, given that São Paulo's citrus sector is strongly threatened by pests and diseases. It was used the Tornqüist Index Method to estimate the citrus industry total productivity of factors - TPF, in order to compare the outcomes with citrus R&D investments. In addition, the net present value - NPV method was used to compare different scenarios, in relation to citrus canker and greening outbreak in São Paulo's groves, to estimate the economic losses avoided due to diseases control. For every R$1 invested in the citrus industry R&D, it was found that R$13,67 is risen on the São Paulo State orange production value. In relation to avoided economic losses due to diseases control, if the citrus canker had not been controlled through tree eradication, the economic losses would have been approximately R$2 billion throughout the past 35 years. Finally, from different levels of citrus greening outbreak simulation, the results suggest that the damage caused by the possible citrus greening outbreak in São Paulo's orchards would be quite significant for the next 20 years, especially if the levels are associated with canker integrated management, regardless of eradication program. Summarizing, the results achieved from this work reinforces the remarkable importance of São Paulo's citrus sector R&D investments.

Os modelos de crescimento econômico mostram que a única forma de promover o crescimento sustentado da economia é por meio do progresso tecnológico. Porém, a manutenção de um fluxo crescente de inovações tecnológicas requer uma forte estrutura de pesquisa e desenvolvimento (P&D), o que implica em uma série de gastos por parte de ambos os setores, público e privado. Conseqüentemente, as instituições de pesquisa são crescentemente cobradas quanto aos resultados dos recursos nelas investidos, o que torna interessante a realização de estudos que procurem avaliar os retornos de tais investimentos e determinar quão proveitosos estes vêm-se revelando para a sociedade. Encontram-se na literatura diversos estudos objetivando medir os retornos econômicos provenientes dos investimentos em P&D na agricultura em geral, porém, existem poucos trabalhos para setores específicos. Desta forma, o presente estudo teve como principal objetivo medir o retorno econômico dos investimentos em pesquisa na citricultura paulista. A justificativa para a escolha do setor analisado é que o Brasil, além de representar sozinho cerca de 80% do total de suco de laranja concentrado e congelado comercializado no mercado internacional, responde por 28% da produção mundial de laranja. O Estado de São Paulo, por sua vez, responde por cerca de 80% da produção nacional de laranja e 98% das exportações brasileiras de suco concentrado. A relevância econômica dessa atividade no Estado tem estimulado inúmeras pesquisas, especialmente em relação aos aspectos fitossanitários, uma vez que a citricultura paulista é fortemente atacada por uma série de pragas e doenças, desde o seu surgimento. A metodologia utilizada para a estimativa do retorno aos investimentos na pesquisa citrícola foi o cálculo da produtividade total dos fatores (PTF), através do Índice de Tornqüist, para posterior comparação com os gastos em pesquisa. Além disso, utilizou-se o método do valor presente líquido (VPL) para comparação entre diferentes cenários com relação à contaminação dos pomares, de modo a estimar o dano econômico evitado pela descoberta e pelo controle das doenças. Dentre os principais resultados encontrados verificou-se que para cada R$1,00 investido na pesquisa citrícola obtém-se um aumento de R$ 13,67 no valor da produção de laranja no Estado de São Paulo. Com relação às perdas de produtividade evitadas graças ao controle das principais doenças, verificou-se que se o cancro cítrico não fosse rigorosamente controlado através da erradicação, o prejuízo teria sido ao redor de R$ 2 bilhões nos últimos 35 anos. Finalmente, a partir da simulação de diferentes níveis de contaminação pelo greening os resultados sugeriram que o prejuízo causado pela possível disseminação desta doença nos pomares paulistas seria bastante significativo nos próximos 20 anos, especialmente se estiverem associados a um cenário de manejo integrado do cancro cítrico, ao invés da erradicação. Em outras palavras, os resultados do estudo corroboram a importância dos investimentos em P&D na citricultura paulista. / The economic growth models show that the only way to promote a sustained growth of the economy is through technological progress. However, keeping a growing technological innovation flow requires a strong research and development - R&D structure, demanding expenses from both public and private sectors. Consequently, the research institutions have been increasingly charged about the outcomes from applied funds, which turn necessary to conceptualize and evaluate such R&D investment returns, as well as to highlight what benefits they can reveal to society. There are several studies aiming to assess the whole agriculture R&D investments returns, whereas only few studies has been doing towards to specific sectors. Thus, the general objective of this study is to assess the economic return from R&D investments in São Paulo State's citrus sector over the last 35 years. Brazil represents 80% of the total frozen concentrated orange juice - FCOJ commercialized in the international market, and 28% of the world orange production. São Paulo State is responsible by 80% of national orange production and 98% of Brazilian FCOJ exportation. The economic importance of this industry has been stimulating several research activities, especially related to plant disease aspects, given that São Paulo's citrus sector is strongly threatened by pests and diseases. It was used the Tornqüist Index Method to estimate the citrus industry total productivity of factors - TPF, in order to compare the outcomes with citrus R&D investments. In addition, the net present value - NPV method was used to compare different scenarios, in relation to citrus canker and greening outbreak in São Paulo's groves, to estimate the economic losses avoided due to diseases control. For every R$1 invested in the citrus industry R&D, it was found that R$13,67 is risen on the São Paulo State orange production value. In relation to avoided economic losses due to diseases control, if the citrus canker had not been controlled through tree eradication, the economic losses would have been approximately R$2 billion throughout the past 35 years. Finally, from different levels of citrus greening outbreak simulation, the results suggest that the damage caused by the possible citrus greening outbreak in São Paulo's orchards would be quite significant for the next 20 years, especially if the levels are associated with canker integrated management, regardless of eradication program. Summarizing, the results achieved from this work reinforces the remarkable importance of São Paulo's citrus sector R&D investments.

Made available in DSpace on 2015-03-26T13:37:36Z (GMT). No. of bitstreams: 1 texto completo.pdf: 4434052 bytes, checksum: e6bd53f46965b44a3dcdbb9a5a63b4ff (MD5) Previous issue date: 2008-02-19 / Conselho Nacional de Desenvolvimento Científico e Tecnológico / This work aimed to select tomato prokaryotic phylloplane residents for biocontrol purposes, by using Xanthomonas campestris pv. vesicatoria and Alternaria solani as challenging pathogens. Additionally, to perform a screening test using detached leaves. Consequently, leaves from healthy tomato plants were collected at Viçosa, MG and epiphytic prokaryote populations were extracted by shacking them in PBS (0,85% NaCl in 0,1M phosphate buffer saline, pH 7.0) following sonication (60Hz, 20 minutes) and serial dilution and plating in culture medium. Three hundred bacterial isolates were obtained and preserved in refrigerator, with periodical tubetube transfer, in a refrigerator. The screening was performed in two selection cycles. In the first selection in greenhouse, with three replicates per isolate and Xanthomonas campestris pv. vesicatoria as challenging pathogen, 79 isolates were able to reduce disease by 82%. In a second selection cycle, using 6 replicates per isolate, the 33 isolates were able to reduce severity by 50% in average. The 33 isolates had their antagonistic potential tested against the tomato fungal and bacterial pathogens Pseudomonas syringae pv. tomato, Pseudomonas corrugata, Xanthomonas campestris pv. vesicatoria, Clavibacter michiganensis subsp. michiganensis, Alternaria solani and Corynespora cassiicola in vitro conditions. Out of the 33 isolates, the isolate RFK-24 inhibited growth of Xathomonas campestris pv. vesicatoria, Clavibacter michiganensis subsp. michiganensis and Alternaria solani while isolate RFS-183 was able to inhibit Alternaria solani, Xanthomonas campestris pv. vesicatoria, Pseudomonas syringae pv. tomato e Pseudomonas corrugata, but none of them inhibited Corynespora cassiicola. Being RFK-24 and RFS- 183 the ones with a wider antagonistic potential out of the 33 previously selected, they were chosen for continuing the research with detached leaves and the challenging pathogens Alternaria solani e Xanthomonas campestris pv. vesicatoria. There was a positive correlation between disease severity reduction in plants the greenhouse and in detached leaves, for both antagonists. For situations involving large number of isolates to undergo mass screening, the approach with detached organs may replace the laborious and time consuming greenhouse screening. / O presente trabalho teve como objetivo isolar procariotos residentes de filoplano de tomateiro como agentes de biocontrole contra o patógeno Xanthomonas campestris pv. vesicatoria. 300 isolados foram obtidos a partir de folhas sadias de tomateiros coletadas na micro-região de Viçosa-MG, as quais foram submetidas à extração das populações procarióticas epifíticas em solução de tampão fosfato (PBS) com emprego de ultra-som (60Hz, 20 min.) e semeadura de diluições em série em placas de Petri contendo meio 523. Em casa-de-vegetação, numa primeira etapa 79 isolados apresentaram capacidade de reduzir, em média, 82 % da severidade de doença. Desses, numa segunda etapa, 33 isolados apresentaram redução média da severidade de doença em 50 %. Os 33 isolados foram submetidos a testes de antibiose in vitro, em que foi avaliada a potencialidade em inibir o crescimento bacteriano e fúngico dos seguintes patógenos do tomateiro: Pseudomonas syringae pv. tomato, Pseudomonas corrugata, Xanthomonas campestris pv. vesicatoria, Clavibacter michiganensis subsp. michiganensis, Alternaria solani e Corynespora cassiicola. Dos 33 isolados pré- selecionados verificou-se que o isolado RFK-24 foi capaz de inibir o crescimento dos patógenos, Xanthomonas campestris pv. vesicatoria, Clavibacter michiganensis subsp. michiganensis e Alternaria solani e o RFS-183 em inibir o crescimento dos patógenos Alternaria solani, Xanthomons campestris pv. vesicatoria, Pseudomonas syringae pv. tomato e Pseudomonas corrugata, mas ambos não inibiram o crescimento da Corynespora cassiicola. Os dois isolados foram selecionados para ensaios in vivo e em folíolos destacados, contra os patógenos desafiantes Alternaria solani e Xanthomons campestris pv. vesicatoria. Para ambos foi observada a correlação entre a severidade de doença apresentada em casa-de-vegetação e os resultados em folíolos destacados, como a inibição da germinação de conídios do patógeno fúngico e a supressividade em meio semi-seletivo dos antagonistas sob o patógeno bacteriano. Os resultados obtidos com o método com folíolos destacados e sua correlação com os dados de biocontrole experimental em casa-de-vegetação permitem deduzir que o uso de folíolos destacados pode ser utilizado como método para seleção massal.