Polyclonal antibodies to the coat protein of Apple stem grooving virus expressed in Escherichia coli: production and use in immunodiagnosis

The coat protein gene of Apple stem grooving virus (ASGV) was amplified by RT-PCR, cloned, sequenced and subcloned in the expression vector pMal-c2. This plasmid was used to transform Escherichia coli BL21c+ competent cells. The ASGV coat protein (cp) was expressed as a fusion protein containing a fragment of E. coli maltose binding protein (MBP). Bacterial cells were disrupted by sonication and the ASGVcp/MBP fusion protein was purified by amylose resin affinity chromatography. Polyclonal antibodies from rabbits immunized with the fusion protein gave specific reactions to ASGV from infected apple (Malus domestica) cv. Fuji Irradiada and Chenopodium quinoa at dilutions of up to 1:1,000 and 1:2,000, respectively, in plate trapped ELISA. The ASGVcp/MBP fusion protein reacted to a commercial antiserum against ASGV in immunoblotting assay. The IgG against ASGVcp/MBP performed favorably in specificity and sensitivity to the virus. This method represents an additional tool for the efficient ASGV-indexing of apple propagative and mother stock materials, and for use in support of biological and molecular techniques.

Detecção e epidemiologia da podridão branca da maçã

Embora a 'Podridão branca' causada por Botryosphaeria dothidea seja uma das principais doenças de verão da macieira (Malus domestica), há pouca informação no Brasil sobre o patógeno e a doença. Este trabalho objetivou definir métodos para a produção de inóculo, para avaliação da patogenicidade de B. dothidea em maçãs sem ferimentos e para desinfestação das maçãs visando a detecção de infecções quiescentes. Caracterizou-se, também, o progresso temporal e o padrão espacial de frutos e de árvores doentes em pomar comercial. A produção de inóculo em batata-dextrose-ágar com papel de filtro e a inoculação de maçãs submetidas previamente a dois dias de câmara úmida, com conídios aderidos em papel toalha, foram eficazes na produção de inóculo e o desenvolvimento da doença, respectivamente. O melhor método de detecção de infecção latente foi a desinfestação das maçãs com uma mistura de NaOCl 1,25% de cloro ativo e álcool 9,6º GL por 2 min. A produção de inóculo de B. dothidea nos ramos de poda foi observado em meses diferentes em cada ciclo. No ciclo 1999, verificou-se infecção das maçãs por B. dothidea, mesmo com proteção química do pomar e não houve diferença na incidência entre frutas oriundas de diferentes posições na árvore. A análise do padrão de distribuição da doença mostrou agregação distinta das árvores doentes e agregação baixa, dos frutos sintomáticos em cada macieira.

Análise temporal da sarna da macieira nas cultivares Royal Gala e Fuji sob os sistemas convencional e orgânico de produção

A sarna da macieira, causada por Venturia inaequalis, é uma das principais doenças da cultura. Devido à grande preocupação com os riscos de contaminação ambiental e intoxicação humana, o cultivo orgânico surge como uma alternativa, buscando preservar a qualidade do produto final em relação ao sistema convencional. Com o objetivo de avaliar a epidemiologia da sarna da macieira nos sistemas convencional e orgânico de produção, foi empregada a técnica de análise da dinâmica temporal, nas cultivares Royal Gala e Fuji, nas condições edafo-climáticas do Sul do Brasil. A incidência foi quantificada semanalmente, gerando 16 mapas em cada sistema de produção e quinzenalmente, a severidade da doença foi avaliada em 100 folhas, distribuídas em quatro ramos, de acordo com escala diagramática específica, com 12 repetições. Foram plotadas curvas de progresso da doença e as epidemias comparadas em relação a) início do aparecimento dos sintomas (IAS); b) tempo para atingir a máxima intensidade da doença (TAMID); c) valor máximo de severidade da doença (y max) e d) área abaixo da curva de progresso da doença (AACPD). Os dados de incidência foram analisados por meio de análise de regressão linear simples, ajustados para três modelos empíricos, Logístico, Monomolecular e Gompertz. As cultivares avaliadas foram suscetíveis à V. inaequalis, com diferenças significativas entre as cultivares e entre os sistemas de produção. Houve maiores níveis de intensidade de doença e taxa de progresso da doença (r) no sistema orgânico que no sistema convencional de produção, sendo que o modelo mais apropriado para descrever a curva de progresso da doença foi o Logístico.

Caracterização morfocultural e molecular de isolados de Colletotrichum gloeosporioides patogênicos ao mamoeiro

Vinte e nove culturas monospóricas de Colletotrichum, isoladas de frutos e pecíolos de mamoeiro (Carica papaya), foram caracterizadas quanto à morfologia dos conídios e apressórios, coloração e crescimento das colônias, sensibilidade ao benomyl, presença de setas e do teleomorfo, PCR com primers taxon-específicos e análise de PCR-RFLP da região ITS. Os 29 isolados foram identificados como C. gloeosporioides com base na morfologia dos conídios e apressórios, tendo a maioria dos isolados conídios cilíndricos e/ou obclavados e apressórios lobados ou fracamente lobados, em contraste com C. acutatum, isolado de morango (Fragaria x ananassa), que apresentou conídios fusiformes e apressórios circulares e lisos. Presença de setas e do teleomorfo, cor de colônia, sensibilidade ao benomyl e velocidade de crescimento variaram conforme o isolado e sofreram influência do meio de cultura usado. Todos os isolados de mamão e quatro de outras hospedeiras, manga (Mangifera indica), morango e maçã (Malus domestica), foram patogênicos a frutos de mamão cv. Sunrise Solo, mas com variabilidade em agressividade. PCR com o primer específico para C. gloeosporioides, CgInt, confirmou a identidade de apenas quatro isolados de mamão e dois isolados apresentaram reação positiva com o primer CaInt2, específico para C. acutatum. A maioria dos isolados de mamão (23) não reagiu com nenhum dos primers. Por outro lado, a análise de restrição da região ITS do rDNA, com RsaI, gerou perfis distintos entre C. gloeosporioides e C. acutatum e mostrou uniformidade entre os isolados de mamão.

Modo de ação de fosfitos de potássio no controle da podridão olho de boi em maçã

A podridão olho de boi causada por Cryptosporiopsis perennansé a principal doença de maçãs em pós-colheita no Brasil. Objetivou-se neste trabalho avaliar a) o uso de sais de fosfito de potássio em associação ou não ao fungicida captan no controle da doença em condições de campo e in vitro, b) as modificações físico-químicas nos frutos e, c) o efeito desses sais na atividade da peroxidase e da fenilalanina amonia-liase em maçãs inoculadas artificialmente. As aplicações dos sais de fosfito de potássio e captan foram realizadas na safra 2007/08, município de Vacaria, em pomar da cv. Pink Lady(r) a partir dos 45 dias antes da colheita ou 24 h antes da colheita. A atividade da peroxidase e da fenilalanina amônia-liase foi determinada em frutos 24 h e 14 dias após as inoculações com o patógeno. Aplicações de fosfito de potássio + captan reduziram em 60% os danos pela podridão olho de boi em pós-colheita, além de reduzir 66% da quantidade de inóculo na superfície das maçãs em aplicação realizada no dia anterior à colheita. O fosfito de potássio inibiu o desenvolvimento in vitrodo fungo sem alterar a atividade das enzimas peroxidase e fenilalanina amônia-liase. Os fosfitos de potássio mostraram ação fungicida erradicante, com potencial de uso no manejo integrado da podridão olho de boi.

Mathematical modeling of dehydration of 'Fuji' and 'Gala' apples slices using infrared

The objective of this work was to determine and model the infrared dehydration curves of apple slices - Fuji and Gala varieties. The slices were dehydrated until constant mass, in a prototype dryer with infrared heating source. The applied temperatures ranged from 50 to 100 °C. Due to the physical characteristics of the product, the dehydration curve was divided in two periods, constant and falling, separated by the critical moisture content. A linear model was used to describe the constant dehydration period. Empirical models traditionally used to model the drying behavior of agricultural products were fitted to the experimental data of the falling dehydration period. Critical moisture contents of 2.811 and 3.103 kgw kgs-1 were observed for the Fuji and Gala varieties, respectively. Based on the results, it was concluded that the constant dehydration rates presented a direct relationship with the temperature; thus, it was possible to fit a model that describes the moisture content variation in function of time and temperature. Among the tested models, which describe the falling dehydration period, the model proposed by Midilli presented the best fit for all studied conditions.

Estimate of respiration rate and physicochemical changes of fresh-cut apples stored under different temperatures

In this study, the influence of storage temperature and passive modified packaging (PMP) on the respiration rate and physicochemical properties of fresh-cut Gala apples (Malus domestica B.) was investigated. The samples were packed in flexible multilayer bags and stored at 2 °C, 5 °C, and 7 °C for eleven days. Respiration rate as a function of CO2 and O2 concentrations was determined using gas chromatography. The inhibition parameters were estimated using a mathematical model based on Michaelis-Menten equation. The following physicochemical properties were evaluated: total soluble solids, pH, titratable acidity, and reducing sugars. At 2 °C, the maximum respiration rate was observed after 150 hours. At 5 °C and 7 °C the maximum respiration rates were observed after 100 and 50 hours of storage, respectively. The inhibition model results obtained showed a clear effect of CO2 on O2 consumption. The soluble solids decreased, although not significantly, during storage at the three temperatures studied. Reducing sugars and titratable acidity decreased during storage and the pH increased. These results indicate that the respiration rate influenced the physicochemical properties.

Development and optimization of a HPLC-RI method for the determination of major sugars in apple juice and evaluation of the effect of the ripening stage

The sugars in apple juice prove its authenticity and its sensory and nutritional properties. The aim of this study was to develop and validate a simple analytical method using high performance liquid chromatography with refractive index detection (HPLC-RI) to determinate and quantify the sugars sucrose, D-glucose, D-fructose, and D-sorbitol polyol in apple juices, as well as to analyze the juices from the Fuji suprema and Lis Gala cultivars at three ripening stages. The analytical performance parameters evaluated indicated that the method was specific for the compounds analyzed, and the linearity of the calibration curves of sugars showed high correlation coefficients (close to 1.0). The limits of detection and quantification are consistent with recommendations available in the literature for this type of matrix. Sample preparation is simple and generates small amount of residues. Over 70% of the sugars were determined in the juices of apples at the pre-ripe stage, with an increase during senescence. This method is applicable for the determination of sugars in juices and evaluation of apple ripening.

Incidence of resistance to benzimidazole fungicides in a field population of Venturia inaequalis /

The allele-specific polymerase chain reaction (PCR) was used to screen for the presence of benomyl resistance, and to characterize their levels and frequencies in field populations of Venturia inaequalis during two seasons. Three hundred isolates of V. inaequalis were collected each season from infected leaves of MalusX domestica. Borkh c.v. Mcintosh. The trees used were sprayed in the year prior to collection with five applications of benomyl, its homologue Azindoyle, or water. Monoconidial isolates of V. inaequalis were grown on 2% potato dextrose agar (PDA) for four weeks. Each isolate was taken from a single lesion from a single leaf. Total genomic DNA was extracted from the four week old colonies of V. inaequalis, prepared and used as a template in PCR reactions. PCR reactions were achieved by utilizing allele-specific primers. Each primer was designed to amplify fragments from a specific allele. Primer Vin was specific for mutations conferring the ben^^"^ phenotype. It was expected to amplify a 171 bp. DNA fragment from the ben^"^ alleles only. Primers BenHR and BenMR were specific for mutations conferring the ben"" and ben'^'' phenotypes, respectively. They were expected to amplify 172 bp. and 165 bp. DNA fragments from the ben"" and ben"^" alleles, respectively. Of the 953 isolates tested, 414 (69.9%) were benomyl sensitive (ben^) and 179 (30.1%) were benomyl resistant. All the benomyl resistant alleles were ben^"", since neither the ben"" nor the ben"" alleles were detected. Frequencies of benomyl resistance were 23%, 24%, and 23% for the 1997 collections, and were 46%, 26% and 38% for the 1998 collections for benomyl, Azindoyle and water treatments, respectively. Growth assay was performed to evaluate the applicability of using PCR in monitoring benomyl resistance in fungal field populations. Tests were performed on 14 isolates representing the two phenotypes (ben^ and ben^"'' alleles) characterized by PCR. Results of those tests were in agreement with PCR results. Enzyme digestion was also used to evaluate the accuracy and reliability of PCR products. The mutation associated with the ben^"'' phenotype creates a unique site for the endonuclease enzyme Bsh^236^ allowing the use of enzyme digestion. Isolates characterized by PCR as ben^'^'^ alleles had this restriction site for the SsA7l2361 enzyme. The most time consuming aspect of this study was growing fungal isolates on culture media for DNA extraction. In addition, the risk of contamination or losing the fungus during growth processes was relatively high. A technique for extracting DNA directly from lesions on leaves has been used (Luck and Gillings 1 995). In order to apply this technique in experiments designed to monitor fungicide resistance, a lesion has to be homogeneous for fungicide sensitivity. For this purpose, PCR protocol was used to determine lesion homogeneity. One hundred monoconidial isolates of V. inaequalis from 10 lesions (10-conidia/ lesion) were tested for their phenotypes with respect to benomyl sensitivity. Conidia of six lesions were homogeneous, while conidia of the remaining lesions were mixtures of ben^ and ben^ phenotypes. Neither the ben" nor the ben' phenotype was detected.

Protokoll der Gewässerschau in Kassel am 19. April 2005

Bei der Begehung von Olebach, Losse und Alte Losse wurden der ökomorphologische Zustand von Sohle, Böschung, Linienführung, Verzahnung von Wasser und Land und der Ufergehölze sowie die Landnutzung notiert. Einleitungen von Abwässern und die Ablagerung von Abfällen auf Böschung und Ufer wurde protokolliert. Wassertemperatur und elektrische Leitfähigkeit wurden an mehreren Punkten gemessen. Die Leitfähigkeitswerte werden vor dem Hintergrund der Geologie der Einzugsgebiete interpretiert und zur chemischen Indikation von diffusen und punktförmigen Einleitungen genutzt. Es werden Maßnahmen zur Renaturierung, Maßnahmen gegen die Ablagerung von Gartenabfällen auf Ufer und Böschung, Maßnahmen gegen die ackerbauliche Nutzung und Bebauung im Gewässerrandstreifen vorgeschlagen.

PGR forum: serving the crop wild relative user community

The Euro-Mediterranean region is an important centre for the diversity of crop wild relatives. Crops, such as oats (Avena sativa), sugar beet (Beta vulgaris), apple (Malus domestica), annual meadow grass (Festuca pratensis), white clover (Trifolium repens), arnica (Arnica montana), asparagus (Asparagus officinalis), lettuce (Lactuca sativa), and sage (Salvia officinalis) etc., all have wild relatives in the region. The European Community funded project, PGR Forum (www.pgrforum.org) is building an online information system to provide access to crop wild relative data to a broad user community; including plant breeders, protected area managers, policy-makers, conservationists, taxonomists and the wider public. The system will include data on uses, geographical distribution, biology, population and habitat information, threats (including IUCN Red List assessments) and conservation actions. This information is vital for the continued sustainable utilisation and conservation of crop wild relatives. Two major databases have been utilised as the backbone to a Euro-Mediterranean crop wild relative catalogue, which forms the core of the information system: Euro+Med PlantBase (www.euromed.org.uk) and Mansfeld’s World Database of Agricultural and Horticultural Crops (http://mansfeld.ipk-gatersleben.de). By matching the genera found within the two databases, a preliminary list of crop wild relatives has been produced. Around 20,000 of the 30,000+ species listed in Euro+Med PlantBase can be considered crop wild relatives, i.e. those species found within the same genus as a crop. The list is currently being refined by implementing a priority ranking system based on the degree of relatedness of taxa to the associated crop.

Characterization of the lipoxygenase (LOX) gene family in the Chinese white pear (Pyrus bretschneideri) and comparison with other members of the Rosaceae

Background Lipoxygenases (LOXs), a type of non-haem iron-containing dioxygenase, are ubiquitous enzymes in plants and participate in the formation of fruit aroma which is a very important aspect of fruit quality. Amongst the various aroma volatiles, saturated and unsaturated alcohols and aldehydes provide the characteristic aroma of the fruit. These compounds are formed from unsaturated fatty acids through oxidation, pyrolysis and reduction steps. This biosynthetic pathway involves at least four enzymes, including LOX, the enzyme responsible for lipid oxidation. Although some studies have been conducted on the LOX gene family in several species including Arabidopsis, soybean, cucumber and apple, there is no information from pear; and the evolutionary history of this gene family in the Rosaceae is still not resolved. Results In this study we identified 107 LOX homologous genes from five Rosaceous species (Pyrus bretschneideri, Malus × domestica, Fragaria vesca, Prunus mume and Prunus persica); 23 of these sequences were from pear. By using structure analysis, phylogenic analysis and collinearity analysis, we identified variation in gene structure and revealed the phylogenetic evolutionary relationship of this gene family. Expression of certain pear LOX genes during fruit development was verified by analysis of transcriptome data. Conclusions 23 LOX genes were identified in pear and these genes were found to have undergone a duplication 30–45 MYA; most of these 23 genes are functional. Specific gene duplication was found on chromosome4 in the pear genome. Useful information was provided for future research on the evolutionary history and transgenic research on LOX genes.

Enraizamento de plantas cultivadas in vitro

The adventitious rooting process of in vitro cultured plantlets is a technique that has been employed for the vegetative propagation of a significant number of native and exotic species. Many factors are associated with the rooting stage influencing positive and/or negatively the establishment of micropropagation protocols. The objective of this work was a literature review of the main inherent factors concerning in vitro rooting process including the correlation among others the endogenous and exogenous auxins levels, juvenility, genotype, mineral nutrition, culture medium conditions, addition of growth regulators and other substances as phenolic compounds and active coal besides growth environmental conditions of in vitro cultures. Although the complete elucidation of all processes involved with rooting of in vitro cultured plants has not been achieved so far, a comprehensive study of the main factors that interfere on rooting is fundamental for the establishment of new researches that might contribute for the rooting of economically important plants.

Maçã 'Royal Gala' revestida com quitosana estocada à temperatura ambiente

The aim of this work was to evaluate apples covered with chitosan during storage at ambient temperature. 'Royal Gala' apples were covered with chitosan (MRQ), immersed in acetic acid solution (MB) or did not receive any treatment (MC) and subsequently analyzed physicochemical, sensorial andinstrumentally, as well as for rotten occurrence, at zero, 7, 14, 21, 28, 35, 42 and 46 days of storage. The ratio, pH and reducing sugar contents increased while total titratable acidity, total sugars, total solids and firmness were reduced during storage. Was observed a drastically reduction of ascorbic acid and slight increase on soluble solids, except in the apples that were not pretreated (MC). Luminosity and chromaticity ratio values improved with time, with higher intensity in MRQ. Rotten occurrence was not expressive. The acceptance of appearance, color, global impression, aroma and flavor decreased with time, and appearance and texture were considered the most important sensory attributes for apples. The use of chitosan delayed ripening, reduced losses in firmness and presented a more intense bright, extending shelf life period up to seven days.

Potencial de resíduos agroindustriais como fontes de compostos bioativos

Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)