Plants respond to insect and pathogen attack by inducing and accumulating a large set of defense proteins. Colonization of sugarcane stalk by opportunistic fungi, such as Fusarium verticillioides and Colletotrichum falcatum, usually occurs after Diatraea saccharalis (Lepidoptera: Cambridae) caterpillars attack increasing the damage caused by the borer. Two homologous of BARWIN protein were identified in sugarcane, SUGARWIN1 and SUGARWIN2. Their gene expression is induced in response to wound and Diatraea saccharalis damage. However, the recombinant SUGARWIN protein does not affect insect development; but promotes significant morphological and physiological changes in Fusarium verticillioides and Colletotrichum falcatum, which lead to fungal cell death via apoptosis, indicating that SUGARWINs may work as a first layer of defense against the fungi infection. In this study, we deepen our understanding of the role of SUGARWINs in plant defense and the molecular mechanisms by which these proteins affect fungi by elucidating their molecular targets. Our results show that SUGARWINs play an important role in plant defense against opportunistic pathogens. We demonstrated that SUGARWINs are induced by C. falcatum, and the induction of SUGARWINs can vary among sugarcane varieties. The sugarcane variety exhibiting the highest level of SUGARWIN induction exhibited a considerable reduction in C. falcatum infection. Furthermore, SUGARWIN1 exhibited ribonuclease and chitinase activity, whereas SUGARWIN2 exhibited only chitinase activity. This variable enzymatic specificity seems to be the result of divergent amino acid composition within the substrate-binding site. Additionally, plants attacked by insects and pathogens display profound physiological, morphological and chemical changes or adaptations, which may result in organism attraction or avoidance. In this study, we also aimed to understand the insect-fungi association in sugarcane and the role of fungal volatile compounds in this association. Our results have shown that D. saccharalis positively influences C. falcatum infection on sugarcane, inducing a fast growing when compared to C. falcatum treatment without D. saccharalis attack. In addition, both fungi, C. falcatum and F. verticillioides, have been shown a double effect on D. saccharalis caterpillar, they promoted a strong attraction for insects due volatile organic compound emission and positively influenced D. saccharalis feeding and weight gain in diets supplemented with fungi. Fungal volatile organic compounds from C. falcatum and F. verticillioides were identified and quantified; acoradiene and acorenol were specifically induced by the fungi. These data suggest a synergistic interaction, mediated by organic volatile compounds, between D. saccharalis and the fungi C. falcatum and F. verticillioides in sugarcane. / As plantas respondem ao ataque de insetos e patógenos induzindo e acumulando um grande conjunto de proteínas de defesa. A colonização do caule de cana por fungos oportunistas, como Fusarium verticillioides e Colletotrichum falcatum, geralmente ocorre após o ataque de lagartas de Diatraea saccharalis (Lepidoptera: Cambridae), resultando no aumento do dano causado pelo inseto. Dois homólogos da proteína BARWIN foram identificados em cana-de-açúcar, SUGARWIN1 e SUGARWIN2. A expressão desses genes é induzida em resposta ao ferimento mecânico e ao ataque de Diatraea saccharalis, entretanto, a proteína não afeta o desenvolvimento do inseto, mas promove alterações morfológicas e fisiológicas significativas em Fusarium verticillioides e Colletotrichum falcatum, causando a morte destes fungos por apoptose. Esses dados indicam que as SUGARWINs podem funcionar como uma defesa inicial contra a infecção fúngica. Neste estudo, aprofundamos nosso entendimento do papel das SUGARWINs na defesa de plantas e os mecanismos moleculares pelos quais essas proteínas afetam os fungos, elucidando seus alvos moleculares. Nossos resultados mostraram que as SUGARWINs desempenham um papel importante na defesa da planta contra patógenos oportunistas. Foi demonstrado que essas proteínas também são induzidas por C. falcatum em cana-de-açúcar, e sua indução pode variar entre as variedades de cana-de-açúcar. A variedade de cana-de-açúcar que apresentou o maior nível de indução de SUGARWINs apresentou uma redução considerável na infecção por C. falcatum. Além disso, SUGARWIN1 exibiu atividade de ribonuclease e quitinase, enquanto que SUGARWIN2 exibiu apenas atividade de quitinase. Esta especificidade enzimática parece ser o resultado da composição divergente de aminoácidos no sítio de ligação do substrato. Além disso, as plantas atacadas por insetos e patógenos exibem profundas alterações fisiológicas, morfológicas e químicas ou adaptações, que podem resultar em atração ou repelência do organismo, dessa forma, estudamos também a associação inseto-fungos na cana-de-açúcar, e o papel dos compostos voláteis fúngicos nessa associação. Nossos resultados mostraram que D. saccharalis influencia positivamente a infecção por C. falcatum em cana-de-açúcar, induzindo crescimento rápido do fungo quando comparado ao tratamento com C. falcatum sem ataque de D. saccharalis. Além disso, ambos os fungos, C. falcatum e F. verticillioides, mostraram um efeito duplo sobre lagartas de D. saccharalis, promovendo uma forte atração desses insetos devido à emissão de compostos orgânicos voláteis e influenciando positivamente a alimentação de D. saccharalis e ganho de peso em dietas suplementadas com fungos. Os compostos orgânicos voláteis fúngicos de C. falcatum e F. verticillioides foram identificados e quantificados; acoradieno e acorenol foram especificamente induzidos pelos fungos. Estes dados sugerem uma interação sinergistica, mediada por compostos orgânicos voláteis, entre D. saccharalis e os fungos C. falcatum e F. verticillioides em cana-de-açúcar.

Made available in DSpace on 2016-12-08T16:44:49Z (GMT). No. of bitstreams: 1 PGPV14MA158.pdf: 622907 bytes, checksum: 4bef911008ae5cffa26706180f710db4 (MD5) Previous issue date: 2014-07-31 / Coordenação de Aperfeiçoamento de Pessoal de Nível Superior / The addition and incorporation of organic matter to the soil, besides favoring crops by improving soil physical, can increase nutrients and add specific biochemicals capable of renewing the native microflora and microfauna. These compounds may, depending on the organic material to act as a suppressant effect and biocontrol. The aim of this study was to evaluate the effect of swine manure compost (DSC) in an orchard of apple, pear and grape vines on the population dynamics of Verticillium dahliae, Fusarium solani, Fusarium oxysporum, Fusarium verticillioides and Trichoderma sp. Soil samples for quantification of fungal colonies and nutrient analysis were collected at a depth of 0-10 cm soil of the orchard with apple, pear and grape vines. The population of pathogenic soil fungi and Trichoderma sp. were obtained by dilution and plating of 10 g of soil samples from soil orchard who received two doses of DSC (50 to 100%) compost and two (50 and 100%), using two culture media (BDA potato-dextrose-agar) and Sabouraud-ágar-chloramphenicol. The application of different doses of DSC and chemical fertilizer began in December 2012, repeated at intervals of 60 days until the 2014 harvest analysis of macronutrients (nitrogen, phosphorus, potassium, calcium and magnesium) and micronutrients (iron, copper, zinc and Manganese) DSC and chemical fertilizer were run using Mehlich-1, spectrophotometry, acid-base titration and Kjeldahl method, all described by Tedesco et al. (1995). Results in the concentration of nutrients was related to the population of Verticillium dahliae, Fusarium solani, Fusarium oxysporum, Fusarium verticillioides and Trichoderma sp. The experimental design was completely randomized, factorial 2 x 5, repeated in time (months). The data were analyzed using the MIXED procedure of SAS (SAS Inst. Inc., Cary, NC, v.9.2) and mean comparisons using Tukey least significant difference p ≤ 0.05. In the apple orchard, Fusarium oxysporum and Fusarium solani showed higher populations in Q100 treatments (0-110 x 103 CFU / g of soil) and Q50 (0-70 x 103 CFU/g of soil) respectively. There were differences in the population periods. Phosphorus, Potassium and Sodium showed significant differences among the treatments tested. In the orchard of pear trees the largest population of Fusarium solani was the S100 treatment (0-50 x 103 CFU/ g of soil). Treatments Q50 and Q100 had higher populations of Verticillium dahliae, Fusarium oxysporum and Fusarium verticillioides in different periods. Concentrations of Nitrogen and Potassium differ between treatments tested. In vineyards the largest populations of Fusarium solani and Fusarium oxysporum were found in December-2012 periods (0-70 x 103 CFU / g of soil) and August 2013 (0-60 x 103 CFU / g of soil) respectively. Concentrations of potassium, phosphorus and sodium were higher in treatment S50 and S100. The orchard of apple, pear and grape vines have different response to chemical and organic fertilization. The intensity of response to fertilization has little influence population dynamics of plant pathogens in soil and Trichoderma / A adição e incorporação de matéria orgânica ao solo, além de favorecer as culturas pela melhoria física do solo, podem potencializar nutrientes e adicionar compostos bioquímicos específicos capazes de renovar a microfauna e microflora nativas. Estes compostos podem, dependendo do material orgânico, agir como efeito supressor e como biocontrole. O objetivo deste trabalho foi avaliar o efeito da aplicação de dejeto suíno compostado (DSC) em pomar de macieiras, pereiras e videiras, sobre a dinâmica populacional de Verticillium dahliae, Fusarium solani, Fusarium oxysporum, Fusarium verticillioides e Trichoderma sp. Amostras de solo para quantificação de colônias fúngicas e análise de nutrientes foram retiradas na profundidade 0-10 cm de solo do pomar de macieiras, pereiras e videiras. A população de fungos fitopatogênicos de solo e Trichoderma sp. foram obtidas pela diluição e plaqueamento de 10 g de amostras de solo provenientes do solo do pomar que receberam duas doses de DSC (50 e 100%) e duas de adubo químico (50 e 100%), utilizando dois meios de cultura, BDA (batata-dextrose-agar) e Sabouraud ágar-cloranfenicol. A aplicação das diferentes doses de DSC e adubo químico tiveram início em dezembro-2012, repetidas em intervalos de 60 dias até a safra 2014. A análise dos macronutrientes (nitrogênio, fósforo, potássio, cálcio e magnésio) e micronutrientes (ferro,cobre,zinco e Manganês) do DSC e da adubação química foram realizados pelos métodos de Mehlich -1, espectrofotometria, titulação ácido-base e método Kjeldahl, todas descritas por Tedesco et al. (1995). Resultados da concentração de nutrientes foi relacionado à população de Verticillium dahliae, Fusarium solani, Fusarium oxysporum, Fusarium verticillioides e Trichoderma sp. O delineamento experimental foi inteiramente casualisado, em arranjo fatorial 2 x 5, repetidos no tempo (meses). Os dados foram analisados pelo procedimento MIXED do SAS (SAS Inst. Inc., Cary, NC, v.9.2) e as comparações de médias usando a diferença mínima significativa de Tukey p ≤ 0,05. No pomar de macieiras, Fusarium oxysporum e Fusarium solani apresentaram maiores populações nos tratamentos Q100 (0-110 x 103 UFC/g de solo) e Q50 (0-70 x 103 UFC/g de solo) respectivamente. Houve diferenças da população nos períodos avaliados. Fósforo, Potássio e Sódio apresentaram diferenças significativas entre os tratamentos testados. No pomar de pereiras a maior população de Fusarium solani foi ao tratamento S100 (0-50 x 103 UFC/g de solo). Os tratamentos Q50 e Q100 apresentaram maiores populações de Verticillium dahliae, Fusarium oxysporum e Fusarium verticillioides em diferentes períodos de avaliação. Concentrações de Potássio e Nitrogênio apresentaram diferenças nos tratamentos testados. Na cultura da videira as maiores populações de Fusarium solani e Fusarium oxysporum foram encontradas nos períodos dezembro-2012 (0-70 x 103 UFC/g de solo) e agosto-2013 (0-60 x 103 UFC/g de solo) respectivamente. Concentrações de Potássio, Fósforo e Sódio foram superiores nos tratamento S50 e S100. O pomar de macieiras, pereiras e videiras apresentam diferentes resposta a adubação química e orgânica. A intensidade de resposta da adubação pouco influencia a flutuação da população de fitopatógenos de solo e Trichoderma

Fusarium verticillioides è responsabile della fusariosi della spiga in mais e della contaminazione della granella con micotossine. Sono state individuate le regioni geniche e i geni candidati per la resistenza a Fusarium dal confronto tra una linea di mais resistente (CO441) e una suscettibile (CO354), impiegando tre diversi approcci: analisi QTL, analisi trascrittomica (RNASeq) e analisi metabolomica. 184 famiglie F2:3 (CO441xCO354) sono state valutate in due diversi ambienti nell’anno 2011 e inoculate artificialmente con due diverse tecniche (forchetta e stuzzicadente). E’ stata rilevata una significativa variazione genotipica in risposta all’infezione. Sulla base di una mappa preliminare di linkage molecolare contenente 74 marcatori microsatelliti polimorfici, sono stati determinati 8 QTLs comuni alla resistenza alla fusariosi della spiga e alla riduzione della contaminazione da fumonisine. Sono stati considerati geni candidati per la resistenza i geni differenzialmente espressi, risultanti dall’ RNASeq, in semi di mais CO441 prima e 72 ore dopo l’infezione. I metaboliti putativi correlati alla resistenza sono stati rilevati tramite high resolution LC-MS in entrambe le linee di mais. I geni candidati e i metaboliti mappano in pathway coinvolti nei meccanismi di difesa: fenilalanina, tirosina e triptofano biosintesi, fenilpropanoidi e flavonoidi biosintesi, metabolismo dell’acido linoleico e α-linolenico. Abbondanti trascritti derivano dalla biosintesi dei terpenoidi e dei diterpenoidi. Nei geni candidati verranno ricercati polimorfismi fra le due linee di mais e che andranno ad arricchire la mappa di linkage molecolare. / Fusarium verticillioides is responsible for Fusarium ear rot in maize and mycotoxin contamination of grain. Genomic regions and candidate genes for resistance to Fusarium were detected through the comparison of resistant (CO441) and susceptible (CO354) maize lines, by following three different approaches: Quantitative Trait Locus (QTL), transcriptomic (RNASeq) and metabolomic analyses. 184 F2:3 families (CO441xCO354) were evaluated in two different environments in 2011 and artificially infected with two side-needle inoculation methods (pin-bar and toothpick). Significant genotypic variation in response to infection was detected. On the basis of a genetic draft map containing 74 polymorphic SSRs markers, 8 common QTLs for Fusarium ear rot resistance and fumonisin contamination reduction were revealed. Candidate genes for resistance resulted from differentially expressed genes before and 72 hours post infection of CO441 kernels through RNASeq technology. Putative metabolites related to resistance were detected through high resolution LC-MS in both maize lines. Candidate genes and metabolites mapped in pathways involved in defense mechanism: phenylalanine, tyrosine and tryptophan biosynthesis, phenylpropanoid and flavonoid biosynthesis, linoleic and α-linolenic acid metabolism. Abundant genic transcripts derived from terpenoid and diterpenoid biosynthesis. Candidate genes will be screened for polymorphisms between the two maize lines in order to enrich the linkage map.

Potential vegetative and reproductive increases in yield, as well as the biological efficacy against Fusarium verticillioides and F. proliferatum causing ear and stem rot in maize crops of commercially-formulated micro-organism formulation T-Gro (Trichoderma harzianum isolate DB103 WP) combined with Spartacus (Beauveria bassiana isolate DB 105 WP), T-Gro combined with Armenius (Bacillus subtilis isolate DB 109 WP), T-Gro combined with Maximus (Bacillus subtilis isolate DB 108 WP), T-Gro combined with Shelter (Bacillus subtilis isolate DB 101), T-Gro combined with Bismarck (Microbacterium maritypicum isolate DB 107 WP), as well as individual treatments of T-Gro, Armenius, Bismarck, Maximus and Shelter, were investigated when applied to maize seed and soil under field conditions. All the micro-organism treatments were compared with Thiram 750WP (750g/kg thiram WP) and an untreated control. The micro-organism treatments showed an increase in vegetative as well as reproductive yields when compared to the reference product Thiram 750 WP and the untreated control. There were no observations of adverse effects on the germination of maize seed in all the treatments that were applied. The three isolates B. subtilis, T. harzianum, and M. maritypicum, showed a significant reduction in vascular tissue discolouration of the main and ear stems, indicating a potential to be used in the reduction and control of diseases caused by Fusarium spp. Results also showed poor to very good increases of stem and foliage biomass as well as cob yield per plant produced by the micro-organism treatments when compared to the untreated control. The highest cob yield per plant that differed significantly from the untreated control was produced by T-Gro and Shelter. No phytotoxicity of any kind was observed with the application of the micro-organism formulations and they could therefore be deemed suitable to be used for the treatment of maize seed. The micro-organism formulations containing fungal and bacterial biological control agents have the potential to be used in commercial maize production to increase vegetative and reproductive yields and reduce the severity of ear and stem rot in maize. / Agriculture Animal Health & Human Ecology / M.Sc. (Agriculture)

Em cana-de-açúcar, a colonização do caule por fungos oportunistas, como Fusarium verticillioides e Colletotrichum falcatum, geralmente ocorre após o ataque da lagarta de Diatraea saccharalis. As plantas respondem ao ataque de insetos pela indução e acúmulo de um grande conjunto de proteínas de defesa. Dois homólogos de uma proteína da cevada induzida por ferimento (BARWIN) são conhecidos em cana-de-açúcar, SUGARWIN1 e SUGARWIN2 (sugarcane wound-inducible proteins). Embora a função da proteína BARWIN não tenha sido totalmente estabelecida, propriedades antifúngicas foram descritas para uma série de homólogos. As SUGARWINs estão localizadas no retículo endoplasmático e no espaço extracelular. A indução de seus transcritos ocorre em resposta ao ferimento mecânico, ataque de D. saccharalis e tratamento com metil jasmonato, mas não pela infecção de patógenos. Os transcritos de SUGARWIN são induzidos tardiamente em cana-de-açúcar e são restritos aos locais onde ocorrem os danos. Apesar de transcritos de SUGARWIN1 e 2 serem altamente induzidos pelo ataque de D. saccharalis a proteína em si não possui efeito sobre o desenvolvimento do inseto. Neste trabalho demonstra-se que SUGARWIN2 recombinante causa alterações na morfologia de F. verticillioides e C. falcatum, produzindo aumento da vacuolização, vários pontos de fratura, liberação de material intracelular e formando calos na região dos séptos, culminando na morte destes fungos por apoptose. A SUGARWIN2 recombinante não apesenta efeito em outros fungos, como Saccharomyces cerevisiae e Aspergillus nidulans. Os resultados sugerem que, no curso da evolução genes de SUGARWIN foram recrutados pela cana-de-açúcar para se proteger de fungos oportunistas que, geralmente, penetram na cana após o dano causado pelo inseto. / In sugarcane fields, colonization of the stalk by opportunistic fungi, like Fusarium verticillioides and Colletotrichum falcatum, usually occurs after the attack of Diatraea saccharalis caterpillars. Plants respond to insect attack by inducing and accumulating a large set of defense proteins. Two homologues of a barley wound-inducible protein (BARWIN) are known in sugarcane, SUGARWIN1 and SUGARWIN2 (sugarcane wound-inducible proteins). Although BARWIN protein function has not been fully established, antifungal properties have been described for a number of homologues. The SUGARWINs are located in the endoplasmic reticulum and in the extracellular space of sugarcane plants. The induction of SUGARWIN transcripts occurs in response to mechanical wounding, D. saccharalis damage, and methyl jasmonate treatment but not to pathogen infection. SUGARWIN transcripts are late induced and are restricted to the wound site. Although SUGARWIN transcripts increased after insect attack, the protein itself did not show any effect on insect development. This work shows that recombinant SUGARWIN2 altered F. verticillioides and C. falcatum morphology increasing vacuolization, points of fractures, leaking of intracellular material, leading to the apoptosis of the germlings. Recombinant SUGARWIN2 do not show any effect in other fungus like Saccharomyces cerevisiae and Aspergillus nidulans. The results suggest that, in the course of evolution, SUGARWIN genes were recruited by sugarcane to protect the plant from fungi that typically penetrate the plant stalk after insect damage.

Potential vegetative and reproductive increases in yield, as well as the biological efficacy against Fusarium verticillioides and F. proliferatum causing ear and stem rot in maize crops of commercially-formulated micro-organism formulation T-Gro (Trichoderma harzianum isolate DB103 WP) combined with Spartacus (Beauveria bassiana isolate DB 105 WP), T-Gro combined with Armenius (Bacillus subtilis isolate DB 109 WP), T-Gro combined with Maximus (Bacillus subtilis isolate DB 108 WP), T-Gro combined with Shelter (Bacillus subtilis isolate DB 101), T-Gro combined with Bismarck (Microbacterium maritypicum isolate DB 107 WP), as well as individual treatments of T-Gro, Armenius, Bismarck, Maximus and Shelter, were investigated when applied to maize seed and soil under field conditions. All the micro-organism treatments were compared with Thiram 750WP (750g/kg thiram WP) and an untreated control. The micro-organism treatments showed an increase in vegetative as well as reproductive yields when compared to the reference product Thiram 750 WP and the untreated control. There were no observations of adverse effects on the germination of maize seed in all the treatments that were applied. The three isolates B. subtilis, T. harzianum, and M. maritypicum, showed a significant reduction in vascular tissue discolouration of the main and ear stems, indicating a potential to be used in the reduction and control of diseases caused by Fusarium spp. Results also showed poor to very good increases of stem and foliage biomass as well as cob yield per plant produced by the micro-organism treatments when compared to the untreated control. The highest cob yield per plant that differed significantly from the untreated control was produced by T-Gro and Shelter. No phytotoxicity of any kind was observed with the application of the micro-organism formulations and they could therefore be deemed suitable to be used for the treatment of maize seed. The micro-organism formulations containing fungal and bacterial biological control agents have the potential to be used in commercial maize production to increase vegetative and reproductive yields and reduce the severity of ear and stem rot in maize. / Agriculture Animal Health and Human Ecology / M.Sc. (Agriculture)

Conselho Nacional de Desenvolvimento Científico e Tecnológico / Cordia americana (L.) Gottschling & J.S.Mill. (guajuvira) is a forest species widely distributed in Brazil with importance in its economics and environment. Therefore, there is a great demand of research in seed technology for this species, as for the physiological and sanitary quality of seeds and, mainly, for the association of Fusarium spp. The objectives of this study were to determine the physiological maturity and seed collecting system to be more favorable to sanitary and physiological quality of the C. americana seeds and evaluate the pathogenicity of Fusarium sp. associated with them, morphological and molecularly characterizing the pathogen on the species level. The phenology of this species was closely observed during the development stages and, after the maturation onset of its fruits, they were collected in the plant and its soil. After the picking, it was determined the moisture content of the seeds, the period of maximum dry weight mass accumulation, maximum germination and vigor, besides, sanitary quality ratings. Through the transmission tests, it was found to occur Fusarium spp. causing disease symptoms in seedlings. After obtaining four isolates of the fungus, they were inoculated in seeds to prove its pathogenicity. The morphological characterization was performed using an identification key for the genus Fusarium and, for the molecular identification, genomic regions ITS were sequenced with its elongation factor 1-α. The results may conclude that physiological maturation in C. americana seeds occurs at 52 days after anthesis and the species F. acuminatum and F. verticillioides were pathogenic to species, causing damping-off in pre and post emergence. / Cordia americana (L.) Gottschling & J. S. Mill. (guajuvira) é uma espécie florestal amplamente distribuída no território brasileiro e que apresenta importância econômica e ambiental. Portanto, existe uma grande demanda por pesquisas na área de tecnologia de sementes para essa espécie, quanto à qualidade fisiológica e sanitária de suas sementes e, principalmente pela associação de Fusarium spp. Os objetivos do presente trabalho consistiram em determinar o ponto de maturação fisiológica e o sistema de coleta de sementes mais favorável à qualidade fisiológica e sanitária de sementes de C. americana, além de avaliar a patogenicidade de Fusarium sp. associado a sementes, caracterizando de forma morfológica e molecular o patógeno em nível de espécie. A fenologia da espécie foi acompanhada durante os estágios de desenvolvimento e, após o início da maturação dos frutos, foram realizadas coletas desses diretamente na planta e no solo. Após as coletas, foi determinado o teor de umidade das sementes, o período de máximo acúmulo de matéria seca, máxima germinação e vigor, além de avaliações da qualidade sanitária. Através dos testes de transmissão, foi verificada a ocorrência de Fusarium spp., causando sintomas de doença nas plântulas. Foram obtidos quatro isolados do fungo que foram inoculados nas sementes para provar a sua patogenicidade. A caracterização morfológica foi realizada através de uma chave de identificação para o gênero Fusarium e, para a identificação molecular, foram sequenciadas as regiões genômicas ITS e fator de elongação 1- α. Os resultados obtidos permitem inferir que o ponto de maturidade fisiológica nas sementes de C. americana ocorre aos 52 dias após antese e que as espécies F. acuminatum e F. verticillioides foram patogênicas a espécie, causando tombamento em pré e pós-emergência.

Em cana-de-açúcar, a colonização do caule por fungos oportunistas, como Fusarium verticillioides e Colletotrichum falcatum, geralmente ocorre após o ataque da lagarta de Diatraea saccharalis. As plantas respondem ao ataque de insetos pela indução e acúmulo de um grande conjunto de proteínas de defesa. Dois homólogos de uma proteína da cevada induzida por ferimento (BARWIN) são conhecidos em cana-de-açúcar, SUGARWIN1 e SUGARWIN2 (sugarcane wound-inducible proteins). Embora a função da proteína BARWIN não tenha sido totalmente estabelecida, propriedades antifúngicas foram descritas para uma série de homólogos. As SUGARWINs estão localizadas no retículo endoplasmático e no espaço extracelular. A indução de seus transcritos ocorre em resposta ao ferimento mecânico, ataque de D. saccharalis e tratamento com metil jasmonato, mas não pela infecção de patógenos. Os transcritos de SUGARWIN são induzidos tardiamente em cana-de-açúcar e são restritos aos locais onde ocorrem os danos. Apesar de transcritos de SUGARWIN1 e 2 serem altamente induzidos pelo ataque de D. saccharalis a proteína em si não possui efeito sobre o desenvolvimento do inseto. Neste trabalho demonstra-se que SUGARWIN2 recombinante causa alterações na morfologia de F. verticillioides e C. falcatum, produzindo aumento da vacuolização, vários pontos de fratura, liberação de material intracelular e formando calos na região dos séptos, culminando na morte destes fungos por apoptose. A SUGARWIN2 recombinante não apesenta efeito em outros fungos, como Saccharomyces cerevisiae e Aspergillus nidulans. Os resultados sugerem que, no curso da evolução genes de SUGARWIN foram recrutados pela cana-de-açúcar para se proteger de fungos oportunistas que, geralmente, penetram na cana após o dano causado pelo inseto. / In sugarcane fields, colonization of the stalk by opportunistic fungi, like Fusarium verticillioides and Colletotrichum falcatum, usually occurs after the attack of Diatraea saccharalis caterpillars. Plants respond to insect attack by inducing and accumulating a large set of defense proteins. Two homologues of a barley wound-inducible protein (BARWIN) are known in sugarcane, SUGARWIN1 and SUGARWIN2 (sugarcane wound-inducible proteins). Although BARWIN protein function has not been fully established, antifungal properties have been described for a number of homologues. The SUGARWINs are located in the endoplasmic reticulum and in the extracellular space of sugarcane plants. The induction of SUGARWIN transcripts occurs in response to mechanical wounding, D. saccharalis damage, and methyl jasmonate treatment but not to pathogen infection. SUGARWIN transcripts are late induced and are restricted to the wound site. Although SUGARWIN transcripts increased after insect attack, the protein itself did not show any effect on insect development. This work shows that recombinant SUGARWIN2 altered F. verticillioides and C. falcatum morphology increasing vacuolization, points of fractures, leaking of intracellular material, leading to the apoptosis of the germlings. Recombinant SUGARWIN2 do not show any effect in other fungus like Saccharomyces cerevisiae and Aspergillus nidulans. The results suggest that, in the course of evolution, SUGARWIN genes were recruited by sugarcane to protect the plant from fungi that typically penetrate the plant stalk after insect damage.

No description available.

This Project identified the relationships between storage conditions, dry matter losses (DMLs) caused by Fusarium species in cereal grains and mycotoxin contamination and assessed novel control strategies for post-harvest grain management including chemical control and ozone. F. graminearum, F. verticillioides and F. langsethiae were inoculated on wheat, maize and oats and stored under environmental conditions where marginal to optimum spoilage and mycotoxin contamination can occur. DMLs were calculated from the CO2 produced and were significantly correlated with deoxynivalenol (DON), zearalenone (ZEA), fumonisins (FUMs) and T-2 and HT-2 toxins respectively. Mycotoxin levels in wheat and maize exceeded the EU legislative limits with 0.9-1% DMLs. Therefore, CO2 monitoring during storage can indicate the level of contamination in a stored batch. Using CO2 production data at different water activity (aw) and temperature conditions, the environmental regimes at which F. langsethiae can grow and contaminate oats with T-2 and HT-2 toxins were identified for the first time. Five acids were examined in vitro and little effect was observed on Fusarium growth, in the aqueous form, while the effect on mycotoxin production varied. Dissolved in ethanol, adipic, fumaric and ferulic acids inhibited fungal growth and controlled DON and FUMs, but T-2 toxin was stimulated by the ethanol. Two garlic essential oils, propyl-propylthiosulfinate (PTS) and propyl propylthiosulfonate (PTSO) were studied for the first time. In vitro, 200 ppm reduced fungal growth (50-100%) and mycotoxin production by >90%. The efficacy was species-dependent. In naturally contaminated oats of 0.93 aw stored for 20 days, 16 ppm PTSO reduced T-2 and HT-2 toxins by 66% and ochratoxin A (OTA) by 88%, while 200 ppm PTS reduced OTA by 95%. In wheat, 100 ppm PTS reduced DON and ZEA and 300 ppm PTS reduced fumonisins by 40-80%. PTSO:PTS (1:1) at 400 and 600 ppm was very effective against DON and ZEA in wheat of 0.92 aw. Ozone (O3) exposure at 200 ppm for 30 min delayed Fusarium spore germination on media of 0.98 aw and inhibited germination at 0.94 aw. O3 was more effective against fungal spores than mycelium and little effect was observed on growing cultures. In vitro, mycotoxin production after exposure depended on the stage of life of the fungi. O3 reduced fungal populations in grains. Mycotoxin production in wet grains treated with 100-200 ppm O3 for 60 min and stored for up to 30 days was reduced or completely inhibited, depending on the species and the exposure system. Simultaneous drying of the grain due to the O3 passage was observed.