O desenvolvimento e implementação de uma estratégia de manejo integrado de doenças na cultura do girassol (Helianthus annuus) requer informações precisas e acuradas sobre os danos causados pela mancha de Alternaria, causada por Alternaria helianthi. Os objetivos desta tese foram: elaborar e validar uma escala diagramática para avaliação da mancha de Alternaria; determinar a influência da temperatura (15-32,5 °C) e da duração do período de molhamento foliar (2-24 h) nos componentes monocíclicos da doença; comparar a relação de variáveis relacionadas à doença ou à área foliar com os componentes de produção de girassol, em experimentos de campo. A escala diagramática, com níveis de severidade de: 0,03; 0,2; 0,6; 3; 7; 12; 25; 40 e 66% da área foliar lesionada, foi validada por três avaliadores experientes. A escala permitiu que se fizesse uma avaliação acurada e precisa, quando se utilizou folhas desenhadas ou folhas verdes com sintomas. O erro absoluto ao estimar a severidade foi menor que 13% e a reprodutibilidade das avaliações foi alta. Em ambiente controlado, verificou-se que a densidade relativa de lesões e a severidade foram influenciadas pela temperatura e pela duração do período de molhamento foliar. A doença foi mais severa na temperatura de 25 °C. A temperatura mínima para desenvolvimento da doença foi de 13,0 °C e a máxima foi de 35,8 °C. A doença aumentou com o aumento da duração do período de molhamento foliar. A temperatura mínima estimada para a taxa de crescimento micelial foi de 5,5 °C e a máxima foi de 32,9 °C. A temperatura mínima estimada para germinação de conídios foi de 7,9 °C e a máxima foi de 40,0 °C. Experimentos foram conduzidos nas safras de 1997/1998, 1998/1999 e 1999/2000 para avaliar a relação entre severidade da mancha de Alternaria, área sob a curva de progresso da doença (AUDPC), índice de área foliar sadia em determinado dia (HLAI), duração da área foliar sadia (HAD), radiação interceptada pela área foliar sadia em determinado dia (HRI), absorção da área foliar sadia (HAA) e componentes de produção de girassol, semeado em quatro épocas. A AUDPC teve relação com rendimento de aquênios, com ajuste do modelo exponencial negativo em duas safras. A relação rendimento x HAD foi linear para os três experimentos. Para as variáveis integrais estudadas, a melhor relação com rendimento foi verificada para HAA, com ajuste do modelo exponencial. A taxa da relação severidade x rendimento tendeu a se estabilizar a partir da fase de desenvolvimento R1. A taxa da relação linear entre HLAI e rendimento estabilizou-se entre as fases R1 a R6. A taxa da regressão entre HRI e rendimento foi bastante variável e não mostrou tendência de estabilização. Verificou-se que plantas que apresentaram severidade maior que 10% na fase de desenvolvimento R3 produziram rendimentos inferiores a 500 kg/ha, para as três safras, independentemente da época de semeadura, o que pode ser utilizado como um limiar de dano. A severidade pode ser usada como uma variável independente para um sistema de manejo da mancha de Alternaria em girassol. / The development and implementation of integrated disease-management system on sunflower (Helianthus annuus) require precise and accurate information on the damage caused by Alternaria leaf spot (Alternaria helianthi). The objectives of this thesis were: to develop and to validate a diagrammatic scale for Alternaria leaf spot; to determine the influence of temperature (15-32.5 °C) and leaf wetness duration (2-24 h) on the monocycle of the disease; and to compare the relationship between variables related to Alternaria leaf spot or to leaf area and yield, in field experiments. The diagrammatic scale with levels representing: 0.03; 0.2; 0.6; 3; 7; 12; 25; 40; and 66% of diseased leaf area was validated by three experienced raters. The scale permitted assessments to be accurate and precise, when drawn or detached leaves were used. The absolute error in estimating disease severity was lower than 13% and the reproduc ibility of assessments was high. In controlled environment studies, relative lesion density and severity were influenced by temperature and leaf wetness duration. The disease was more severe in the temperature of 25 °C. The minimum temperature for disease development was 13.0 °C and the maximum was 35.8 °C. The disease increased with increasing periods of leaf wetness. The estimated minimum temperature for mycelial growth rate was 5.5 °C and the maximum was 32.9 °C. The estimated minimum temperature for conidia germination was 7.9 °C and the maximum was 40.0 °C. Three field experiments were carried out in 1997/1998, 1998/1999 and 1999/2000 to investigate the relationship between severity of Alternaria leaf spot, area under disease progress curve (AUDPC), healthy leaf area index at any given day (HLAI), healthy leaf area duration (HAD), radiation intercepted by healthy leaf area at any given day (HRI), total healthy leaf area absorption (HAA), and yield components of sunflower, sowed in four dates. AUDPC showed relationship with yield in two years, following the negative exponential model. The yield x HAD relationship was linear for each of three trials. Among the integral variables analyzed, the yield x HAA relationship was the best one, fitted by the exponential model. The slope of the yield-severity relationship was stable from R1 growth stage. The yield-HLAI relationship was stable between R1 and R6. The yield-HRI relationship was variable and not stable. The yield-severity relationship in R3 growth stage proved that plants with severity higher than 10% had yield lower than 500 kg/ha, despite the sowing date, which can be used as a damage threshold. Severity can be used as an independent variable to a system of sunflower Alternaria leaf spot management.

Atualmente, a produtividade da cultura da batata (Solanum tuberosum L.) pode ser reduzida devido à ocorrência de doenças em todo o território nacional, destacando-se a pinta preta, causada por Alternaria spp. Historicamente se acreditava que A. solani (AS) era a principal causadora mas, atualmente, descobriram outras espécies associadas, sendo predominante hoje, em território nacional a espécie A. grandis (AG). O objetivo deste trabalho foi avaliar a sensibilidade in vitro de três isolados de A. grandis e um isolado de A. solani frente a onze fungicidas amplamente utilizados no manejo desta doença em campo. Foi avaliado o efeito dos fungicidas sobre o crescimento micelial dos isolados, utilizando-se do método da incorporação do fungicida no meio de cultura fundente. As diluições dos fungicidas foram realizadas em água destilada e esterilizada, de acordo com a metodologia descrita por Camargo (2013) e Ishizuka (2016). Para os fungicidadas com mistura de ingrediente ativo, considerou-se a soma das concentrações no cálculo. Inicialmente, as concentrações do meio de cultura, foram 100, 10, 1, 0,1, 0,01 e 0,001 mg L-1. Após, as doses foram ajustadas conforme a CI50 de cada fungicida. Quantificou-se também a inibição sobre a germinação dos conídios nas contrações discriminatórias de 0; 0,1; 1; 10 e 100 mg.L-1 .24 horas após o preparo do meio as placas foram observadas em microscópio óptico, determinando-se a germinação dos conídios em 300 unidades observadas por placa de Petri. Todos os isolados testados foram altamente sensíveis aos ingredientes ativos fluazinam, pirimetanil, procimidona e piraclostrobina. Não houve diferenças de comportamento de sensibilidade entre as espécies de A. solani e A grandis que possam caracterizar uma perda de sensibilidade por conta da predominância da espécie de A. grandis associada à pinta preta da batata no Brasil. Encontraram-se evidências de insensibilidade dos isolados de A. grandis e A. solani aos ingredientes ativos Clorotalonil e Boscalida. / Currently, the productivity of potato crop (Solanum tuberosum L.) may be reduced due to the occurrence of disease throughout the country, especially due to early blight, caused by Alternaria spp. Historically it was believed that A. solani (AS) was the main cause, most currently was founded others associated species, being predominant today the occurrence of the disease associated with the species A. grandis (AG). The objective of this study was to evaluate the in vitro susceptibility of three isolates of A. grandis and one isolate of A. solani for eleven fungicides widely used in the management of this disease in the field, was evaluated the effect of fungicides on mycelial growth using the fungicidal method that incorporate it in the medium culture. Dilutions of fungicides were performed in sterile distilled water according to the methodology described by Camargo (2013) and Ishizuka (2016). For the fungicidates with active ingredient mixture, was considered the sum of the both concentrations in the calculation.The concentrations of the culture medium were 100, 10, 1, 0.1, 0.01 and 0.001 mg L-1. The doses were adjusted according to the CI50 of each fungicide, was quantified also the inhibition of spore germination, and the fungicides were incorporated into the agar medium - water in discriminatory contractions of 0; 0.1; 1; 10 and 100 mg.L-1 . Then, 24 hours after preparation of the medium the plates were observed under an optical microscope, determining the spore germination observed at 300 units per plate. All tested isolates were highly sensitive to the active ingredients fluazinam, pyrimethanil, procymidone and pyraclostrobin. There was no sensitivity behavioral differences between species of A. solani and A. grandis which may characterize a loss of sensitivity due to the predominance of species of A. grandis associated with early blight in Brazil, was found evidences of insensitivity of isolates of A. grandis and A. solani to the active ingredients Chlorothalonil and Boscalida.

<p>Potreba za potpunijom informacijom o važnosti kontaminacije useva Alternaria<br />toksinima su nedavno dobili na značaju. Imajući u vidu toksičnost metabolita koje<br />produkuju pojedine vrste gljive iz roda Alternaria u sistemu od njive do trpeze uticalo<br />je na to da se u sklopu istraživanja u oblasti mikotoksikologije registruje porast<br />interesovanja za Alternaria toksine i gljive koje ih produkuju. S obzirom na činjenicu<br />da sa na&scaron;ih prostora nema dostupnih podataka o prirodnoj kontaminaciji p&scaron;enice sa<br />Alternaria toksinima, a o pona&scaron;anju Alternaria toksina tokom procesa prerade p&scaron;enice nema dostupnih podataka u naučnoj literaturi, proizi&scaron;li su ciljevi ove doktorske disertacije. Osnovni cilj planiranih istraživanja u ovoj doktorskoj disertaciji je bio sagledavanje uticaja konvencionalno primenjenih mehaničkih (či&scaron;ćenje i suvo<br />mlevenje) i hidrotermičkih (ekstrudiranje) tehnolo&scaron;kih postupaka na redukciju<br />prisutnih Alternaria toksina u p&scaron;enici. Primenom postupaka či&scaron;ćenja p&scaron;enice<br />uobičajenih u praksi rada silosne i mlinske čistione postiže se značajna redukcija<br />prisustva svih ispitivanih Alternaria toksina u zrnenoj masi.U postupku mlinskeprerade p&scaron;enice, čak i u slučaju prerade p&scaron;enice u kojoj je sadržaj pojedinih Alternaria toksina ispod granice detekcije u frakcijama mlevenja p&scaron;enice u koje dospevaju periferni delovi p&scaron;eničnog zrna, odnosno kasnijim prolazi&scaron;tima krupljenja i mlevanja, kao i sporednim proizvodima meljave, može se očekivati povi&scaron;en sadržaj Alternaria toksina. Optimalni efekti procesa ektrudiranja u slučaju kori&scaron;ćenja jednopužnog ekstrudera ostvaruju se u slučaju visoke vlage polaznog materijala (w=24%) velike brzine protoka materijala (q=25 kg/h) i srednje brzine obrtanja puža ekstrudera (v=390 obrtaja/min) pri čemu se postiže redukcija TeA=60-65 %, AOH=90 % i AME=95 %. Dodatni cilj je bio da se sagleda učestalost pojave Alternaria toksina, i stekne preliminarni uvid u pojavu i opseg koncentracije Alternaria toksina u p&scaron;enici sa teritorije Vojvodine sa ciljem da se dobiju preliminarni podaci o uticaju proizvodne<br />godine, lokaliteta i najkarakterističnijih klimatskih uslova na pojavu Alternaria toksina<br />u p&scaron;enici, kao i da se sagleda uticaj uslova introdukovane stimulacije i inhibicije<br />kontaminacije p&scaron;enice sa Alternaria spp. na sadržaj Alternaria toksina u p&scaron;enici. Analiza učestalosti incidencije Alternaria toksina u p&scaron;enici sa područja Vojvodine ukazuju na evidentnu pojavu značajnog broja partija p&scaron;enice kontaminirane sa TeA, praćenu sporadičnom pojavom partija kontaminiranih sa AOH i AME. Kako prosečne, tako i maksimalne koncetracije TeA u p&scaron;enici iz Vojvodine proizvedene u trogodi&scaron;njem periodu od 2011. do 2013. godine bile su vi&scaron;e od zvanično objavljenih vi&scaron;egodi&scaron;njih podataka od strane Evropske agencije za bezbednost hrane (EFSA). Utvr&ntilde;ene koncentracije AME i AOH u p&scaron;enici iz Vojvodine su daleko niže od koncentracije TeA kao najče&scaron;ćeg kontaminenta iz grupe Alternaria toksina. Preliminarno ima indicija da se najvi&scaron;e, ekstremne koncentracije ispitivanih Alternaria toksina u p&scaron;enici iz pojedinih proizvodnih godina i iz najugroženijih regiona mogu povezati sa vi&scaron;im prosečnim dnevnim temperaturama sa jedne i povećanom sumom padavina u maju i junu sa druge strane u odnosu na dugogodi&scaron;nji prosek (1981-2010). Inokulacija p&scaron;enica sa Alternaria tenuissima dovodi po povećanja koncentracije TeA u zrnenoj masi u odnosu na netretiranu p&scaron;enicu, dok tretman iste p&scaron;enice fungicidom utiče na smanjenje prisutva ovog Alternaria toksina &scaron;to ukazuje da infekcija prodire u zrno u većem obimu kod inokulisanih uzoraka p&scaron;enice, dok je u netretiranim i za&scaron;tićenim manje izražena. Koncetracija Alternaria toksina u plevi p&scaron;enice je značajno vi&scaron;a u odnosu na zrnenu masu ovr&scaron;ene p&scaron;enice &scaron;to ukazuje na izvesnu za&scaron;titnu funkciju pleve od prodiranja Alternaria toksina u unutra&scaron;nji deo endosperma zrna p&scaron;enice. Krajnji cilj istraživanja je bio da se na osnovu dobijenih podataka o efikasnosti primenjenih tehnolo&scaron;kih postupaka u pogledu redukcije Alternaria toksina u p&scaron;enici i podataka u učestalosti i uzrocima pojave Alternaria toksina u p&scaron;enici da okvirna procena mogućnosti smanjenja rizika od pojave visokih udela Alternaria toksina u proizvodima prerade p&scaron;enice na trži&scaron;tu. U lancu proizvodnje, prometa, skladi&scaron;tenja i prerade p&scaron;enice kao proizvodi sa niskim rizikom u pogledu pojave i koncentracije Alternaria toksina mogu se istaći bela bra&scaron;na i hidrotermički obra&ntilde;eni proizvodi, kao &scaron;to su naprimer ekstrudirani proizvodi od p&scaron;enice. Kao proizvodi visokog rizika u pogledu pojave i koncentracije Alternaria toksina ističu se pleva dobijena tokom žetve p&scaron;enice, nečistoće dobijene u procesu silosnog postupka či&scaron;ćenja p&scaron;enice, tamne frakcije bra&scaron;na dobijene u postupku mlevenja p&scaron;enice i sporedni prozvodi mlevenja p&scaron;enice &ndash; mekinje i osevci.</p> / <p><span style="font-size:11px;">The need for more complete information about the importance of contamination of crops with Alternaria toxins recently gained in importance. The toxicity of metabolites produced by certain species from Alternaria genus in the whole supply chain resulted in the area of mycotoxicological research in increased interest for Alternaria toxins and fungi producing them. Having in mind that there are no available data about natural contamination of wheat with Alternaria toxins in the region of Serbia, and not any data is available in academic literature about the influence of processing procedures on Alternaria toxins in wheat, the aims of this PhD thesis have been defined. The basic aim of this dissertation was to investigate the influence of conventional mechanical (cleaning and dry milling) and hydrothermal (extrusion) technological processes on reduction of Alternaria toxins present in wheat. The application of wheat cleaning processes common in wheat warehouses and mills significant reduction of concentration of all investigated Alternaria toxins is achieved. In wheat milling process, even for wheat with concentration of Alternaria toxins under the limit of detection, in fraction containing peripheral kernel parts, including last break and milling flows and by products, increased concentration of Alternaria toxins can be expected. Optimal effects of extrusion process with application of single screw extruder are achieved in the case of high raw material moisture (w=24%), high capacity (q=25 kg/h) and medium speed of extruder screw rotation (v=390 rpm) when reduction of TeA=60-65%, AOH=90% and AME=95% is achieved. Additional aim was to obtain data about the frequency of Alternaria toxins including occurrence and ranges of concentration of Alternaria toxins in wheat for the region of Vojvodina with the aim to obtain preliminary data about the influence of production season, locality and the most characteristic climatic conditions on occurrence of Alternaria toxins in wheat, as well as to perceive the influence of introduced </span>stimulation and inhibition of wheat contamination with Alternaria spp. on concentration of Alternaria toxins in wheat. Analysis of frequency of occurrence of Alternaria toxins in wheat from the region of Vojvodina points out at evident incidence of significant number of wheat lots contaminated with TeA accompanied with sporadic occurrence of lots contaminated with AOH and AME. Both, average and maximal concentrations of TeA in wheat produced in Vojvodina in the period from 2011 to 2013 were above the official multiannual data published by European Food Safety Agency (EFSA). Concentrations of AME and AOH in wheat from Vojvodina were far under the concentrations of TeA as the most frequent contaminant from the group of Alternaria toxins. Preliminary there are indications that the highest concentrations of analysed Alternaria toxins in wheat from certain production seasons and the most affected production regions can be related with higher average daily temperatures at one, and increased sum of precipitation at the other hand in comparison to multiannual average (1981-2010). Inoculation of wheat with Alternaria tenuissima resulted in increased concentration of TeA in comparison to non-treated wheat, while the treatment of the same wheat with fungicide influences at decrease of the same Alternaria toxins, pointing out that the infection penetrates to higher extent into inoculated wheat while in non-treated and protected wheat this process is less expressed. Concentration of Alternaria toxins in wheat chaff is much higher in comparison to harvested wheat pointing out at certain protective function of chaff in the process of penetration of Alternaria toxins into inner kernel parts. Final aim of the research was to provide the general assessment of possibilities for reduction of risk of occurrence of high Alternaria toxins concentrations in wheat products at the market based on obtained data about efficiency of applied technological processes for reduction of Alternaria toxins in wheat products and obtained data on frequency of occurrence of Alternaria toxins. In the wheat supply chain white flours and hidrothermically processed like extruded wheat products can be emphasized as the products with low risk regarding Alternaria toxins. As the high risk products regarding Alternaria toxins chaff obtained during harvesting, impurities obtained in wheat cleaning processes, dark flours obtained in wheat milling process and wheat milling by-products, shorts and bran, can be emphasized.</p>

Call number: LD2668 .T4 1978 A43 / Master of Science

Submitted by PPG Biologia Celular e Molecular (bcm@pucrs.br) on 2018-03-06T18:10:51Z No. of bitstreams: 1 ANDRESSA_VON_WURMB_HELRIGUEL_LEITE_DIS.pdf: 1584575 bytes, checksum: 5f9a44ec42089e160df54cc84eaadb9a (MD5) / Approved for entry into archive by Caroline Xavier (caroline.xavier@pucrs.br) on 2018-03-07T14:04:49Z (GMT) No. of bitstreams: 1 ANDRESSA_VON_WURMB_HELRIGUEL_LEITE_DIS.pdf: 1584575 bytes, checksum: 5f9a44ec42089e160df54cc84eaadb9a (MD5) / Made available in DSpace on 2018-03-07T14:11:58Z (GMT). No. of bitstreams: 1 ANDRESSA_VON_WURMB_HELRIGUEL_LEITE_DIS.pdf: 1584575 bytes, checksum: 5f9a44ec42089e160df54cc84eaadb9a (MD5) Previous issue date: 2017-08-25 / Coordena??o de Aperfei?oamento de Pessoal de N?vel Superior - CAPES / Tomato (Solanum lycopersicum) cultures are frequently attacked by many pathogens, leading to yield losses. Among the pathogens, the fungus Alternaria solani (?Early blight? disease) can cause significant yield reductions and decreases the fruit quality. Due to the high susceptibility of tomato to fungal pathogens, the agricultural practices include the intensive use of agrochemicals, which may result in fruits with high levels of residual pesticides and higher production costs. The promotion of plant defense system represents an additional tool for disease control and plant growth management. Some rhizobacteria have been shown the ability to affect plant development and modulate plant defense against pathogens. Among those bacteria, the genus Streptomyces is able to synthesize molecules that directly interfere with growth, through auxin and ACC deaminase production, and indirectly, through antibiotics and siderophores synthesis. The aim of this work was to select Streptomyces (CLV04, CLV05 and CLV07) with the ability to promote seed germination and plant growth, as well as improve tomato plant resistance against A. solani. Results showed that CLV07 delayed the initial seed germination, but it did not impair the final germination process. However, CLV04, CLV05 and CLV07 reduced the early root and shoot growth. All these rhizobacteria produced high auxin levels. CLV07 was efficient for inducing plant resistance against A. solani. Furthermore, all CLVs showed antagonism by competition, decreasing the A. solani growth. Taking together all results, Streptomyces tested were deleterious for seed and initial plant growth, but CLV07 showed a potential to be used for inducing plant resistance. However, the inoculation of these rhizobacteria on seeds is not recommended. / O tomateiro (Solanum lycopersicum) ? constantemente atacado por diversos pat?genos, levando a perdas de produtividade. Dentre os principais pat?genos, destaca-se o fungo Alternaria solani que causa a doen?a ?pinta-preta? (?Early blight?), capaz de reduzir a produ??o e/ou levar ? forma??o de frutos de baixa qualidade. Devido ? grande susceptibilidade do tomateiro a pat?genos, as pr?ticas agr?colas incluem o uso intensivo de agroqu?micos que resultam, frequentemente, em tomates com altos n?veis residuais de pesticidas. A promo??o do crescimento e da defesa vegetal utilizando microrganismos representa uma ferramenta para o manejo do tomateiro. Dentre as diversas rizobact?rias com capacidade de modular o crescimento e a defesa vegetal, o g?nero Streptomyces vem se destacando. Estas bact?rias s?o capazes de sintetizar mol?culas que interferem tanto diretamente no crescimento, como a auxina e a ACC desaminase, quanto indiretamente, atrav?s da produ??o de antibi?ticos e sider?foros. Pretendeu-se, neste trabalho, selecionar isolados de Streptomyces (CLV04, CLV05 e CLV07) com capacidade de promover a germina??o e o crescimento de plantas de tomate, al?m de aumentar a resist?ncia vegetal contra A. solani. Os resultados demonstram que o CLV07 causou retardo na germina??o, mas n?o reduziu a taxa final de germina??o. Apesar das rizobact?rias CLV04, CLV05 e CLV07 produzirem elevados n?veis de auxina, elas causaram a redu??o do crescimento tanto da raiz quanto da parte a?rea de pl?ntulas de tomate cultivadas in vitro. O CLV07 foi eficiente na promo??o da defesa de plantas de tomate contra A. solani. Todas as rizobact?rias apresentaram antagonismo por competi??o, reduzindo o crescimento do mic?lio do fungo A. solani. Considerando todos os resultados, as diferentes bact?rias Streptomyces foram prejudiciais ? germina??o e ao crescimento inicial de pl?ntulas de tomate. Desta forma, n?o ? recomend?vel a utiliza??o destas bact?rias em sementes e pl?ntulas jovens. Contudo, o CLV07 apresenta potencial para ser utilizado para a indu??o de resist?ncia de plantas de tomate contra o pat?geno A. solani.

Doctor of Philosophy(PhD), / Airborne fungi are ubiquitous in the environment and human exposure is inevitable. Such fungi differ greatly in their taxonomic, physical, ecological and pathogenic characteristics. Currently, 69 000 species have been taxonomically classified and more than 80 of these are recognised to be aeroallergen sources. Many strategies have evolved to sample, identify and interpret fungal exposure to these species, however no strategy serves all purposes as exposure is a complex and dynamic process confounded by spatial, temporal and geographic variations in airborne counts, in addition to the inadequacies of the immunodiagnostic techniques available. To date, the interpretation of personal exposure and sensitisation to fungal allergens has been restricted to a few select species and the contribution of other genera, airborne hyphae and fragmented conidia to allergic disease are all poorly understood. The aim of the thesis was to utilize the Halogen Immunoassay (HIA) to diagnose fungal allergic sensitisation, to investigate the distribution and factors influencing allergens of fungi in the air and to understand what is actually inhaled in exposure settings. The novelty of the HIA derives from its unique ability to provide allergen sources that are actively secreted by the collected fungal spores and hyphae, which are bound to protein binding membranes (PBM) and then immunoprobed. In Chapter 2, the HIA was compared to the commercial in vitro Pharmacia UniCap assay (CAP) and the in vivo skin prick test (SPT), using 30 sera from subjects SPT positive to Aspergillus fumigatus and/or Alternaria alternata and 30 who were SPT negative to these fungi but sensitised to non-fungal allergens. Sera were analysed by CAP and the HIA against A. alternata, A. fumigatus, Cladosporium herbarum and Epicoccum purpurascens and compared statistically. Between 3% and 7% of SPT negative sera were identified to have specific IgE towards A. fumigatus and A. iv alternata, respectively. For the SPT positive sera, significant associations were found between the HIA and CAP scores for all fungal species tested (P<0.0001). Correlations between the HIA and SPT however, were weakly correlated for A. alternata (rs = 0.44, P<0.05) but not for A. fumigatus. In Chapter 3, personal exposure to indoor fungal aerosols was examined using the HIA to identify the fungal components that people were allergic to. Personal air sampling pumps (PASs) collected airborne fungal propagules onto PBMs for 2.5 hours indoors (n=21). Collected fungi were incubated overnight in a humid chamber to promote the germination of conidia. The membranes were then immunostained with pooled human Alternaria species-positive sera. All air samples contained fungal hyphae that expressed soluble allergens and were significantly higher in concentration than counts of conidia of individual well-characterised allergenic genera. Approximately 25% of all hyphae expressed detectable allergen compared to non-stained hyphae (P<0.05) and the resultant localisation of immunostaining was heterogeneous among hyphae. Fungal conidia of ten genera that were previously uncharacterised as allergen sources accounted for 8% of the total conidia that demonstrated IgE binding. In Chapter 4, the number and identity of fungi inhaled by 34 adults in an outdoor community setting was measured over 2 hour periods by people wearing Intra-nasal air samplers (INASs) and compared to fungal counts made with a Burkard spore trap and filter air samplers worn on the lapel. Using INAS, the most prevalent fungi inhaled belonged to soil borne spores of Alternaria, Arthrinium, Bipolaris, Cladosporium, Curvularia, Epicoccum, Exserohilum, Fusarium, Pithomyces, Spegazzinia, Tetraploa and Xylariaceae species, in addition to hyphal fragments. These results showed that inhaled exposure in most people varied in a 2-fold range with 10-fold outliers. In addition, the INAS and personal air filters agreed more with each other than with Burkard spore trap counts. The analysis was further confounded by different sampling efficiencies, locations of devices and ability to visualise and count fungal propagules. In Chapter 5, a double immunostaining technique based on the HIA was developed and applied to the conidia, hyphae and fungal fragments of A. alternata, A. fumigatus and Penicillium chrysogenum to discriminate between sources of allergens, v using IgE and to identify the fungi, using a fungal-specific antibody. The localisation of immunostaining was heterogeneous between both conidia and the state of germination with greater concentrations of double immunostaining detected following germination for each fungal species (P<0.0001). Fragmented A. alternata hyphae and morphologically indiscernible fragments could be identified for the first time using this technique. In Chapter 6, the factors affecting the release of allergen from the spores of eleven different species were studied. For nine of eleven species, between 5.7% and 92% of spores released allergen before germination. Ungerminated spores of P. chrysogenum and Trichoderma viride did not release detectable allergen. After germination, all spores that germinated eluted allergen from their hyphae. Upon germination there was a significant increase in the percentage of spores eluting detectable allergen (P<0.0001) and the localisation of allergen along the hyphae varied between species. Increased elution of allergen post germination might be a common feature of many species of allergenic fungi following inhalation. Additionally, Chapter 6 explored the extent to which inhaled spores or hyphae germinate after deposition in the nasal cavity and thus cause exposure to allergens. Twenty subjects had their noses lavaged at three separate intervals, (1) at the beginning of the experiment, (2) after one hour indoors and (3) after one hour outdoors. The recovery of spores and hyphal fragments from the nasal cavity varied between individuals and was significantly greater after outdoor exposures. Germinated fungal spores were recovered often in high concentrations for Aspergillus-Penicillium species, however the proportion between ungerminated and germinated spores were much lower for other genera recovered. Conclusions: Our analysis of cultured and wild-type fungi presents a new paradigm of natural fungal exposure, which in addition to commonly recognized species, implicates airborne hyphae, fragmented conidia and the conidia of a much more diverse range of genera as airborne allergens. Exposure is heterogeneous between individuals in the same geographic locality and the spectrum of fungal genera inhaled differs with the method of analysis. Many of the spores inhaled are likely to be allergenic, however upon germination there is an increased elution of allergen and this might be a common vi feature of many fungal species following inhalation. This project also provides novel techniques to diagnose fungal allergy by immunostaining wild-type fungi to which a patient is exposed with the patient’s own serum. Such an immunoassay combines environmental with serological monitoring on a patient specific basis and potentially avoids many problems associated with extract variability, based on the performance of current diagnostic techniques for fungal allergy.

Thesis (M.S.)--West Virginia University, 2002. / Title from document title page. Document formatted into pages; contains viii, 77 p. : ill. (some col.). Includes abstract. Includes bibliographical references (p. 71-77).

Red kidney bean seeds (Phaseolus vulgaris) contain a variety of bioactive proteins including lectins, enzyme inhibitors, hydrolytic enzymes and antifungal proteins. The aim of this research was to investigate activities of selected low pH and heat-stable bioactive proteins extracted from different parts of red kidney bean seed, seedling and pod as well as seed and root exudates. Crude red kidney bean seed extracts inhibited growth of Alternaria alternata as well as its protease activity, but not its amylase activity. A protein with inhibitory activity against growth of A. alternata was purified from extracts of the red kidney bean cotyledons and embryonic axis. This purified bean protein was devoid of chitinase and β-1, 3- glucanase activities. Also, it did not inhibit porcine pancreatic α-amylase, bovine trypsin, amylase and protease of A. alternata suggesting that the antifungal activity of the protein is not related to these activities. Proteinaceous extracts of red kidney bean cotyledons induced melanin and conidia formation in mycelium of A. alternata. A protein responsible for this conidiation inducing effect was shown for the first time to be a mannose-binding lectin which is also known as PvFRIL (Phaseolus vulgaris fetal liver tyrosine kinase 3-receptor interacting lectin). An unexpected finding was that extracts of the embryonic axis stimulated rather than inhibited porcine α-amylase activity. Phytohemagglutinin (PHA-L in particular), co-extracted with α-amylase inhibitor from red kidney bean seeds, was implicated as an α-amylase stimulator with the potential of greatly assisting digestion of starch. In cotyledonary extracts, amylase stimulatory activity was masked by amylase inhibitory activity that was inactivated when the extracts were boiled for 10 min. An in-gel non-denaturing electrophoretic method was used to show presence of porcine α-amylase isoinhibitors in extracts of the cotyledons and embryonic axis. All other seedling parts as well as seed and root exudates had amylase stimulatory activity. Another improved non-denaturing electrophoretic method with immobilized azoalbumin was developed for in-gel detection of isoinhibitors of bovine trypsin in seed parts. It eliminates the need for both time-consuming and labourious staining, destaining or renaturation steps used in other methods. Accumulation of most of the selected bioactive proteins during seed development in different seed parts appeared to start at 20 days after flower abscission. The activities of these proteins decreased to lower levels after 11 days of germination. Besides these observed developmental changes, under abiotic (UV-C irradiation) and biotic (seedlings co-cultured with A. alternata) stress, increased activity of some of the selected bioactive proteins were detected. In conclusion, this study has contributed to a better understanding of antifungal activity and the selected bioactive proteins in extracts of red kidney bean.

Doctor of Philosophy(PhD), / Airborne fungi are ubiquitous in the environment and human exposure is inevitable. Such fungi differ greatly in their taxonomic, physical, ecological and pathogenic characteristics. Currently, 69 000 species have been taxonomically classified and more than 80 of these are recognised to be aeroallergen sources. Many strategies have evolved to sample, identify and interpret fungal exposure to these species, however no strategy serves all purposes as exposure is a complex and dynamic process confounded by spatial, temporal and geographic variations in airborne counts, in addition to the inadequacies of the immunodiagnostic techniques available. To date, the interpretation of personal exposure and sensitisation to fungal allergens has been restricted to a few select species and the contribution of other genera, airborne hyphae and fragmented conidia to allergic disease are all poorly understood. The aim of the thesis was to utilize the Halogen Immunoassay (HIA) to diagnose fungal allergic sensitisation, to investigate the distribution and factors influencing allergens of fungi in the air and to understand what is actually inhaled in exposure settings. The novelty of the HIA derives from its unique ability to provide allergen sources that are actively secreted by the collected fungal spores and hyphae, which are bound to protein binding membranes (PBM) and then immunoprobed. In Chapter 2, the HIA was compared to the commercial in vitro Pharmacia UniCap assay (CAP) and the in vivo skin prick test (SPT), using 30 sera from subjects SPT positive to Aspergillus fumigatus and/or Alternaria alternata and 30 who were SPT negative to these fungi but sensitised to non-fungal allergens. Sera were analysed by CAP and the HIA against A. alternata, A. fumigatus, Cladosporium herbarum and Epicoccum purpurascens and compared statistically. Between 3% and 7% of SPT negative sera were identified to have specific IgE towards A. fumigatus and A. iv alternata, respectively. For the SPT positive sera, significant associations were found between the HIA and CAP scores for all fungal species tested (P<0.0001). Correlations between the HIA and SPT however, were weakly correlated for A. alternata (rs = 0.44, P<0.05) but not for A. fumigatus. In Chapter 3, personal exposure to indoor fungal aerosols was examined using the HIA to identify the fungal components that people were allergic to. Personal air sampling pumps (PASs) collected airborne fungal propagules onto PBMs for 2.5 hours indoors (n=21). Collected fungi were incubated overnight in a humid chamber to promote the germination of conidia. The membranes were then immunostained with pooled human Alternaria species-positive sera. All air samples contained fungal hyphae that expressed soluble allergens and were significantly higher in concentration than counts of conidia of individual well-characterised allergenic genera. Approximately 25% of all hyphae expressed detectable allergen compared to non-stained hyphae (P<0.05) and the resultant localisation of immunostaining was heterogeneous among hyphae. Fungal conidia of ten genera that were previously uncharacterised as allergen sources accounted for 8% of the total conidia that demonstrated IgE binding. In Chapter 4, the number and identity of fungi inhaled by 34 adults in an outdoor community setting was measured over 2 hour periods by people wearing Intra-nasal air samplers (INASs) and compared to fungal counts made with a Burkard spore trap and filter air samplers worn on the lapel. Using INAS, the most prevalent fungi inhaled belonged to soil borne spores of Alternaria, Arthrinium, Bipolaris, Cladosporium, Curvularia, Epicoccum, Exserohilum, Fusarium, Pithomyces, Spegazzinia, Tetraploa and Xylariaceae species, in addition to hyphal fragments. These results showed that inhaled exposure in most people varied in a 2-fold range with 10-fold outliers. In addition, the INAS and personal air filters agreed more with each other than with Burkard spore trap counts. The analysis was further confounded by different sampling efficiencies, locations of devices and ability to visualise and count fungal propagules. In Chapter 5, a double immunostaining technique based on the HIA was developed and applied to the conidia, hyphae and fungal fragments of A. alternata, A. fumigatus and Penicillium chrysogenum to discriminate between sources of allergens, v using IgE and to identify the fungi, using a fungal-specific antibody. The localisation of immunostaining was heterogeneous between both conidia and the state of germination with greater concentrations of double immunostaining detected following germination for each fungal species (P<0.0001). Fragmented A. alternata hyphae and morphologically indiscernible fragments could be identified for the first time using this technique. In Chapter 6, the factors affecting the release of allergen from the spores of eleven different species were studied. For nine of eleven species, between 5.7% and 92% of spores released allergen before germination. Ungerminated spores of P. chrysogenum and Trichoderma viride did not release detectable allergen. After germination, all spores that germinated eluted allergen from their hyphae. Upon germination there was a significant increase in the percentage of spores eluting detectable allergen (P<0.0001) and the localisation of allergen along the hyphae varied between species. Increased elution of allergen post germination might be a common feature of many species of allergenic fungi following inhalation. Additionally, Chapter 6 explored the extent to which inhaled spores or hyphae germinate after deposition in the nasal cavity and thus cause exposure to allergens. Twenty subjects had their noses lavaged at three separate intervals, (1) at the beginning of the experiment, (2) after one hour indoors and (3) after one hour outdoors. The recovery of spores and hyphal fragments from the nasal cavity varied between individuals and was significantly greater after outdoor exposures. Germinated fungal spores were recovered often in high concentrations for Aspergillus-Penicillium species, however the proportion between ungerminated and germinated spores were much lower for other genera recovered. Conclusions: Our analysis of cultured and wild-type fungi presents a new paradigm of natural fungal exposure, which in addition to commonly recognized species, implicates airborne hyphae, fragmented conidia and the conidia of a much more diverse range of genera as airborne allergens. Exposure is heterogeneous between individuals in the same geographic locality and the spectrum of fungal genera inhaled differs with the method of analysis. Many of the spores inhaled are likely to be allergenic, however upon germination there is an increased elution of allergen and this might be a common vi feature of many fungal species following inhalation. This project also provides novel techniques to diagnose fungal allergy by immunostaining wild-type fungi to which a patient is exposed with the patient’s own serum. Such an immunoassay combines environmental with serological monitoring on a patient specific basis and potentially avoids many problems associated with extract variability, based on the performance of current diagnostic techniques for fungal allergy.

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