FundaÃÃo Cearense de Apoio ao Desenvolvimento Cientifico e TecnolÃgico / O cajueiro tem uma grande importÃncia sÃcio-econÃmica e cultural para a regiÃo Nordeste, por gerar emprego para os agricultores, sendo por muitas vezes a sua Ãnica fonte de renda. Assim, as pesquisas com esta fruteira tÃm aumentado bastante nos Ãltimos anos, visando primariamente, reduzir os danos fitossanitÃrios e elevar a produtividade. As doenÃas, como nas demais espÃcies cultivadas, causam significativos danos. Dentre as doenÃas do cajueiro, a resinose e a podridÃo preta das hastes, ambas causadas pelo fungo Lasiodiplodia theobromae, se destacando entre as principais doenÃas em algumas regiÃes do semi-Ãrido nordestino. PorÃm, ainda hà poucos estudos sobre a biologia, morfologia, caracterizaÃÃo genÃtica desse patÃgeno, fato que dificulta o seu manejo. Este estudo objetivou caracterizar a diversidade genÃtica e patogÃnica de uma populaÃÃo com 22 isolados de L. theobromae associada ao cajueiro, utilizando marcadores microssatÃlites e inoculaÃÃes em plantas de cajueiro. Para a caracterizaÃÃo molecular, extraiu-se o DNA de cada isolado a partir do micÃlio do fungo crescido em meio lÃquido. Cada amostra foi submetida à reaÃÃo de polimerase em cadeia (PCR) com 15 pares de iniciadores desenvolvidos para flanquear regiÃes especificas contendo sequÃncias simples repetidas (microssatÃlites) dessa espÃcie. Os produtos amplificados foram observados em gel de eletroforese corados com brometo de etÃdio, e os dados tabulados em planilha binÃria e analisados pelo mÃtodo de agrupamento nÃo balanceado baseado na mÃdia aritmÃtica (UPGMA), utilizando o programa MVSP. As similaridades genÃticas foram estimadas pelo coeficiente de Nei e Li. Os resultados indicaram uma grande variabilidade genÃtica da populaÃÃo avaliada, representada por diversos grupos de similaridade variando de 22,2% a 100%. TrÃs destes isolados apresentaram entre si, 100% de similaridade genÃtica com os marcadores testados. No entanto, eram morfologicamente diferentes e um deles era procedente de localidade diferente que os demais. Os dados mostraram que a similaridade genÃtica por si sà nÃo deve ser aplicado como Ãnico fator de diversidade entre isolados. Para se testar a patogenicidade, os isolados foram inoculados em mudas de cajueiro, analisando-se o tamanho da lesÃo e a taxa de crescimento do fungo. Os resultados mostraram nÃo haver diferenÃas estatÃsticas entre os isolados, fato que demonstra que, independente do local de origem, todos os isolados mostraram-se patogÃnicos, e a sua taxa de crescimento decresce, isto Ã, a planta deve produzir algum tipo de defesa contra o patÃgeno, o que dificulta a sua infecÃÃo. / The cashew has a major socio-economic and cultural development for the Northeast region, by generating employment for farmers and is frequently their only source of income. Thus, research on this fruit has increased greatly in recent years, aimed primarily to reduce damage and increase plant productivity. Diseases, as in other crops, cause significant damage. Among cashew diseases, gummosis and black stem rot, both caused by the fungus Lasiodiplodia theobromae, stand out among the major diseases in some regions of the semi-arid. However, there are few studies on the biology, morphology, genetic characterization of this pathogen, a fact that burden their management. This study aimed to characterize the genetic and pathogenic diversity of a population of 22 isolates of L. theobromae associated with cashew, using microsatellite markers and inoculations in cashew plants. For molecular characterization, DNA was extracted from mycelium of each isolated of the fungus grown in liquid medium. Each DNA sample was subjected to a polymerase chain reaction (PCR) with 15 pairs of primers designed to flank specific regions containing simple repeated sequences (microsatellites) in this species. The amplified products were observed in gel electrophoresis stained with ethidium bromide, and the data recorded on a binary and analyzed by grouping unbalanced based on arithmetic mean (UPGMA) using the program MVSP. Genetic similarities were estimated by the coefficient of Nei and Li The results indicated a large genetic variability of the population, represented by different groups of similarity ranging from 22.2% to 100%. Three of these isolates showed 100% genetic similarity with the markers tested. However, they were morphologically differences and one of them was originated from different location than the others. The data showed that the genetic similarity alone should not be applied as the only factor of diversity among isolates. To test the pathogenicity, each isolate was inoculated on cashew seedlings. Lesion development size and recovery of the fungus were recorded. The results showed no statistical differences between the isolates, which demonstrates that, regardless of place of origin, all isolates were pathogenic, and its growth rate decreases, i. e, the plant must produce some form of defense against pathogen, which hinders their infection.

Made available in DSpace on 2014-07-29T16:11:53Z (GMT). No. of bitstreams: 1 DISSERTACAO DEFINITIVA c ficha catalografica.pdf: 2149699 bytes, checksum: ebcbff0f45d07e12d12a9708947a59eb (MD5) Previous issue date: 2009-07-15 / Tyrosinase (E.C.1.14.18.1) is an enzyme of industrial interest that catalyses the ohydroxylation of monophenols (monophenolase activity) and the oxidation of o-diphenols to reactive o-quinones (diphenolase activity), both reactions using molecular oxygen. Pycnoporus sanguineus (L. ex Fries) Murril, is a white rot fungi capable of producing tyrosinase and widely distributed in nature. It is found in regions of mild climate and in tropical forest. The production and characterization of tyrosinase from P. sanguineus were investigated. The selection of inductors, determination of the luminosity influence, biomass and culture media in the production of tyrosinase and the effect of inhibitors on enzyme activity were determined. The fungus produced intracellular tyrosinase and the higher activity was observed using 0.15% L-tyrosine as inducer, in the presence of light, with inoculum of 10 mycelium discs, medium malt extract broth 2%, incubation at 30°C, and constant agitation of 150 rpm, during 2 days. 6 mmol.L-1 salicylhydroxamic acid (SHAM) and 6 mmol.L-1 phenylthiourea (PTU) inhibited 100% of the tyrosinase activity. 0.1 mmol.L-1 sodium azide inhibited 4.15% of tyrosinase activity, while no inhibition was observed after addition of 0.1 mmol.L-1 of phenylmethanesulfonyl fluoride (PMSF). Using L-dopa as substrate, the intracellular crude extract presented optimum pH of 6,6, optimum temperature of 45°C, low stability at 50°C, maintaining about 50% of the activity after 15 min of incubation. The tyrosinase production was confirmed by non-denaturing polyacrylamide gel electrophoresis, using commercial fungal tyrosinase as positive control / A tirosinase (E.C.1.14.18.1), também conhecida como polifenoloxidase ou catecolase, é uma enzima de interesse industrial que catalisa a o-hidroxilação de monofenóis (atividade monofenolase) e a subseqüente oxidação do o-difenol resultante em o-quinonas reativas (atividade difenolase), usando oxigênio molecular. O Pycnoporus sanguineus (L. ex Fries) Murril, um fungo de decomposição branca (white rot) capaz de produzir a tirosinase, é amplamente distribuído na natureza, sendo encontrado em regiões de clima mais ameno e em florestas tropicais. A produção e caracterização da tirosinase produzida por P. sanguineus foi investigada. Foi realizada seleção de indutores, determinação da influência da luminosidade, biomassa e meios de cultura na produção de tirosinase, bem como o efeito de inibidores sobre a atividade enzimática. O extrato bruto enzimático foi caracterizado quanto ao pH ótimo, temperatura ótima e termoestabilidade. Os resultados obtidos demonstraram que o fungo produziu tirosinase intracelular e a mais alta atividade ocorreu com a utilização de 0,15% de L-tirosina como indutor, na presença de luz, com inóculo de 10 discos de micélio fúngico, meio caldo extrato de malte 2%, incubação à 30°C e agitação de 150 rpm, durante 2 dias. Ácido salicilhidroxâmico (SHAM) 6 mmol.L-1 e feniltiouréia (PTU) 6 mmol.L-1 inibiram 100% da atividade de tirosinase. Azida sódica 0,1 mmol.L-1 inibiu 4,15% da atividade de tirosinase, enquanto nenhuma inibição foi observada após adição de fluoreto de fenilmetanosulfonil (PMSF) 0,1 mmol.L-1. Utilizando a L-dopa como substrato, o extrato bruto intracelular apresentou pH ótimo de 6,6, temperatura ótima de 45°C, baixa estabilidade à temperatura de 50°C, mantendo apenas cerca de 50% de atividade após 15 minutos de incubação. A produção da tirosinase foi comprovada através de eletroforese em gel de poliacrilamida não desnaturante, tendo como controle positivo tirosinase fúngica comercial

Este trabalho objetivou caracterizar os danos pós-colheita em frutos de laranja &#39;Valência&#39; e de tangor &#39;Murcott&#39;, destinados à exportação, e em frutos de laranjas &#39;Pêra&#39;, &#39;Lima&#39; e &#39;Natal&#39; e de tangor &#39;Murcott&#39;, destinados ao mercado interno, após diferentes etapas do beneficiamento em packinghouse, em 2004 e 2005 e na Ceagesp-SP em 2006; identificar os pontos críticos e a magnitude de impacto em linhas de beneficiamento de citros dos packinghouses; e caracterizar a micoflora ambiental nos packinghouses e nos pontos de revenda de atacadistas da Ceagesp, assim como a micoflora da superfície de equipamentos e instalações e a presença de isolados de Penicillium spp. resistentes a fungicidas nos packinghouses. Frutos cítricos foram coletados na chegada ao packinghouse, após a pré-lavagem, após o desverdecimento, na banca, no palete e na Ceagesp, e armazenados durante 14 a 21 dias a 25&#176;C e 85-90% de UR. A incidência de injúrias foi avaliada visualmente a cada três dias. Para a avaliação da magnitude de impactos nos pontos de transferência da linha de beneficiamento empregou-se uma esfera instrumentada com registrador de aceleração (G). A micoflora ambiental foi amostrada mediante o método gravimétrico, com placas de Petri, contendo meio batata-dextrose-ágar, abertas por dois minutos. A micoflora de superfície de equipamentos e instalações foi amostrada mediante placas Rodac. Observou-se baixa incidência (<3,5%) de podridões nos frutos destinados à exportação. As principais podridões encontradas em laranja &#39;Valência&#39; e tangor &#39;Murcott&#39; foram a podridão peduncular de Lasiodiplodia e a antracnose, respectivamente. Nos frutos para o mercado interno a incidência de podridões atingiu valores médios entre 12,8% e 36,2%, ao final do armazenamento, sendo o bolor verde a principal doença pós-colheita nos diferentes frutos cítricos. As injúrias mecânicas de oleocelose foram crescentes com o beneficiamento até a banca de embalagem e a aplicação de cera reduziu a incidência de frutos murchos. Na avaliação dos impactos na linha de beneficiamento do packinghouse de exportação, 95% dos impactos variaram entre 30-95 G. Já no packinghouse de mercado interno, 94% dos impactos variaram entre 24-131 G. A população fúngica no ambiente e na superfície do packinghouse de exportação foi de 12,3 e 52,3 ufc/placa, respectivamente, enquanto a população fúngica no packinghouse de mercado interno foi de 46,3 e 68,2 ufc/placa, respectivamente. Posição intermediária aos packinghouses foi observada na Ceagesp, com uma população fúngica ambiental de 25,3 ufc/placa. Cladosporium e Penicillium foram os gêneros fúngicos mais abundantes. A contaminação de &#34;zonas limpas&#34; nos packinghouses (lavagem dos frutos, banca, caixa e contêiner) não foi substancialmente menor que nas &#34;zonas sujas&#34; (recepção dos frutos e primeira seleção). A porcentagem de isolados de P. digitatum resistentes a tiabendazol foi de 25,9 no ambiente e de 30,1 na superfície do packinghouse de exportação, enquanto que para imazalil foi de 1,5 no ambiente e de 16,0 na superfície. No packinghouse de mercado interno, a porcentagem de isolados de P. digitatum resistentes a tiabendazol foi de 51,9 no ambiente e de 39,2 na superfície, enquanto que para imazalil foi de 0,1 e 0,9, respectivamente. / The purposes of this work were a) to characterize postharvest injuries in &#39;Valência&#39; oranges and &#39;Murcott&#39; tangors aimed at the external market, and in &#39;Pêra&#39;, &#39;Lima&#39;, &#39;Natal&#39; oranges and &#39;Murcott&#39; tangors aimed at the internal market after different processing stages in a packinghouse in 2004 and 2005, and at Ceagesp-SP in 2006; b) to identify critical points and impact extent on processing lines in packinghouses; c) to characterize the environmental mycoflora in packinghouses and in retail points at Ceagesp; d) to characterize the superficial mycoflora of equipment and facilities, and e) to characterize the presence of isolated Penicillium spp. resistant to fungicides in packinghouses. Fruits were collected at the packinghouse, at their arrival, after pre-washing and degreening, from the packing table, from the pallet and at Ceagesp. They were stored for 14 to 21 days at 25&#176;C and 85-90% RH. The incidence of injuries was visually evaluated every three days. An instrumented sphere with acceleration register (G) was used to evaluate the extent of impacts at the transference points of the citrus processing line. The environmental mycoflora was sampled according to the gravimetric method, using Petri dishes containing potato-dextrose-agar medium opened for two minutes. The superficial mycoflora on equipment and facilities was sampled with Rodac plates. There was low rot incidence (under 3.5%) in fruits aimed at the external market. The main disease affecting &#39;Valência&#39; oranges and &#39;Murcott&#39; tangors were Lasiodiplodia stem-end rot and anthracnose, respectively. Rots averaged between 12.8% and 36.2% at the end of the storage period in fruits aimed at the internal market, and green mold was the main postharvest disease. Oleocellosis increased along the processing stages until the arrival of fruits at the packing table. Wax application reduced the incidence of wilted fruits. Ninety-five percent of the impacts in the packinghouse processing line in fruits aimed at the external market ranged between 30 and 95 G, while 94% of the impacts in fruits aimed at the internal market ranged between 24 and 131 G. The environmental and the packinghouse superficial fungal population in fruits aimed at the external market were 12.3 and 52.3 cfu/plate, respectively, while these populations in fruits aimed at the internal market were 46.3 and 68.2 cfu/plate, respectively. Intermediate values in relation to packinghouses were observed at Ceagesp, where the environmental fungal population was 25.3 cfu/plate. Cladosporium and Penicillium were the most prevailing genera of fungi. The contamination of &#34;clean zones&#34; in the packinghouses (washing of fruits, packing table, boxes and containers) was not substantially lower than contamination in &#34;dirty zones&#34; (reception of fruits and first selection). The percentage of P. digitatum isolates resistant to thiabendazole was 25.9 in the environment and 30.1 on the packinghouse surface for fruits aimed at the external market, while the corresponding data concerning isolates resistant to imazalil were 1.5 and 16.0, respectively. In the packinghouse of fruits aimed at the internal market, the percentage of isolates of P. digitatum resistant to thiabendazole was 51.9 in the environment and 39.2 on the packinghouse surface, while the corresponding data concerning isolates resistant to imazalil were 0.1 and 0.9, respectively.

O gênero Fusarium é um grupo de fungos de importância mundial, não só por causar patologias em plantas, mas também porque abriga espécies toxigênicas. Dentre estas, Fusarium verticillioides, Fusarium proliferatum e Fusarium subglutinans, cujos teleomorfos se agrupam no complexo Gibberella fujikuroi, estão associadas a patologias em milho. O presente estudo caracterizou uma coleção de 100 isolados de Fusarium spp. tanto do ponto de vista morfológico como molecular com vistas a identificá-los em nível de espécie. Para isto, foram usados como marcadores morfológicos a presença/ausência de clamidósporos, o tipo de célula conidiogênica e a disposição dos microconídios sobre a célula conidiogênica. Os isolados foram também identificados em espécie baseado em informações disponíveis na literatura acerca de marcadores moleculares desenvolvidos a partir da reação de PCR e primers espécie-específicos. A ausência de clamidósporo permitiu alocar a totalidade dos isolados no complexo G. fujikuroi. Os demais critérios morfológicos permitiram identificar 77 isolados como F. verticillioides, 20 como F. proliferatum, 2 como F. subglutinans. Apenas 1 isolado não foi possível identificar em espécie. Análises moleculares concordaram em 100% dos casos em que os isolados foram identificados como F. verticillioides e F. subglutinans. Porem, no caso dos 20 isolados identificados morfologicamente como F. proliferatum apenas 4 foram confirmados na análise molecular; os demais foram identificados como F. verticillioides. A diversidade genética estudada por AFLP ratificou a separação das espécies F. verticillioides e F. proliferatum, com um índice de similaridade de 0,40. Marcadores AFLP também evidenciaram alta diversidade genética de F. verticillioides. Todos os isolados causaram podridão do colmo em três híbridos comerciais de milho e não variaram em agressividade, independente do nível de resistência dos híbridos. / The genus Fusarium is an important group of fungi worldwide, not only for its capability to cause disease but because it also contains species which produce toxins. Among these, Fusarium verticillioides, Fusarium proliferatum and Fusarium subglutinans, which teleomorphs are grouped in the Gibberella fujikuroi group are associated with diseases in maize. This study characterized a collection of 100 isolates of Fusarium spp. under morphological and molecular criteria to identify them at the species level. For this purpose, morphological markers such as presence/absence of chlamydospores, conidiogenou cell type and microconidia arrangement on conidiogenou cell were assessed. The isolates were also identified at the species level based upon available information about molecular markers developed from the PCR reaction using speciesspecific primers. The absence of chlamydospores in all of the isolates placed them within the G. fujikuroi complex, and based on the other morphological criteria, 77 isolates were identified as F. verticillioides, 20 as F. proliferatum, 2 as F. subglutinans and one isolate remained unidentified at the species level. The molecular analyses agreed with the morphological identification of all F. verticillioides and F. subglutinans. However, in the case of 20 isolates identified morphologically as F. proliferatum only 4 were confirmed, the rest being identified as F. verticillioides. The genetic diversity based on AFLP confirmed the separation of F. verticillioides and F. proliferatum in two groups, with a similarity index of 0.40. AFLP markers also showed high genetic diversity within F. verticillioides. All isolates were caused stalk rot on three commercial hybrids and did not vary in aggressiveness regardless of the resistance level of the hybrids.

Esse trabalho teve dois objetivos distintos, quantificar e caracterizar os danos pós-colheita em frutos de ameixa e nectarina na Companhia de Entrepostos e Armazéns Gerais de São Paulo (CEAGESP) e testar produtos que possam ser utilizados no controle das doenças pós-colheita podridão parda (Monilinia fructicola) e podridão mole (Rhizopus stolonifer) nestes dois frutos. Durante a safra 2003 e 2004, foram realizados 20 levantamentos de quantificação de danos em frutos de nectarina e 11 em frutos de ameixa. Em cada levantamento foram avaliados 1% do total de caixas comercializados em cinco permissionários da CEAGESP. A amostragem foi estratificada por variedade, calibre e procedência dos frutos, sendo quantificados danos mecânicos, fisiológicos e doenças pós-colheita. Para o controle da podridão parda e mole, o efeito curativo e protetor do CaCl2, cera e luz UV-C, em diferentes concentrações e o efeito curativo do tratamento com ozônio foram avaliados. A incidência de danos na CEAGESP em frutos de ameixa variou de 5 a 47%, e em nectarina entre 14 e 54%, considerando as safras 2003 e 2004. Tanto em ameixa como em nectarina os danos mais freqüentes foram os mecânicos, com variação de 2 a 25% em frutos de ameixa e de 7 a 30% em frutos de nectarina. Danos fisiológicos variaram de 1 a 22% e de 2 a 18% em frutos de ameixa e nectarina, respectivamente. Danos provocados por doenças variaram de 0 a 13% em frutos de ameixa e de 1 a 9% em frutos de nectarina. Entre os patógenos mais constatados figuraram fungos do gênero Rhizopus, Monilinia, Geotrichum, Cladosporium, Fusarium e Alternaria, além de bactérias. Correlação entre a incidência de frutos com dano mecânico e a incidência de frutos doentes foi constatada na safra 2004, nos dois frutos. Em ameixa, a variedade Gulfblazer apresentou maior incidência a danos mecânicos e fisiológicos. Não foi observada diferença na incidência de danos mecânicos e de doenças, entre as variedades de nectarina avaliadas na safra 2003. Na safra 2004, a incidência de danos mecânicos e fisiológicos foi superior na variedade Sunraycer comparada às demais variedades. Em relação aos produtos testados, no geral, CaCl2 a 1%, mostrou potencial de controle das duas doenças nos dois frutos, aplicado como protetor ou curativo. A cera ECF 124 a base de carnaúba foi o produto mais eficiente, com redução de até 70% das duas doenças em ambos os frutos, principalmente quando aplicada de maneira protetora, a 9%. As doses (1,4 e 5,2 kJm-2) de UV-C testadas não controlaram as doenças com resultados praticamente iguais à testemunha (dose 0,0 kJm-2). A presença de ozônio não reduziu nenhuma das doenças em nenhum dos dois frutos avaliados. / This study had two distinct objectives, to quantify and characterize the postharvest damages in plums and nectarines in the wholesale market of São Paulo (CEAGESP), and to test products that can be used in the control of the postharvest diseases such as the brown rot (Monilinia fructicola) and soft rot (Rhizopus stolonifer) in these two fruits species. Twenty assessments in nectarine and eleven in plums were carried out in 2003 and 2004. In each year 1% of the total of boxes commercialized in five outlets of CEAGESP was assessed, considering mechanical and physiological damages and postharvest diseases. The selection of samples was conducted through stratified sampling, taking fruit cultivar, place of origin and fruit size as the stratification criteria. Curative and protective control measures of brown and soft rot were analyzed testing CaCl2, wax and UV-C light, in different concentrations. The ozone was studied only as curative treatment. The incidence of damages in plums at CEAGESP ranged from 5 to 47%, and in nectarines from 14 to 54%. Either in plums or in nectarines, postharvest mechanical injuries were the most frequent damages, varying from 2 to 25% in plums and from 7 to 30% in nectarines. Physiological damages ranged from 1 to 22% and from 2 to 18% in plums and nectarines, respectively. Postharvest diseases ranged from 0 to 13% in plums and from 1 to 9% in nectarines. The most frequent postarvest pathogens were Rhizopus, Monilinia, Geotrichum, Cladosporium, Fusarium and Alternaria, besides bacteria. A correlation between the incidence of mechanical damages and the incidence of postharvest diseases in 2004 in both fruits was noticed. Cultivar Gulfblazer (plum) showed more incidence of mechanical and physiological damages than Reubennel. No differences were observed in the incidence of mechanical and physiological damages among the cultivars of nectarines assessed in 2003. No difference in the susceptibility to postharvest diseases among the nectarine cultivars Sunred, Sunripe, Sunraycer and Colombina in 2003 was verified. In 2004 cv. Sunraycer showed incidence of mechanical damages significantly higher than the other varieties. Regarding the products tested, as a whole, CaCl2 at 1% showed potential of controlling the two diseases in both kind of fruits, applied as a protective or curative measure. The carnauba wax (ECF 124) was the most efficient product, with a reduction of up to 70% of both diseases in both kind of fruits, mainly when applied as a protective agent, at 9%. The doses (1,4 e 5,2 kJm-2) of UV-C tested did not control the diseases with results practically similar to the reference dose (0,0 kJm-2). The presence of ozone did not reduce either of the diseases in any of the fruits evaluated.

Colletorichum acutatum é o agente causal da Podridão Floral dos Citros (PFC), doença que em determinadas condições ambientais constitui-se em fator limitante à produção citrícola em várias regiões produtoras do mundo. Além da PFC, esse fungo causa antracnose em outros hospedeiros, sendo um dos patógenos que mais acarreta danos em frutíferas tropicais, subtropicais e temperadas no mundo. O trabalho teve como objetivos estudar a especificidade patogênica e compatibilidade vegetativa entre isolados de C. acutatum dos citros e de outros hospedeiros como: goiaba, pimentão, morango e pêssego. Para os estudos de especificidade patogênica, foram realizadas inoculações cruzadas entre isolados de citros e dos outros hospedeiros visando verificar se os diferentes isolados são capazes de causar sintomas de PFC em flores de citros e antracnose em frutos. Foram também obtidos, a partir dos mesmos isolados, mutantes deficientes na absorção de nitrogênio (mutantes nit). Esses foram caracterizados fenotipicamente e pareados a fim de verificar por meio de estudos de compatibilidade vegetativa a capacidade de recombinação entre si, gerando heterocários com patogenicidade alterada. Para verificar a ocorrência de possíveis alterações na patogenicidade dos heterocários formados, foi feita a inoculação dos heterocários e dos isolados parentais nos seus respectivos hospedeiros de origem. Nos ensaios de inoculação cruzada, houve grande variação quanto à patogenicidade dos isolados inoculados. Isolados provenientes de citros e de goiaba causaram lesões em flores de citros, isso demonstra a ausência de especificidade entre isolados dos dois hospedeiros. Porém, isolados provenientes de pimentão, pêssego e morango não foram capazes de causar sintomas em flores de citros o que indica a existência de especificidade desses isolados. Os isolados provenientes dos citros e de outros hospedeiros foram capazes de causar antracnose em goiaba, morango e pêssego, mas apenas os isolados de pimentão causaram antracnose em pimentão. Alguns isolados de citros foram capazes de recombinar entre si e com isolados de goiaba, pimentão e morango. Dos heterocários formados, dois foram caracterizados quanto a sua patogenicidade, Het 3 e Het 5. Como resultado, o heterocário proveniente do isolado de citros com goiaba (Het 5) comportou-se de forma semelhante à um de seus parentais. O heterocário proveniente de citros com pimentão (Het 3), mostrou-se mais agressivo do que seus parentais quando inoculados em pimentão. Com esses estudos pode-se concluir que existe especificidade patogênica entre isolados de C. acutatum de diferentes hospedeiros, entretanto, isolados de diferentes hospedeiros podem recombinar entre si e gerar heterocários com características patogênicas alteradas. / Colletorichum acutatum is the causal agent of postbloom fruit drop (PFD).This disease is a limiting factor for citrus production under specifics environmental conditions in several regions of the world. In addition to the PFD, this fungus causes anthracnose on other hosts. It is one of the pathogens that cause more damage in tropical, subtropical and temperate fruit around the world. This work aimed to study the specificity pathogenic and vegetative compatibility among isolates of C. acutatum from citrus and other hosts: guava, pepper, strawberry and peach. For studies of pathogenic specificity, cross inoculations were performed among isolates from citrus and other hosts in order to verify whether different strains are capable of causing symptoms of PFD in citrus flowers and fruit anthracnose. Furthermore, it was obtained from the same isolates, nitrate-nonutilizing mutants (nit mutants). They were phenotypically characterized and paired to verify, by means of vegetative compatibility studies, the ability of recombination between them, generating heterokaryons with altered pathogenicity. In order to verify the occurrence of possible changes in the pathogenicity of the heterokaryons formed, parental isolates and heterokaryons were inoculated in their respective original hosts. In cross-inoculation tests, there was a great variation in the isolates pathogenicity. Isolates from citrus and guava caused lesions on citrus blossoms; this demonstrates the absence of pathogenic specificity between isolates of the two hosts. However, isolates from pepper, peach and strawberry were unable to induce symptoms on citrus flowers showing the existence of specificity of these isolates. The strains from citrus and other hosts were able to cause anthracnose on guava, strawberry and peach, but only isolates of pepper caused anthracnose on pepper. Some isolates from citrus were able to recombine among themselves and with isolates from guava, peppers and strawberries. From the heterokaryons obtained, two of them had their pathogenicity characterized: Het 3 and Het 5. As a result, the heterokaryon derived from citrus and guava (Het 5) behaved similarly to one of their parental isolates. The heterokaryon derived from citrus and pepper (Het 3) was more aggressive than their parental isolates when inoculated in pepper. With these results we can conclude that there is specificity between pathogenic strains of C. acutatum from different hosts. However, isolates from different hosts can recombine with each other and generate heterokaryons with altered pathogenic characteristics.

Nos últimos anos, o plantio mecanizado da cana-de-açúcar vem sendo cada vez mais utilizado no Brasil. Tal sistema possui menor custo sobre o convencional e viabiliza o plantio durante o ano todo. Entretanto, a maior quantidade de ferimentos nas mudas colhidas mecanicamente e a realização de plantios em períodos frios e com umidade do solo inadequada têm aumentado a incidência da podridão abacaxi, doença causada pelo fungo Thielaviopsis paradoxa. Áreas onde esta doença é problemática apresentam muitas falhas, exigindo, em alguns casos, replantio. Thielaviopsis paradoxa penetra nas mudas através de ferimentos, o que é importante em cana-de-açúcar, uma vez que os colmos são seccionados durante o plantio. Medidas que estimulem a brotação rápida ou que protejam os ferimentos das mudas produzem excelentes resultados no controle da doença. Com o objetivo de avaliar o controle da podridão abacaxi da cana-de-açúcar por meio da pulverização de fungicidas em mudas no sulco de plantio, foram instalados quatro experimentos, sendo dois em casa de vegetação e dois no campo. Para os ensaios em casa de vegetação, utilizou-se um substrato esterilizado, composto por uma mistura de solo, areia e esterco de gado. Uma suspensão de T. paradoxa na concentração de 103 esporos/g de substrato foi incorporada dois dias antes do plantio. As avaliações foram realizadas em um período de 45 dias, determinando-se o número de plantas, a velocidade de brotação, a biomassa da parte aérea e de raízes, e a severidade da doença. No campo, um dos experimentos foi realizado sem a inoculação do patógeno, com a colheita das mudas e plantio mecanizados. No outro experimento, uma suspensão do fungo foi inoculada com pulverizador costal manual na concentração de 9 x 104 esporos/m2, e o plantio foi realizado manualmente. As avaliações foram realizadas em um período de 13 meses, determinando-se o número de plantas, a velocidade de brotação, a biomassa da parte aérea, o teor de açúcares totais recuperáveis e o rendimento. Nos quatro experimentos, os fungicidas foram pulverizados sobre as mudas imediatamente após o plantio, com pulverizador costal pressurizado com CO2. Os dados das avaliações foram submetidos à análise de variância e à análise de contrastes ortogonais. Além disso, os tratamentos foram comparados com a testemunha através do Teste de Dunnett a 5% de significância. Os resultados mostraram que a pulverização de fungicidas nas mudas controlou a podridão abacaxi da cana-de-açúcar, sendo que os efeitos dessa prática foram mais positivos em condições mais favoráveis à manifestação da doença. Os fungicidas Azoxistrobina + Ciproconazol; Azoxistrobina + Fluodioxonil + Metalaxil- M; Propiconazol; Piraclostrobina e Piraclostrobina + Epoxiconazol foram eficientes no controle da podridão abacaxi da cana-de-açúcar. Por outro lado, Carboxina + Tiram, comercialmente utilizado com essa finalidade, não foi efetivo em controlar a doença, tanto em casa de vegetação como no campo. / In the last few years, mechanized planting of sugarcane has been frequently used in Brazil. This system has a lower cost than the conventional system and enables planting throughout the year. However, the higher quantity of injuries in seedpieces harvested mechanically and the planting at low temperatures and with inadequate soil humidity have increased the incidence of pineapple disease, caused by the fungus Thielaviopsis paradoxa. Areas where this disease is problematic present stand reduction, requiring, in some cases, replanting. Thielaviopsis paradoxa penetrates the seedpieces through wounds caused due to stalk sectioning during planting. Measures which increase shoot emergence or which protect seedpieces wounds produce excellent results in the disease control. In order to assess control of pineapple disease of sugarcane through in-furrow fungicide spray application on seedpieces, four experiments were installed, two in greenhouse and two under field conditions. For the experiments in the greenhouse, substrate consisting of a mixture of soil, sand and cattle manure was used. Suspension of T. paradoxa at the concentration of 103 spores/g of substrate was incorporated two days before planting. Evaluations were done in a period of 45 days assessing number of shoots, germination speed, biomass of shoots and roots, and disease severity. In the field, one of the experiments was performed with no inoculation of the pathogen, and the seedpieces harvesting and planting were mechanized. For the other experiment, a suspension of T. paradoxa spores at the concentration of 9 x 104 spores/m2 was sprayed in the plots and the planting was performed manually. Evaluations were done in a period of 13 months assessing number of shoots, germination speed, biomass of shoots, total recoverable sugar content and yield. In the four experiments, fungicides were sprayed on seedpieces immediately after the planting with a CO2 pressurized sprayer. Data were submitted to analysis of variance and to analysis of orthogonal contrasts. Moreover, treatments were compared to control by Dunnett´s Test at 5% of significance. Results showed that in-furrow spray application on seedpieces controlled pineapple disease of sugarcane, and the effects of this practice were more positive under more favorable conditions for the disease manifestation. The fungicides Azoxystrobin + Cyproconazole; Azoxystrobin + Fluodioxonil + Metalaxyl-M; Propiconazole; Pyraclostrobin and Pyraclostrobin + Epoxiconazole were efficient for controlling pineapple disease of sugarcane. On the other hand, Carboxin + Thiram, commercially used for this purpose, was neither effective in the greenhouse nor in the field to control the disease.

The damping-off and stem rot disease syndrome is harmful to many cultivated crops. Damping-off and stem rot caused by Sclerotium rolfsii Sacc. on cowpea results in yield losses with serious socio-economic implications. The objectives of the present study were to investigate the occurrence of the diseases in Benin, study etiology and factors influencing the diseases, and develop strategies for the control of the diseases in the field. Results showed that the diseases are distributed countrywide. Sclerotium rolfsii was the main causal agent but minor pathogens, namely Pythium sp., Rhizoctonia solani Kühn and Phoma pomorum Thüm were also recorded. In the Ouémé valley, the diseases were favoured by soil moisture and S. rolfsii initial inoculum that were higher closer to the river. Sclerotium rolfsii isolates collected in the valley showed genetic diversity in terms of pathogenicity, mycelial compatibility groups and ITS rDNA sequences. A paper-based screening method was found to be a rapid laboratory method for screening for resistance in cowpea cultivars. Furtheremore, Moringa oleifera L. leaf extracts, Trichoderma Kd 63 and Trichoderma IITA 508 significantly reduced the disease incidence. The best disease control was recorded in the field when M. oleifera seed treatment was integrated with a soil sprinkle of Trichoderma. The present work provides information on damping-off and stem rot of cowpea in Benin and control strategies for ecologically sustainable cowpea production. / Thesis (DPhil (Microbiology and Plant Pathology))--University of Pretoria, 2006. / Microbiology and Plant Pathology / unrestricted

In Waldökosystemen ist Totholz von zentraler Bedeutung, indem es zahlreichen Organismen einen Lebensraum bietet oder als Substrat dient, Bestandteil des Kohlenstoff- und Nährstoffkreislaufs ist sowie als ein wichtiges strukturelles Element fungiert. Für seine Zersetzung ist die Überwindung der Ligninbarriere von besonderer Bedeutung. Dazu sind lediglich saprobionte Pilze aus den Phyla der Basidiomycota und Ascomycota in der Lage, die verschiedene Strategien – die Fäuletypen – entwickelt haben, um Lignin abzubauen oder zu modifizieren und somit Zugang zu den vom Lignin inkrustierten Polysachariden (Zellulose und Hemizellulosen) zu erhalten. Eine besondere Rolle spielen dabei Weißfäulepilze, die mit ihren extrazellulären oxidativen Enzymen, wie Laccasen und verschiedenen Peroxidasen, Lignin komplett bis zum Kohlendioxid (CO2) mineralisieren. Trotz der Bedeutung des Ligninabbaus für die Totholzzersetzung sind extrazelluläre oxidative Enzyme im natürlichen Totholz kaum erforscht. Ziel dieser Arbeit war es, die Rolle der oxidativen Enzyme für die Totholzzersetzung unter Realbedingungen zu verifizieren, ihre räumlichen und zeitlichen Muster zu beschreiben und ihre Abhängigkeiten von verschiedenen Totholzvariablen sowie der pilzlichen Artengemeinschaft in und auf Totholz zu ermitteln. Weiter wurde die Veränderung der Totholzvariablen über den Zersetzungsprozess für unterschiedliche Baumarten vergleichend beschrieben und der Einfluss der Waldbewirtschaftung auf den Prozess untersucht. Dazu wurden 197 natürliche Totholzstämme (coarse woody debris, CWD) von Fagus sylvatica (Rotbuche), Picea abies (Gemeine Fichte) und Pinus sylvestris (Gemeine Kiefer) in unterschiedlich stark bewirtschafteten Wäldern in Deutschland untersucht. Insgesamt wurden 735 Proben genommen und darin die Aktivität von Laccase (Lacc), Genereller Peroxidase (GenP) und Mangan-Peroxidase (MnP) gemessen. Weiterhin wurden Variablen wie Dichte, Wassergehalt, pH-Wert, wasserlösliche Ligninfragmente, die Gehalte an Lignin und Extraktiven sowie an Nährstoffen und Metallen (N, Al, Ca, Cu, K, Mg, Mn und Zn) ermittelt. Die pilzliche Artengemeinschaft wurde anhand genetischer Fingerprints (F-ARISA) und mittels Fruchtkörperkartierung erfasst. In 79 % der untersuchten Totholzproben wurden oxidative Enzymaktivitäten festgestellt. Sie waren hoch variabel über den Zersetzungsverlauf sowie in Bezug auf die Probenahmepositionen innerhalb der einzelnen Stämme. Generell waren die Aktivitäten im F.-sylvatica-Totholz höher als im Koniferentotholz. Lineare und logistische Modelle zeigten, dass die pilzliche Artengemeinschaft, gefollgt von den wasserlöslichen Ligninfragmenten, die wichtigste Einflussgröße hinsichtlich der oxidativen Enzyme war. Ein saurer pH-Wert unterstützte die Funktion von Lacc und MnP; Mangan, Eisen und Kupfer waren in ausreichenden Konzentrationen vorhanden, um die Funktion und Bildung der Enzyme zu gewährleisten. Die holzabbauenden Pilze erwiesen sich als optimal an das niedrige Stickstoffangebot im Totholz angepasst, sodass ein erhöhter Stickstoffeintrag über zwei Jahre die oxidativen Enzymaktivitäten nicht weiter beeinflusste. Der pH-Wert sowie die Gehalte an Lignin, Extraktiven und Nährstoffen waren im Vergleich der drei Baumarten signifikant verschieden, obwohl die zeitlichen Veränderungen der Variablen über den Zersetzungsprozess vergleichbar waren. Die Anzahl operativer taxonomischer Einheiten (OTUs ~ molekulare Artenzahl) nahm im Verlauf der Holzzersetzung zu, während die Zahl fruktifizierender Arten für mittlere Zersetzungsgrade am höchsten war. Beide Artenzahlen nahmen zusammen mit dem Stammvolumen zu. Die Weißfäulepilze dominierten über den gesamten Zersetzungsprozess die fruchtkörperbasierte Artenzahl aller drei Baumarten, was mit dem Vorhandensein oxidativer Enzymaktivitäten einhergeht. Generell nahmen der massebezogene Gehalt des Lignins, der Extraktive und der Nährstoffe über die Zersetzung zu, während der volumenbezogene Gehalt abnahm. Der pH-Wert im Holz aller drei Baumarten sank kontinuierlich im Verlauf der Zersetzung. Eine Erhöhung der Waldbewirtschaftungsintensität hatte einen negativen Effekt auf das Stammvolumen und darüber vermittelt auf die Zahl fruktifizierender Pilzarten, jedoch kaum auf andere untersuchte Totholzvariablen. Aufgrund des häufigen Vorkommens von Weißfäulepilzen, der gleichzeitigen Präsenz oxidativer Enzymaktivitäten und des substanziellen Ligninabbaus kann auf eine fundamentale Bedeutung von Laccasen und Peroxidasen für die Zersetzung des Totholzes geschlossen werden. Nicht zuletzt die charakteristische Molekularmassenverteilung der wasserlöslichen Ligninfragmente deutete darauf hin, dass die Mn-oxidierenden Peroxidasen (MnPs) die dominierenden oxidativen Enzyme des Ligninabbaus sind. Das hoch variable Muster der oxidativen Enzymaktivitäten ist jedoch das Resultat eines komplexen Zusammenspiels der Holzeigenschaften und der pilzlichen Artengemeinschaft. Die dabei bestehenden funktionellen Abhängigkeiten müssen weiter im Detail in zukünftigen Studien analysiert und aufgeklärt werden. / In forest ecosystems, deadwood is an important component that provides habitat and substrate for numerous organisms, contributes to the carbon and nutrient cycle as well as serves as a structural element. Overcoming the lignin barrier is a key process in deadwood degradation. Only specialized saprotrophic fungi of the phyla Basidiomycota and Ascomycota developed different strategies – the rot types – to degrade lignin or to modify it in way, which allows them to get access to the polysaccharides (cellulose and hemicelluloses) that are incrusted within the lignocellulosic complex. In this context, basidiomycetous white rot fungi secreting oxidative enzymes (especially laccases and peroxidases) are of particular importance, since they are the only organisms that are able to substantially mineralize lignin to carbon dioxide (CO2). Although lignin degradation is such an important process for deadwood degradation, oxidative enzyme activities have been only poorly studied under natural conditions in deadwood. The aim of this work was to verify the importance of oxidative enzymes for deadwood degradation in the field, to describe their temporal and spatial patterns of occurrence and to identify dependencies from deadwood variables as well as from the fungal community within and on deadwood. Furthermore, the changes of different deadwood variables were studied over the whole period of degradation and compared among three tree species. Last but not least, the influence of forest management intensity on the process of deadwood degradation was evaluated. Therefor, 197 logs of naturally occurring deadwood (coarse woody debris, CWD) of Fagus sylvatica (European beech), Picea abies (Norway spruce) and Pinus sylvestris (Scots pine) were monitored and sampled in forests with different management regimes across three regions in Germany. A total of 735 samples were taken from the logs and analyzed regarding activities of laccase (Lacc), general peroxidase (GenP) and manganese peroxidase (MnP). Wood density, water content, content of lignin and extractives as well as of nutrients and metals (N, Al, Ca, Cu, K, Mg, Mn und Zn) were determined in the samples, too. The fungal community was assessed based on sporocarps (fruiting bodies) and molecular fingerprints (F-ARISA). Oxidative enzyme activities were present in 79 % of all samples. The activities were found to be highly variable both regarding the time course of degradation and their distribution within the logs. Activities were generally higher in wood samples of F. sylvatica than in samples of conifers. Linear and logistic models revealed that the fungal community structure was the most important determinant for oxidative enzyme activities in the samples, followed by the amount of water-soluble lignin fragments. Moreover, the prevalent acidic pH determined in deadwood was suitable to facilitate the function of laccase and peroxidases. Concentrations of metals (manganese, copper, iron) were sufficient to ensure synthesis and functioning of the enzymes. Deadwood-dwelling fungi turned out to be well adapted to low nitrogen concentrations and thus, an elevated nitrogen deposition over a period of two years did not affect the oxidative enzyme activities. The pH as well as the content of lignin, extractives and nutrients significantly differed among the tree species; however, their trend over the course of degradation was rather similar. Molecular species richness (determined by F-ARISA as OTUs) increased over the whole course of degradation, while the number of fruiting species was highest in the intermediate stage of degradation. Both types of species richness increased with increasing volume of the CWD logs. Over the entire degradation period, white rot fungi – based on the identification of sporocarps – were the most abundant group of wood rot fungi in and on all three tree species. This corresponds well with the overall presence of oxidative enzyme activities. During degradation, the mass-related content of lignin, extractives and nutrients frequently increased, although the volume-related content decreased. The pH  of all three tree species decreased in deadwood over the whole period of degradation. Higher forest management intensity had a negative effect on the log volume of deadwood and in consequence on fungal species richness (fruiting bodies), but hardly to other analyzed variables. Based on the widespread occurrence of white rot fungi, the concomitant presence of oxidative enzyme activities as well as the substantial loss of lignin, it can be concluded that laccases and peroxidases are highly relevant for deadwood decomposition. Not least, the detected characteristic molecular size distribution of water-soluble lignin fragments points to a key role of Mn oxidizing peroxidases (MnPs) in enzymatic lignin degradation. The variable patterns of oxidative enzymes observed in wood samples is therefore the result of a complex array of wood variables and the fungal community structure, which will have to be resolved in more detail in future studies.

La connaissance des effets et modes d’action des QTL (Quantitative Trait Loci) de résistance des plantes aux maladies est un enjeu majeur pour l’exploitation durable des résistances génétiques en agriculture. Dans le pathosystème pois/Aphanomyces euteiches, présentant une importance économique majeure, l’objectif de la thèse a consisté à valider les effets de QTL de résistance, seuls ou en combinaison, et à connaitre leurs modes d’action sur les étapes du cycle de l’agent pathogène. Un total de 157 NILs (Near Isogenic Lines) de pois issues d’un programme de Back-cross Assisté par Marqueurs, porteuses d’aucun, un, deux ou trois des sept principaux QTL de résistance préalablement identifiés, a été génotypé à l’aide d’une puce 15K SNPs et évalué pour la résistance. Les NILs porteuses de QTL à effets majeurs, seuls ou en combinaison avec des QTL à effets mineurs,ont présenté des niveaux accrus de résistance partielle en conditions contrôlées et au champ par rapport aux NILs dépourvues de QTL, dans différents fonds génétiques. Certaines NILs comportant des QTL individuels ou combinés à effets mineurs ont également montré un niveau réduit de sévérité de la maladie dans l’une ou les deux conditions de test. La plupart des QTL a présenté des effets significatifs sur le ralentissement de l’infection et/ou de la quantité d’ADN pathogène ayant colonisé la racine pendant sept jours après inoculation. Cette thèse fournit des outils et éléments de choix de QTL à combiner en sélection pour augmenter l’efficacité de la résistance partielle à A. euteiches dans les futures variétés de pois / Knowledge of the effects and action modes of resistance QTL to plant diseases is a major challenge for the durable use of genetic resistances in agriculture. In the pea/Aphanomyces euteiches pathosystem, which has a major economic importance, the aim of this study was to validate the single or combined effects of main resistance QTL, and study their action modes on steps of the pathogen life cycle. A total of 157 pea NILs (Near Isogenic Lines) created by Marker-Assisted Back-crossing and carrying no, one, two or three of the seven main resistance QTL previously identified, was genotyped using a 15K SNPs array and evaluated for resistance. The NILs carrying major-effect QTL, individually or in combination with minor-effect QTL, had increased levels of partial resistance in controlledconditions and in the field compared to NILs without QTL, in different genetic backgrounds. Several NILs carrying single or multiple minor-effect QTL also showed reduced levels of disease severity in one or the two test conditions. Most of the QTL had significant effects on slowing down infection and/or pathogen DNA quantity which had colonized the root for seven days after inoculation. This study gives tools and information for the choice of resistance QTL to use in pyramiding breeding strategies for increasing partial levels of resistance to A. euteiches in future pea varieties.