Vitis labrusca L. germination: influence of treatments to break dormancy, storage and ripening point of fruits

Grapevine seeds have morphophysiological dormancy, which complicates their germination, an important step in obtaining new plants after crossing during breeding. The objective of this work was to establish the ideal conditions for Vitis labrusca L. ‘Isabella’ seed storage conditions, the method for breaking seed dormancy, ripening of fruits at harvest, need for fungicide application before seed sowing and their germination. The seeds were submitted to three tests with (1 and 2) different treatments to break seed dormancy and storage in two conditions for two different times (Isabella), and (3) seeds from unripe and ripe fruits treated or untreated with fungicide. Germination was monitored two times per week. The percentage of germination (%G), germination mean time (GMT), and germination speed index (GSI) were calculated. Data were submitted to analysis of variance (ANOVA) and means were compared using the LSD-Fisher test (p < 0.05). The 12-month storage at a controlled temperature (25±2°C) provided the best germination results (up to 19.5% of G%). Stratification for 90 or 120 days on wet paper or sand at 5±2°C of seeds from mature fruits is the most suitable for the germination of Isabella seeds (65% to 72% of G%, and 1.73 and 1.95 of GSI for the treatments of stratification on wet sand for 120 days). It is also not necessary to apply fungicide on seeds before sowing, as long as disinfestation with 70% alcohol and sodium hypochlorite 1%, and triple washing with water is done (no statistical difference for seeds from ripe fruits treated or not with fungicide, 58% and 64.7% of %G, and 1.37 and 1.22 of GSI, respectively)

Genetics and improvement of veasts for the bioconvertion of H. tuberosus L. extracts

O presente trabalho teve como principais objetivos estabelecer quais as leveduras mais adequadas a fermentação alcoólica e à produção de xaropes de frutose, a partir de inulina de extrato de tubérculos de H. tuberosus. Para tanto foram desenvolvidos experimentos de seleção de leveduras, avaliação da atividade inulinolítica extracelular, estudo de parâmetros cinéticos e estudos genéticos importantes para o melhoramento destes organismos. A seleção de leveduras permitiu estabelecer que as espécies mais adequadas à fermentação de extratos de H. tuberosus são K. marxianus, K. fragilis e C. pseudotropicalis. Com leveduras selecionadas foi possível desenvolver fermentações de 12 horas com 9,87°GL de etanol e rendimentos de 96,56%, com produtividade volumétrica de 7,87 g.l-1.h -1 e produtividade de específica de 2,35.g. g-1.h -1, o que torna o processo de fermentação deste substrato comparável com as fermentações de cana-de-açúcar. As linhagens testadas de K. marxianus, K. fragilis e C. pseudotropicalis exibiram altas atividades inulinolíticas extracelulares, o mesmo não ocorrendo nas linhagens de S. cerevisiae, C. guillemondii e T. colliculosa, nas quais não foi possível detectar atividade extracelular desta enzima. Os dados obtidos mostraram que a inulinase extracelular de K. marxianus não apresenta repressão catabólica, e a sua síntese mesmo sendo constitutiva foi estimulada por inulina, sacarose e celobiose. Por outro lado, a inulinase extracelular de K. fragilis sofre repressão catabólica e retroinibição em concentrações de açúcares redutores superiores a 3x10-3M, sendo a sua síntese induzida por inulina e por ?-fructosídios (incluindo sacarose). Observou-se adicionalmente que os extratos enzimáticos de Kluyveromyces, apresentam dois pH ótimos (p H 4,0·e 7,0) de atividade inulinolítica , e são sensíveis a temperaturas superiores a 50°C. Dentre as linhagens testadas, três de K. marxianus exibiram caráter killer a pH 4,5, sendo a sua toxina muito ativa sobre leveduras do gênero Saccharomyces, mas não sobre o próprio gênero. O caráter killer nestas leveduras está associado à presença de dois plasmídios de DNA (pM1 e pM2) e a resistência à própria toxina deve-se a genes nucleares. Sendo a toxina muito eficiente no controle de contaminantes de Saccharomycesdurante a multiplicação, está levedura pode vir a ser usada onde a presença destas leveduras não for desejada. As linhagens de K. marxianus não exibe reação sexual definida e as taxas de hibridização foram muito reduzidas, tanto em cruzamentos intra, como interespecíficos. Es te aspecto não foi limitante para os estudos genéticos, permitindo a identificação de dois genes inulinolíticos designados lnl1 e lnl2, presentes respectivamente em IZ 1339 e IZ 619. Estes genes estão ligados a 10 cm, e quando os alelos lnl1 e lnl2, estão presentes na mesma linhagem ocorre interação que resulta em atividades muito superiores aos genes individuais. A variação entre segregantes de híbridos de K. marxianus mostrou-se significativa, sendo obtidos segregantes com capacidade fermentativa superior aos parentais e as linhagens selecionadas. As técnicas recombinogênicas empregadas mostraram-se eficientes e este resultado mostra o grande potencial de resposta de métodos que envolvem a recombinação nestas leveduras. Mesmo os métodos não convencionais de cruzamento mostraram-se viáveis, sendo os produtos de fusão entre K. marxianus e K. lactis estáveis, produzindo ascos com esporos reniformes, porém com baixa viabilidade. Estes produtos de fusão apresentaram boa capacidade de fermentação de glicose, extratos de H. tuberosus lactose, exibindo também o caráter killer do parental K. marxianus Os resultados permitem concluir que a fermentação de extratos de H. tuberosus e a produção de enzimas inulinolíticas por apresentarem produtividades e rendimentos elevados que possibilitam a produção econômica de etanol e xarope de frutose via hidrólise enzimática a partir deste vegetal. Através do melhoramento das leveduras do gênero Kluyveromyces e da produção agrícola de H. tuberosus, estes processos podem ser mais econômicos e portanto com maior potencial de competição com outras tecnologias existentes.This work had the main objectives of selection of suitable yeasts for fermen tation.and enzyme production for inulin hydrolysis. Both properties are required to use H. tuberosus (Jerusalem Artichoke) extracts to obtain ethanol and fructose syrups. Kinetic parameters of fermentation and enzyme activities were studied and some genetics of Kluyveromyces yeasts developed as well as the breeding and selection of promising lines. Among different yeasts tested, K. marxianus, K. fragilis and C. pseudotropicalis were considered to be adequate for H. tuberosus extracts fermentation. Fermentations were achived in 12 hours with selected yeasts and 9,87°GL of ethanol production.and a yield of 96,56%. Volumetric productivity reached 7,87 g.l-1.h -1 and the specific productivity of 2,35.g. g-1.h -1. The yeasts Saccharomyces cerevisiae, Candida guillemondii and Torulopsis colliculosa, do not show extra-celular inulinase production but high levels of enzyme activity were found for K. marxianus, K. fragilis and C. pseudotropicalis The extracelular inulinase of K. marxianus, has not presented catabol ic repressionar i d synthesis appeared to be constitutive, but stimulated by inul in, sucrose and cellobiose. On the other hand, the extracelular inulinase of K. fragilis presented catabol ic repression and possibly innactivation at concentrations above 3x10-3M of reduzing sugars. Synthesis was induced by inul in or other ?-fructosídios including sucrose. ln addition it was observed that Kluyveromyces extracelular enzymatic extracts show two peaks of inul inolytic activity, one at pH 4.0 and the other at pH 7.0, and activity was ceased in temperatures above 50°C. Among the yeasts tested two strains of K. marxianus presented the killer character at pH 4.5 with a very active toxin against Saccharomyces yeasts but not to other Kluyveromyces This kil ler character was determined by the presence of two DNA plasmids (pM1 e pM2) and toxfn resistance was proved to be of nuclear nature. Considering the toxin very efficient against Saccharomyces contaminant yeasts during multiplication this ability could .be of interest in fermentations where the presence of Saccharomyces is undesired. The strains of Kluyveromyces did not exibited mating type reaction and hybridization rates were very low in both intra and interespecific crosses. This aspects was not limitant· for the genetic studies and it was possible to identify two genes for inulin utilization designed lnl1and lnl2, which were present respectively in the strains IZ 1339 and IZ 619. The genes are linked at about 10 cM and when both are presented intereaction is observed leaging to enhanced inulinase activity. Variation among segregant strains from the hybrids of K. marxianus was significant and superior strains were obtained compareci to the parental selected lines. The recombinogenic methods have shown to be efficient and such results are an evidence of the great resppnse potential of this methods. Even non conventional methods of intercrossing have shown to be viable. Fusion products among K. marxianus and K. lactis were stable and sporulated. However the spore viability was very low. The fúsion hybrids show good mentation ability of glucose, H. tuberosus extracts and lactose, and show the killer character of the parental K. marxianus strain. The results obtained support the conclusion that H. tuberosus fermentation and the production of inulinolitic enzymes, due to its high productivity and yields, make possiblw the economic production of ethanol and fructose syrups via enzymatic hydrolisis. Consldering the by the breeding of Kluyveromyces yeasts and better agricultural production of H. tuberosus the process could ·be more economical and competitive with other existent technologies

Colletotrichum species associated to ripe rot disease of grapes in the "Serra Gaucha" region of Southern Brazil

Ripe rot disease caused by Colletotrichum ( Glomerella) has become a serious problem for viticulture in Southern Brazil. Global warming contributes to the increase of this devastating and difficult to control disease. Several species of Colletotrichum, with different phytopathological characteristics, have been associated with ripe rot disease in different viticultural regions. In this article, a total of 63 fungi were isolated from grapes showing symptoms of ripe rot disease, and classified by sequencing of ITS region, and compared with the sequences deposited in GenBank. The isolates were included in three clades of Colletotrichum: 84.1% belonged to the "gloeosporioides" clade, 3.2% to the "boninense" clade, and 12.7% to the "acutatum" clade. Of the 53 isolates included in the "gloeosporioides" clade, 44.4% were classified as C. viniferum/C. ampelinum, 37.1% as C. fruticola, 13.0% as C. kahawae and 5.5% as a species related to C. fruticola. In turn, the two isolates of the "boninense" clade were classified as C. kartii/C. phyllanthi, and the six "acutatum" isolates were similar to C. acutatum and C. nymphaeae reference materials. The identified species were previously linked to ripe rot disease in other viticulture regions of the world, but the frequency of some species in southern Brazil is particularly different