Com o crescimento populacional acelerado e a diminuição das áreas para cultivo, o aumento da produtividade de gêneros alimentícios de todos os tipos é essencial para o atendimento da população mundial, no entanto as perdas desde o momento de colheita até a chegada ao consumidor limita a oferta de frutas ao consumidor. Estas perdas na pós-colheita de maçã, morango e pêssego, são na sua maioria causadas por fitopatógenos causando podridões. Estas podridões são causadas por vários fungos, dentre estes estão o Colletotrichum sp., causador da podridão amarga em maçãs, Botrytis cinerea agente causador do mofo cinzento em morangos e Monilinia fructicola em pêssegos o qual causa a podridão parda. Usualmente o controle destes fungos é efetivado com a utilização de fungicidas, no entanto, a utilização de químicos neste processo preocupa os consumidores, pois, podem existir resíduos nas frutas e no ambiente. Desta forma métodos alternativos como a indução de resistência podem ser alternativas para o controle dos microrganismos causadores de doenças na pós-colheita de frutos. A indução de resistência consiste em estimular através de moléculas elicitoras (indutoras) as defesas vegetais, ou seja, a síntese de composto que atuem diretamente sobre o fitopatógeno como as proteínas-RPs, ou que promovam o reforço estrutural nos tecidos adjacentes ao sítio de infecção do fungo. A quitosana retirada da carapaça de crustáceos é uma alternativa de molécula elicitora de baixo custo e sem riscos ao consumidor que vem sendo utilizada na indução de resistência em frutos na pós-colheita. Esse biopolímero apresenta a capacidade de desencadear as respostas defensivas dos frutos; maçã, morango e pêssego contra os fungos, Colletotrichum sp., Botrytis cinerea e Monilinia fructicola respectivamente. Foram desenvolvidos experimentos na Universidade Tecnológica Fededral do Paraná durante os anos de 2013 e 2014, com objetivo de verificar a capacidade da quitosana de induzir as defesas dos frutos da maçãs, morangos e pêssegos em pós-colheita, bem como , a atividade fungistática sobre os fungos Colletotrichum sp., Botrytis cinerea e Monilinia fructicola em condições in vitro. Para os experimentos com os frutos o indutor foi aplicado nas concentrações 0,25; 0,5; 1,0 e 2,0% e a testemunha (água destilada), os tratamentos foram organizados em delineamento inteiramente casualizado com quatro repetições de 20 frutos de maçã, 20 frutos de morango e 15 frutos de pêssego. Os frutos foram selecionados e padronizados, sendo em seguida as maçãs e os pêssegos submetidos ao tratamento por imersão em soluções de quitosana, já os morangos foram tratados pulverizando a solução contendo quitosana diretamente sobre os frutos. Após 24horas, os frutos foram inoculados, com uma suspensão contendo conídios do patógeno Colletotrichum sp. nas maçãs, B. cinerea em morangos e M. fructicola em pêssegos na concentração 5.10-3 conídios/ml diretamente sobre os frutos com auxílio de um borrifador e de uma micropipeta. Após a realização dos tratamentos os frutos foram acondicionados em B.O.D. a 26 ± 1 °C para maçãs e pêssegos e a 10 ± 1 °C para os morangos. Foram realizadas avaliações periódicas após a implantação e no término dos experimentos para os seguintes parâmetros, físicoquímicas (perda de massa, sólidos e solúveis totais, acidez titulável, firmeza de polpa, incidência de podridões) e bioquímicas (proteínas, açúcares redutores, açúcares totais, antocianinas, flavonoides, e a atividade das enzimas fenilalanina mônia-liase(FAL), peroxidases, quitinases e β-glucanases). Nos experimentos in vitro avaliou-se o efeito da quitosana sobre os patógenos (Colletotrichum sp, B. cinerea e M. fructicola) Os fungos previamente cultivados em placas puras forma transferidos para placas contendo meio B.D.A. com as concentrações de quitosana (0; 0,25; 0,5, 1 e 2%). Sendo após 24 e 48 horas realizadas as medições perpendiculares do diâmetro da colônia pra verificar o crescimento micelial. Os dados dos experimentos foram submetidos à análises de normalidade e de variância e as médias comparadas pelo teste de Tukey e de regressão (p=0,05), com auxílio do software Assistat. Os resultados demonstraram a interferência da quitosana sobre a indução de resistência ao controle da incidência da podridão amarga em pós-colheita de maçãs Gala, ativando a PRPs B-1-3 Glucanase e no controle de Colletotrichum sp in vitro com ação fungistática. Já em morango o indutor controlou o mofo cinzento ativando as peroxidases e quitinases e β-1-3-Glucanase e diretamente o fungo B. cinerea in vitro. Em pêssegos a atuação foi sobre a manutenção da qualidade dos frutos, na indução ativando os genes da quitinase, β 1-3 Glucanase e da mesma forma em in vitro sobre M. fructicola. Conclui-se então que a quitosana apresenta um grande potencial na indução de resistência de frutos na pós-colheita, ativando as defesa contra fungos fitopatogênicos e diretamente sobre os mesmo com atividade fungistática e fungitóxica in vitro. Sendo que nos frutos de maçã cv. Gala atuou diretamente sobre os parâmetros físico-químicos reduzindo a perda de massa, mantendo a firmeza de polpa e reduzindo capacidade de esporulação de Colletotrichum sp. confirmando também o efeito indutor pela ativação da enzima β-1,3 glucanase e a atividade fungistática sobre Colletotrichum sp. in vitro com redução do crescimento micelial. Já em morangos cv. Camarosa, ativa a síntese das proteínas-RPs quitinases e β-1-3 glucanases contra o mofo cinzento, mantém a firmeza de polpa em níveis mais elevados e interfere no metabolismo dos açúcares totais e redutores e das flavonoides. E ainda atua diretamente sobre B. cinerea diminuindo crescimento micelial in vitro em relação controle. Nos frutos de pêssego a quitosana atua na indução de resistência a podridão parda em pós-colheita de pêssegos “delicioso” ativando as enzimas FAL, quitinase e β-1-3-glucanases. E Atua sobre M. fructicola in vitro com ação fungistática. / With the rapid population growth and the reduction of areas for cultivation, the increased productivity of foods of all kinds is essential to meet the world's population demand. However post-harvest losses from the time of harvest until the arrival to the consumer, limits the supply of fruits to the consumer. The losses in the post-harvest of apples, strawberries and peaches, caused by the incidence of rot led by phytopathogenic fungi, is responsible for most of the losses. These rots are caused by various fungi, among these are the Colletotrichum sp., cause of bitter rot on apples, Botrytis cinerea causative agent of gray mold on strawberries and M. fructicola on peaches which causes brown rot. These phytopathogens are fungi with high ability to spread infection and therefore cause serious damage to fruits, generating losses in post-harvest. Usually the fungi control is conducted with the use of fungicides. However, the use of chemicals in the process concerns consumers, since there may be residues in fruits and the environment. Therefore, alternative methods like the resistance induction can be used to control the disease-causing microorganisms in the postharvest fruits. The induction consists on stimulating the plants defenses through (inducing) elicitor molecules, specifically the synthesis of compounds that act directly on the pathogen as phenols, protein-RPs, or producing structural reinforcement of tissues adjacent to the site of infection of the fungus. Currently, chitosan extracted from crustacean shells is an alternative elicitor molecule of low cost and no risk to the consumer that has been used in the induction of the resistance in postharvest fruits. This biopolymer has the ability to trigger the defensive responses of the fruits; apple, peach, and strawberry against fungi Colletotrichum sp., Botrytis cinerea and Monilinia fructicola, respectively. The inductor was applied at concentrations of 0.25, 0.5; 1.0 and 2.0% and on the control (distilled water). The treatments were arranged in a completely randomized design with four replications of 20 apple fruits, 20 strawberry fruits and 15 peach fruits. The fruits were selected and standardized, and subsequently the apples and peaches were subjected to treatment by immersion in solutions of chitosan, and strawberries were treated by spraying the chitosan-containing solution directly onto the fruits. After 24 hours, the fruits were inoculated with a solution containing conidia of the phytopathogen Colletotrichum sp. on apples, B. cinerea on strawberries and M. fructicola on peaches, in concentrations of [5.10]^(-3) conidia/ml directly on the fruit, with the help of a spray bottle or with a micropipette in the case of apples, directly inoculating the solution in an wound to the 2 mm bark. After completion of the treatments, the fruits were placed in BOD at 26 ± 1 ° C for apples and peaches, and 10 ± 1 ° C for strawberries, and evaluated after 24 hours to determine the following parameters; weight loss, physical and chemical analysis (solids and total soluble, titratable acidity, firmness, decay incidence) and biochemical (protein, reducing sugars, total sugars, anthocyanins, flavonoids, FAL, peroxidase, chitinase and β-glucanase). An initial sample of the fruits was taken to carry out the initial analyzes, using this data as comparative parameters. In a second experiment, the fungus (Colletotrichum sp, Botrytis cinerea and M. fructicola) were grown in the middle of culture containing the different concentrations of chitosan, to verify the existence of fungitoxic or fungistatic effect of the biomolecule in vitro. The fungi were previously cultivated in clean plates, and subsequently transferred to plates containing PDA medium with the chitosan concentrations (0, 0.25, 0.5, 1 and 2), and after 24 and 48 hours, were performed perpendicular measurements of the diameter of the colony to verify its mycelial growth. Data from experiments were submitted to analysis of normality and variance, and measures were compared by Tukey test and regression test (p = 0.05), with the assistance of Assistat software. The results demonstrated the interference of chitosan on the induction of resistance to control the incidence of bitter rot in postharvest Gala apple, activating the PRPs B-1-3 glucanase and control of Colletotrichum sp in vitro with fungistatic action. On strawberries, the inductor controlled the gray mold by activating the peroxidase, chitinase and β-1-3-glucanase, directly under the fungus B. cinerea in vitro. On peaches, the action was on the maintenance of fruit quality, on the induction, activating genes of chitinase, β 1-3 glucanase, and on the same way, on the in vitro of M. fructicola. Therefere, it has been concluded that chitosan has great potential in the induction of fruit resistance in post-harvest, activating the defense against pathogenic fungi, and directly over the latter with fungistatic activity and in vitro fungitoxic activity.

O mofo cinzento é causado pelo fungo Botrytis cinerea (Pers.: Fr.), causador de grandes perdas econômicas em várias culturas. Este patógeno é de difícil controle, devido à ampla gama de hospedeiros, sua atividade saprófita e por formar estruturas de resistência (escleródios). Uma das formas de controle deste patógeno é a utilização de produtos químicos, no entanto, principalmente na pós-colheita de frutos, a tolerância por resíduos químicos nos alimentos, é cada vez menos desejável do ponto de vista ecológico e de saúde pública. Os tratamentos alternativos, como a utilização de extratos de plantas, vêm sendo pesquisados na fitopatologia, para reduzir o uso dos fungicidas sintéticos. A canola (Brassica napus) é uma planta que possui compostos biocidas, com potencial de controle de pragas e doenças. Este trabalho teve como objetivos avaliar o efeito de diferentes extratos da canola (alcoólico, macerado, aquoso sem tempo de reserva e infusão) no controle de Botrytis cinerea in vitro e em pós-colheita de morangos. Foram realizados dois experimentos in vitro, sendo um para avaliar o crescimento micelial e outro a germinação de conídios. O delineamento experimental para os dois experimentos foi inteiramente casualizado, em esquema fatorial 4 x 5, sendo o fator modos de extração de extratos e concentrações (0, 3, 6, 9 e 12%), em 4 repetições. No ensaio de crescimento micelial, a unidade experimental foi uma placa de Petri, e um tubo de ensaio no teste de germinação de conídios. Em pós-colheita foram testados os quatro tipos de extratos na concentração de 16%. O delineamento foi inteiramente casualizado, com 4 repetições por tratamento, sendo a parcela composta por 10 frutos/bandeja. Os parâmetros físico-quimicos avaliados foram podridões, perda de massa, firmeza de polpa e acidez titulável. As análises bioquímicas avaliadas foram proteínas totais, antocianinas e flavonóides, e a atividade da enzima fenilalanina amônia-liase (FAL) e peroxidases. Os resultados obtidos permitiram concluir que houve redução do crescimento micelial e da germinação de conídios, em função das concentrações, mas não ocorreram diferenças entre os extratos. A maior eficiência dos extratos ocorreu na concentração de 8,31%, na avaliação com 96horas. Para a germinação o comportamento foi linear decrescente, ou seja, o aumento das concentrações influenciou na menor germinação dos conídios. Os extratos: alcoólico, maceração e infusão reduziram as podridões causadas por B. cinerea em pós-colheita de morangos. Os extratos atuaram na alteração do teor de acidez dos frutos, e no comportamento das peroxidases, mas não apresentaram efeito sobre sólidos solúveis totais (SST), firmeza de polpa, perda de massa, antocianinas, flavonoides,e atividade da FAL. Os resultados obtidos neste trabalho, comprovam o potencial da canola no controle do mofo cinzento em pós-colheita de morangos, bem como do fungo Botrytis cinerea. / The gray mold is caused by the fungus Botrytis cinerea (Pers.: Fr.), causer of great economic losses in various cultures. This pathogen is difficult to control, due to the wide range of hosts, its saprophytic activity and by forming structures of resistance (esclerodios). One of the ways to control this pathogen is the use of chemical products, however, mainly in post-harvest fruit, tolerance by chemical residues in foods is increasingly less desirable from an ecological and public health point of view. The alternative treatments, such as the use of plant extracts, are being probed in phytopathology, to reduce the use of synthetic fungicides. The canola (Brassica napus) is a plant that has biocidal compounds, with potential to control pests and diseases. This study aimed to evaluate the effect of different extracts of canola (alcoholic, macerate, aqueous without reserve time and infusion) in the control of Botrytis cinerea in vitro and in post-harvest of strawberries. Two experiments were conducted in vitro, being one to evaluate the mycelial growth and another the germination of conidia. The experimental design for the two experiments was completely randomized, in a 4 x 5 factorial scheme, and the modes of extraction of extracts and concentrations (0, 3, 6, 9 and 12 %), in 4 repetitions. In the trial mycelial growth, the experimental unit was a Petri dish, and a test tube in germination test of conidia. In post-harvest were tested four types of extracts at a concentration of 16 %. The experimental design was completely randomized, with 4 repetitions per treatment, being the plot composed by10 fruits/tray. The physico-chemical parameters evaluated were rotting, weight loss, firmness and titratable acidity. The biochemical analyzes were evaluated total proteins, anthocyanins and flavonoids, and the activity of the enzyme phenylalanine ammonia-lyase (PAL) and peroxidases. The results obtained allowed us to conclude that there was a reduction of mycelial growth and germination of conidia, as a function of the concentrations, but there were no differences between the extracts. The greater efficiency of extracts occurred at a concentration of 8.31 %, in evaluation with 96 hours. For germination, the behavior was decreasing linear, in other words, the increase of concentrations influenced the lower germination of conidia. The extracts: alcoholic, maceration and infusion reduced the rot caused by B. cinerea in post-harvest of strawberries. The extracts operate in fruits acidity modification, and in the behavior of peroxidases, but they had no effect on total soluble solids (TSS), pulp firmness, weight loss, anthocyanins, flavonoids,and activity of the PAL. The results obtained in this work, have demonstrate the potential of canola in the control of gray mold on post-harvest of strawberries, as well as the fungus Botrytis cinerea.

Com o crescimento populacional acelerado e a diminuição das áreas para cultivo, o aumento da produtividade de gêneros alimentícios de todos os tipos é essencial para o atendimento da população mundial, no entanto as perdas desde o momento de colheita até a chegada ao consumidor limita a oferta de frutas ao consumidor. Estas perdas na pós-colheita de maçã, morango e pêssego, são na sua maioria causadas por fitopatógenos causando podridões. Estas podridões são causadas por vários fungos, dentre estes estão o Colletotrichum sp., causador da podridão amarga em maçãs, Botrytis cinerea agente causador do mofo cinzento em morangos e Monilinia fructicola em pêssegos o qual causa a podridão parda. Usualmente o controle destes fungos é efetivado com a utilização de fungicidas, no entanto, a utilização de químicos neste processo preocupa os consumidores, pois, podem existir resíduos nas frutas e no ambiente. Desta forma métodos alternativos como a indução de resistência podem ser alternativas para o controle dos microrganismos causadores de doenças na pós-colheita de frutos. A indução de resistência consiste em estimular através de moléculas elicitoras (indutoras) as defesas vegetais, ou seja, a síntese de composto que atuem diretamente sobre o fitopatógeno como as proteínas-RPs, ou que promovam o reforço estrutural nos tecidos adjacentes ao sítio de infecção do fungo. A quitosana retirada da carapaça de crustáceos é uma alternativa de molécula elicitora de baixo custo e sem riscos ao consumidor que vem sendo utilizada na indução de resistência em frutos na pós-colheita. Esse biopolímero apresenta a capacidade de desencadear as respostas defensivas dos frutos; maçã, morango e pêssego contra os fungos, Colletotrichum sp., Botrytis cinerea e Monilinia fructicola respectivamente. Foram desenvolvidos experimentos na Universidade Tecnológica Fededral do Paraná durante os anos de 2013 e 2014, com objetivo de verificar a capacidade da quitosana de induzir as defesas dos frutos da maçãs, morangos e pêssegos em pós-colheita, bem como , a atividade fungistática sobre os fungos Colletotrichum sp., Botrytis cinerea e Monilinia fructicola em condições in vitro. Para os experimentos com os frutos o indutor foi aplicado nas concentrações 0,25; 0,5; 1,0 e 2,0% e a testemunha (água destilada), os tratamentos foram organizados em delineamento inteiramente casualizado com quatro repetições de 20 frutos de maçã, 20 frutos de morango e 15 frutos de pêssego. Os frutos foram selecionados e padronizados, sendo em seguida as maçãs e os pêssegos submetidos ao tratamento por imersão em soluções de quitosana, já os morangos foram tratados pulverizando a solução contendo quitosana diretamente sobre os frutos. Após 24horas, os frutos foram inoculados, com uma suspensão contendo conídios do patógeno Colletotrichum sp. nas maçãs, B. cinerea em morangos e M. fructicola em pêssegos na concentração 5.10-3 conídios/ml diretamente sobre os frutos com auxílio de um borrifador e de uma micropipeta. Após a realização dos tratamentos os frutos foram acondicionados em B.O.D. a 26 ± 1 °C para maçãs e pêssegos e a 10 ± 1 °C para os morangos. Foram realizadas avaliações periódicas após a implantação e no término dos experimentos para os seguintes parâmetros, físicoquímicas (perda de massa, sólidos e solúveis totais, acidez titulável, firmeza de polpa, incidência de podridões) e bioquímicas (proteínas, açúcares redutores, açúcares totais, antocianinas, flavonoides, e a atividade das enzimas fenilalanina mônia-liase(FAL), peroxidases, quitinases e β-glucanases). Nos experimentos in vitro avaliou-se o efeito da quitosana sobre os patógenos (Colletotrichum sp, B. cinerea e M. fructicola) Os fungos previamente cultivados em placas puras forma transferidos para placas contendo meio B.D.A. com as concentrações de quitosana (0; 0,25; 0,5, 1 e 2%). Sendo após 24 e 48 horas realizadas as medições perpendiculares do diâmetro da colônia pra verificar o crescimento micelial. Os dados dos experimentos foram submetidos à análises de normalidade e de variância e as médias comparadas pelo teste de Tukey e de regressão (p=0,05), com auxílio do software Assistat. Os resultados demonstraram a interferência da quitosana sobre a indução de resistência ao controle da incidência da podridão amarga em pós-colheita de maçãs Gala, ativando a PRPs B-1-3 Glucanase e no controle de Colletotrichum sp in vitro com ação fungistática. Já em morango o indutor controlou o mofo cinzento ativando as peroxidases e quitinases e β-1-3-Glucanase e diretamente o fungo B. cinerea in vitro. Em pêssegos a atuação foi sobre a manutenção da qualidade dos frutos, na indução ativando os genes da quitinase, β 1-3 Glucanase e da mesma forma em in vitro sobre M. fructicola. Conclui-se então que a quitosana apresenta um grande potencial na indução de resistência de frutos na pós-colheita, ativando as defesa contra fungos fitopatogênicos e diretamente sobre os mesmo com atividade fungistática e fungitóxica in vitro. Sendo que nos frutos de maçã cv. Gala atuou diretamente sobre os parâmetros físico-químicos reduzindo a perda de massa, mantendo a firmeza de polpa e reduzindo capacidade de esporulação de Colletotrichum sp. confirmando também o efeito indutor pela ativação da enzima β-1,3 glucanase e a atividade fungistática sobre Colletotrichum sp. in vitro com redução do crescimento micelial. Já em morangos cv. Camarosa, ativa a síntese das proteínas-RPs quitinases e β-1-3 glucanases contra o mofo cinzento, mantém a firmeza de polpa em níveis mais elevados e interfere no metabolismo dos açúcares totais e redutores e das flavonoides. E ainda atua diretamente sobre B. cinerea diminuindo crescimento micelial in vitro em relação controle. Nos frutos de pêssego a quitosana atua na indução de resistência a podridão parda em pós-colheita de pêssegos “delicioso” ativando as enzimas FAL, quitinase e β-1-3-glucanases. E Atua sobre M. fructicola in vitro com ação fungistática. / With the rapid population growth and the reduction of areas for cultivation, the increased productivity of foods of all kinds is essential to meet the world's population demand. However post-harvest losses from the time of harvest until the arrival to the consumer, limits the supply of fruits to the consumer. The losses in the post-harvest of apples, strawberries and peaches, caused by the incidence of rot led by phytopathogenic fungi, is responsible for most of the losses. These rots are caused by various fungi, among these are the Colletotrichum sp., cause of bitter rot on apples, Botrytis cinerea causative agent of gray mold on strawberries and M. fructicola on peaches which causes brown rot. These phytopathogens are fungi with high ability to spread infection and therefore cause serious damage to fruits, generating losses in post-harvest. Usually the fungi control is conducted with the use of fungicides. However, the use of chemicals in the process concerns consumers, since there may be residues in fruits and the environment. Therefore, alternative methods like the resistance induction can be used to control the disease-causing microorganisms in the postharvest fruits. The induction consists on stimulating the plants defenses through (inducing) elicitor molecules, specifically the synthesis of compounds that act directly on the pathogen as phenols, protein-RPs, or producing structural reinforcement of tissues adjacent to the site of infection of the fungus. Currently, chitosan extracted from crustacean shells is an alternative elicitor molecule of low cost and no risk to the consumer that has been used in the induction of the resistance in postharvest fruits. This biopolymer has the ability to trigger the defensive responses of the fruits; apple, peach, and strawberry against fungi Colletotrichum sp., Botrytis cinerea and Monilinia fructicola, respectively. The inductor was applied at concentrations of 0.25, 0.5; 1.0 and 2.0% and on the control (distilled water). The treatments were arranged in a completely randomized design with four replications of 20 apple fruits, 20 strawberry fruits and 15 peach fruits. The fruits were selected and standardized, and subsequently the apples and peaches were subjected to treatment by immersion in solutions of chitosan, and strawberries were treated by spraying the chitosan-containing solution directly onto the fruits. After 24 hours, the fruits were inoculated with a solution containing conidia of the phytopathogen Colletotrichum sp. on apples, B. cinerea on strawberries and M. fructicola on peaches, in concentrations of [5.10]^(-3) conidia/ml directly on the fruit, with the help of a spray bottle or with a micropipette in the case of apples, directly inoculating the solution in an wound to the 2 mm bark. After completion of the treatments, the fruits were placed in BOD at 26 ± 1 ° C for apples and peaches, and 10 ± 1 ° C for strawberries, and evaluated after 24 hours to determine the following parameters; weight loss, physical and chemical analysis (solids and total soluble, titratable acidity, firmness, decay incidence) and biochemical (protein, reducing sugars, total sugars, anthocyanins, flavonoids, FAL, peroxidase, chitinase and β-glucanase). An initial sample of the fruits was taken to carry out the initial analyzes, using this data as comparative parameters. In a second experiment, the fungus (Colletotrichum sp, Botrytis cinerea and M. fructicola) were grown in the middle of culture containing the different concentrations of chitosan, to verify the existence of fungitoxic or fungistatic effect of the biomolecule in vitro. The fungi were previously cultivated in clean plates, and subsequently transferred to plates containing PDA medium with the chitosan concentrations (0, 0.25, 0.5, 1 and 2), and after 24 and 48 hours, were performed perpendicular measurements of the diameter of the colony to verify its mycelial growth. Data from experiments were submitted to analysis of normality and variance, and measures were compared by Tukey test and regression test (p = 0.05), with the assistance of Assistat software. The results demonstrated the interference of chitosan on the induction of resistance to control the incidence of bitter rot in postharvest Gala apple, activating the PRPs B-1-3 glucanase and control of Colletotrichum sp in vitro with fungistatic action. On strawberries, the inductor controlled the gray mold by activating the peroxidase, chitinase and β-1-3-glucanase, directly under the fungus B. cinerea in vitro. On peaches, the action was on the maintenance of fruit quality, on the induction, activating genes of chitinase, β 1-3 glucanase, and on the same way, on the in vitro of M. fructicola. Therefere, it has been concluded that chitosan has great potential in the induction of fruit resistance in post-harvest, activating the defense against pathogenic fungi, and directly over the latter with fungistatic activity and in vitro fungitoxic activity.

Objetivando melhor conhecimento a respeito dos nematóides encontrados associados a fruteiras de clima subtropical e temperado, coletaram-se 149 amostras de solo da rizosfera e de raízes de amoreira-preta, caquizeiro, framboeseira, macieira, marmeleiro, nespereira, nogueira-macadâmia, oliveira, pereira, pessegueiro e umezeiro em áreas de produção do Estado de São Paulo e em uma localidade de Minas Gerais. Como resultado desse levantamento, identificaram-se 11 gêneros e as espécies relacionadas a seguir: Aorolaimus nigeriensis, Discocriconemella degrissei, Helicotylenchus dihystera, H. erythrinae, H. microcephalus, H. pseudorobustus, Helicotylenchus spp., Hemicycliophora poranga, Meloidogyne hapla, M. incognita, M. javanica, Mesocriconema onoense, M. ornata, M. sphaerocephalum, M. xenoplax, Mesocriconema spp., Pratylenchus brachyurus, P. zeae, Rotylenchulus reniformis, Scutellonema brachyurus, Tylenchulus semipenetrans, Xiphinema brevicollum, X. elongatum, X. krugi, X. setariae, X. surinamense e Xiphinema spp.. Os três gêneros mais freqüentes foram Helicotylenchus, Mesocriconema e Xiphinema, presentes em 60,4; 55,0 e 30,2 % das amostras, respectivamente; as espécies mais comuns foram H. dihystera e M. xenoplax, ocorrendo em 49 e 38,3 % das amostras. Contudo, apenas M. incognita e M. javanica estavam associados a danos em pessegueiros cujos porta-enxertos não tinham resistência genética. Em casa de vegetação, avaliaram-se as reações de genótipos das citadas fruteiras, mais goiabeira, frente aos nematóides de galhas Meloidogyne incognita raça 2 e M. javanica. As plantas, individualmente inoculadas com 5000 ovos de cada espécie de nematóide, foram conduzidas em recipientes plásticos durante 120 dias. A caracterização das reações baseou-se na capacidade reprodutiva dos parasitos, determinando-se os índices de massas de ovos e de galhas, bem como os números de nematóides por sistema radicular e por grama de raízes. Apenas a cultivar GF-677 de Prunus persica x P. dulcis comportou-se como suscetível, possibilitando desenvolvimento e multiplicação dos dois nematóides. Os demais genótipos avaliados foram hospedeiros desfavoráveis ao desenvolvimento dos nematóides, sendo considerados resistentes, embora vários deles tivessem proporcionado restritas taxas de reprodução dos parasitos. Estudou-se ainda, também sob condição de casa de vegetação, a patogenicidade de M. incognita raça 2 em caquizeiro 'Kyoto', verificando-se correlação negativa entre os níveis populacionais iniciais utilizados (0, 160, 800, 4 000, 20 000 e 100 000 ovos por parcela) e a altura e a massa seca de raízes das plantas, após seis meses da inoculação. Tendo em vista que a intensa formação de galhas radiculares observada e o efeito negativo sobre os dois parâmetros de crescimento das plantas mostraram-se associados a taxas de reprodução muito baixas do parasito, considerou-se que a reação ocorrida foi de intolerância. / In order to achieve a better knowledge on the nematodes found associated with subtropical and temperate fruits, a total amount of 149 soil and root samples were collected from within apple, blackberry, japanese apricot, loquat, macadamia, olive, peach, pear, persimmon, quince and raspberry orchards located in the states of São Paulo and Minas Gerais, Southeastern Brazil. From this survey, a number of species belonging to eleven genera were identified, namely Aorolaimus nigeriensis, Discocriconemella degrissei, Helicotylenchus dihystera, H. erythrinae, H. microcephalus, H. pseudorobustus, Helicotylenchus spp., Hemicycliophora poranga, Meloidogyne hapla, M. incognita, M. javanica, Mesocriconema onoense, M. ornata, M. sphaerocephalum, M. xenoplax, Mesocriconema spp., Pratylenchus brachyurus, P. zeae, Rotylenchulus reniformis, Scutellonema brachyurus, Tylenchulus semipenetrans, Xiphinema brevicollum, X. elongatum, X. krugi, X. setariae, X. surinamense and Xiphinema spp. The most frequent genera were Helicotylenchus, Mesocriconema and Xiphinema, which occurred in 60.4, 54.4 and 30.2 % of the samples, respectively; the two most common species found were Helicotylenchus dihystera and Mesocriconema xenoplax, detected in 49.0 and 38.8 % of the samples, respectively. However, only Meloidogyne incognita and M. javanica could be associated with peach trees that were stunted or showed general symptoms of decline; in these cases, the rootstocks did not have genetic resistance to root-knot nematodes. The host suitability of several genotypes of the mentioned fruit species, plus guava, were evaluated in relation to Meloidogyne incognita race 2 and M. javanica under greenhouse conditions. The plants were individually inoculated with 5,000 nematode eggs and kept to grow in plastic bags for four months. Nematode reproductive rate was determined with basis on gall index, egg mass index and numbers of nematodes per root system and per gram of roots. Only the cultivar GF-677 of Prunus persica x P. dulcis was susceptible to both nematode species. All other genotypes were poor/resistant hosts, even that some allowed the parasites to reproduce at low rates. The pathogenicity of Meloidogyne incognita race 2 to persimmon cv. Kyoto was also studied under greenhouse conditions. After six months of the inoculation with increasing nematode population levels – 0; 160; 800; 4,000; 20,000; and 100,000 eggs/plant – a negative correlation with plant height and top dry weight values was obtained. As an intense root galling was associated with low nematode reproductive rates and some depressive effects on the plant growth, the reaction was rated as being of intolerance.

Frente à recente detecção em território brasileiro de Monilina laxa, que assim como M. fructicola, é causadora da podridão parda do pessegueiro, e à falta de informações sobre a epidemiologia dos patógenos causadores da doença em regiões subtropicais, surgiu a necessidade deste trabalho, que teve por objetivos determinar as melhores condições de temperatura e molhamento para o desenvolvimento de M. fructicola e M. laxa in vitro e para a infecção e colonização pêssegos por conídios dos patógenos. Quatro isolados de M. fructicola e um isolado de M. laxa, provenientes de plantios do Estado de São Paulo e mantidos no laboratório de Epidemiologia da ESALQ, foram estudados. Foram avaliados os efeitos da temperatura e da duração do molhamento na germinação dos conídios in vitro e o efeito da temperatura no crescimento micelial e na esporulação dos patógenos. Ensaios para determinar a sensibilidade dos patógenos ao fungicida iminoctadina também foram conduzidos. Adicionalmente, pêssegos maduros foram adquiridos, feridos, inoculados com suspensão de conídios e incubados em diferentes temperaturas sob diferentes períodos de câmara úmida. Diariamente incidência e severidade foram avaliadas. A esporulação foi avaliada ao final de cada tratamento. A faixa ótima de temperatura para germinação de conídios de M. laxa, de 5 a 30ºC, é maior que a de M. fructicola, de 10 a 30ºC. O período de molhamento não teve influência na germinação dos conídios. A melhor faixa de temperatura para crescimento micelial tanto de M. fructicola quanto M. laxa de foi de 20 a 25ºC. A esporulação de M. laxa foi inversamente proporcional à temperatura de incubação. Não houve relação entre temperatura e produção de esporos em M. fructicola. Ambas as espécies mostraram-se sensíveis, in vitro, à molécula iminoctadina. A incidência da podridão parda foi superior a 80,9% em todos os tratamentos, para as duas espécies fúngicas. A 10ºC os períodos de incubação e de latência foram menores para M. laxa que para M. fructicola. A faixa ótima de temperatura para o desenvolvimento de lesões variou de 15 a 25ºC para os dois patógenos. A esporulação dos patógenos não mostrou relação com a temperatura nos ensaios in vivo. Diferenças nos sinais da espécie do patógeno foram observados nas lesões. / Due to the recent detection in Brazil of Monilinia laxa, that similar to M. fructicola causes the brown rot of peaches, and the lack of information about the epidemiology of the causal agents of this disease from subtropical regions, raised the need of such a study, which the aims were to determine the best temperature and wetness duration conditions to the development of M. fructicola and M. laxa in vitro and to infection and colonization of peaches by conidia from both pathogens. Four M. fructicola isolates and one M. laxa isolate, collected from infected fruit of São Paulo orchards and maintained at the Epidemiology Laboratory at ESALQ, were studied. Temperature and wetness duration effects on conidia germination in vitro and temperature effects on mycelial growth and sporulation of pathogens were evaluated. Trials to evaluate the sensitivity of isolates to the fungicide iminoctadine were also conducted. Furthermore, mature peaches were wounded on the surface, inoculated with a conidia suspension and incubated at different temperatures during different wet chamber periods. Incidence and severity of the disease were evaluated daily. Sporulation was evaluated at the end of each treatment. The favorable range of temperature to conidia germination of M. laxa, from 5 to 30ºC, was greater than M. fructicola range, from 10 to 30ºC. The wetness duration showed no influence on conidia germination. The best range of temperature for mycelial growth of both M. fructicola and M. laxa was from 20 to 25ºC. Sporulation of M. laxa showed an inverse relationship to the incubation temperature. No relationship between temperature and conidia production was found for M. fructicola. Both species were sensitive to the iminoctadine fungicide in vitro. Incidence of brown rot was greater than 80.9% in all treatments, for both species. At 10ºC incubation and latency periods were shorter for M. laxa than for M. fructicola. The best temperatures for lesion development varied between 15 and 25ºC for both pathogens. Sporulation of the two species showed no relationship to the temperature on the in vivo trials. Visual differences were observed on pathogen symptoms on fruit.

Please read the abstract in the dissertation. Copyright / Dissertation (MSc (Plant Pathology))--University of Pretoria, 2007. / Microbiology and Plant Pathology / unrestricted

Lipopolysaccharides from Gram-negative bacteria are amphipathic, tripartite molecules consisting of a hydrophobic lipid A portion, a core hetero-oligosaccharide and a repetitive hydrophilic O-antigen polysaccharide region. Through cell : cell interactions, plants can come into contact with LPS originating from root-associated rhizobacteria, bacterial endophytes as well as bacterial pathogens. Biologically active LPS molecules have been shown to act as determinants of bacterial virulence but also as determinants of induced systemic resistance (ISR) and activators of the phenotypically related systemic acquired resistance (SAR), characterised by accelerated and enhanced defence responses. LPS as a ¡¥pathogen associated molecular pattern, PAMP¡¦ molecule, has the ability to activate the innate mammalian immunity system and to act as an immunomodulator of immune ¡V and inflammatory systems via the conserved lipid A region. It is thus believed that LPS is able to promote plant disease resistance through activation of ISR and/or SAR; however here, the O-antigen region is also implicated to play a pivotal role in the signal perception and transduction in response to elicitation by this bio-active lipoglycan. LPS was isolated from the cell walls of the endophyte, Burkholderia cepacia, characterised by denaturing electrophoresis and compared to the equivalent from the pathogen Ralstonia solanacearum. When dissolved in the presence of Ca2+ and Mg2+, the LPS attained its biologically active micellar state through complex formation. The former LPS strongly induced the activation of two MAPKs following treatment of Nicotiana tabacum cv Samsun leaves, while comparative inductions with the R. solanacearum counterpart were extremely weak and might be ascribed to it lacking an extensive O-antigen region. No previous reports on LPS-responsive MAP kinases in plant tissues exist in the literature. The time- and dose dependent activation of the two kinases were therefore investigated and their physico-chemical properties compared. A novel 32 kDa MAP kinase was transiently activated in response to exposure to LPS with optimal activation at 7 min post-elicitation with 100 ƒÝg.ml-1 LPS. Its identity as an ERK (extracellular signal-related) MAPK was confirmed by immunodetection with a pTEpY-specific (anti-active) MAPK antibody, tyrosine-phosphorylated association of activation and inhibition of activation by U0126, an inhibitor of upstream MAPKKs. The kinase did not utilise casein, histone or myelin basic protein as substrates and no endogenous substrate could be identified. The activated MAP kinase exhibited a pI of 6.3, but two charge isomers of 32 kDa respectively were found upon two-dimensional electrophoresis. Although loss of the dual-phosphorylated epitope during purification attempts prevented extensive purification, 30% ammonium sulphate fractionation significantly (33 fold) enriched the MAPK. A second, distinct, 30 kDa MAP kinase was transiently activated in response to 125 ƒÝg.ml-1 LPS at 40 min post-elicitation, and its identity as a p38 MAPK, to date not yet found in plants, was confirmed by immunodetection with a pTGpY-specific (anti-active) MAPK antibody, tyrosine-phosphorylation associated with activation and inhibition of activation by SB203580, a direct inhibitor of p38 MAPKs. The kinase did not utilise casein, histone or MBP as substrates and no endogenous substrate could be identified. The kinase displayed a pI of 6.0, but two charge isomers of 30 kDa respectively were found following two-dimensional electrophoresis. Loss of the dual-phosphorylated epitope again prevented significant purification, but the protein was found to be significantly (83 fold) enriched by 30% ammonium sulphate fractionation. Although LPS has been reported to be capable of altering Ca2+ permeability and perturbation of Ca2+ homeostasis across plasma membranes, Ca2+ did not appear to potentiate or reduce the activation of either the 30 or the 32 kDa kinases. To date other MAP kinases have been shown to act either independently or upstream from reactive oxygen intermediates (ROI) produced during the oxidative burst. It was found that peroxide and concomitant ROI is either not generated in leaf tissue in response to LPS elicitation, or if generated, do not trigger the activation of the two kinases. The identification and partial characterisation of these two novel tobacco MAPKs in the signal perception and transduction response to LPS, significantly contributes to understanding the biochemical basis of the mechanism of action of LPS as a ¡¥resistance elicitor¡¦ involved in the triggering of effective plant defence responses and contributes towards relating the activation of mammalian innate immunity to similar responses in plants. / Prof. I.A. Dubery

In response to compatible rhizobia, leguminous plants develop unique plant organs, root nodules, in which rhizobia fix nitrogen into ammonia. During nodule invasion, the rhizobia gain access to newly divided cells, the nodule primordia, in the root inner cortex through plant-derived cellulose tubes called infection threads. Infection threads begin in curled root hairs and bring rhizobia into the root crossing several cell layers in the process. Ultimately the rhizobia are deposited within nodule primordium cells through a process resembling endocytosis. Plant host mechanisms underlying the formation and regulation of the invasion process are not understood. To identify and clone plant genes required for nodule invasion, recent efforts have focused on Medicago truncatula. In a collaborative effort the nodulation defect in the lin (lumpy infections) mutant was characterized. From an EMS-mutagenized population of M. truncatula, two non-allelic mutants nip (numerous infections with polyphenolics) and sli (sluggish infections) were identified with defects in nodule invasion. Infection threads were found to proliferate abnormally in the nip mutant nodules with only very rare deposition of rhizobia within plant host cells. nip nodules were found to accumulate polyphenolic compounds, indicative of a host defense response. Interestingly, nip was also found to have defective lateral root elongation suggesting that NIP has a role in both nodule and lateral root development. NIP was found to map at the upper arm of chromosome 1. In sli, infection threads were observed to bring rhizobia from infection threads to newly divided nodule primordium cells in the roots inner cortex. Polyphenolic accumulation in sli nodule/bumps was found. Lateral roots in sli were found to be clustered at the top of the root, indicating that sli like nip may be defective in lateral root development.

Please read the abstract (Summary) in the section 00front of this document / Dissertation (MSc)--University of Pretoria, 2005. / Microbiology and Plant Pathology / Unrestricted

Fusarium wilt of banana, caused by Fusarium oxysporum f. sp. cubense (Foc), is one of the most destructive plant diseases in recorded history. The disease was first discovered in Australia in 1874 but became renowned for the severe losses it caused to export banana plantations during the 1960s in Central America. The banana export industry was saved only by replacing Gros Michel bananas, the dessert banana grown for the export market, with highly resistant Cavendish banana cultivars. Despite this apparent solution, the fungus was found to attack Cavendish bananas in the sub-tropics, where plants were believed to be predisposed to the disease by the cool winter climate. Good management practices and conventional disease management strategies have not been sufficient to reduce losses and stop the disease from spreading, and today Fusarium wilt can be found in almost all banana-producing countries of the world. Since 1988, Foc has been responsible for significant losses of Cavendish bananas in tropical Asia. The only sustainable control measure, the use of resistant varieties, is not always popular as people prefer to eat locally adopted varieties that, unfortunately, are susceptible to Foc. Sustainable Fusarium wilt management in banana depends on the improvement of existing banana cultivars or the development of novel disease management strategies. Molecular biology and biotechnology provide opportunities to introduce foreign resistance genes into existing cultivars and to develop new, environmentally friendly products that can protect susceptible bananas from Foc. Better knowledge of the Fusarium wilt pathogen, its diversity, and its mechanisms of pathogenesis will contribute significantly to developing these novel approaches for control of the disease. Molecular information on the pathogenicity of Foc, however, is limited, whereas other formae speciales of F. oxysporum have been better studied. In this thesis, Agrobacterium tumefaciens-mediated transformation of (ATMT) was employed to investigate genes responsible for pathogenicity of Foc to banana. Chapter 1 provides an overview of pathogenicity in F. oxysporum. Pathogenic and non-pathogenic forms of the fungus are first introduced to the reader, and then the biology, epidemiology and etiology of pathogenic forms of F. oxysporum are discussed. The genetic make-up and ability of the Fusarium wilt fungus to cause disease in plants concludes the first part of the review. In recent years, there has been a noted increase in the number of techniques available to study hostpathogen interactions. The second part of the review concentrates on these techniques and their applications in studying pathogenicity of the Fusarium wilt pathogen. In Chapter 2, an ATMT and screening system for Foc was developed. Five A. tumefaciens strains were evaluated for their efficiency to transform Foc with a randomly integrating vector that confers hygromycin B resistance and expression of green fluorescent protein (GFP). A small insertion mutant library of Foc was created, and a subset of transformants was characterized by determining the number of T-DNA inserts present, the location and identity of predicted genes disrupted by T-DNA insertion, and whether transformants of Foc were altered in their virulence against susceptible banana plants. In Chapter 3, the role of a known pathogenicity gene, Frp1, of the tomato pathogen F. oxysporum f. sp. lycopersici (Fol) was investigated in Foc. The first objective was to isolate and characterize the Frp1 gene in Foc, and to compare it to the homologous gene in Fol. A vector containing a modified Fol Frp1 gene was then obtained and used for targeted disruption of the gene in Foc via ATMT. Mutants in which the Frp1 gene was disrupted were then analyzed for GFP expression, culture morphology, and alterations in pathogenicity to banana. / Dissertation (MSc)--University of Pretoria, 2008. / Microbiology and Plant Pathology / unrestricted