Entre as hospedeiras de invasão sistêmica do vírus do mosaico do mamoeiro - estirpe mamoeiro (Papaya ringspot virus type P; PRSV-P) encontram-se espécies de cucurbitáceas, cuja suscetibilidade à transmissão experimental, mecânica e com afídeos, é variável. A literatura nacional e internacional apresenta resultados distintos quanto à recuperação desse vírus a partir de cucurbitáceas presentes próximas ou no interior de plantios de mamoeiros infectados com esse vírus. O presente trabalho teve como objetivo avaliar em casa de vegetação a suscetibilidade de quatro espécies de cucurbitáceas a cinco isolados do PRSV-P obtidos de diferentes regiões do Brasil e inoculados de forma mecânica. Visou também estudar a infecção natural de cucurbitáceas cultivadas nas entrelinhas ou próximas de mamoeiros com mosaico. Para o desenvolvimento do trabalho em casa-de-vegetação os isolados do PRSV-P mantidos em mamoeiros, foram inoculados nos cotilédones de abobrinha de moita cv. Caserta, moranga cv. Exposição, pepino híbrido Primepack Plus e melancia cv. Crimson Sweet. As plantas foram avaliadas com base nos sintomas e indexadas por PTA-ELISA, e recuperação biológica do biótipo P do PRSV através de inoculações em mamoeiro. A confirmação da infecção dos mamoeiros foi realizada da mesma forma, por sintomatologia e indexação por PTA-ELISA. A abobrinha de moita foi a espécie mais suscetível aos cinco isolados do PRSV-P, seguida da melancia e do pepino. Não foi possível transmitir o vírus a moranga cv. Exposição. Para estudar a infecção natural realizou-se um ensaio com plantas de abobrinha de moita em Linhares-ES, três ensaios independentes em Rinópolis-SP e quatro em Piracicaba-SP, incluindo-se nessa última localidade a melancia e o pepino. Depois de aproximadamente 40 a 60 dias de exposição em campo coletaram-se amostras individuais ou compostas (de 3 a 5 plantas) das folhas dos ponteiros das plantas para realizar a recuperação biológica do PRSV-P para mamoeiros em casa de vegetação. A presença de afídeos foi monitorada em Piracicaba durante a execução dos experimentos no campo. No único teste de exposição em Linhares, nenhuma planta de abobrinha cultivada entre mamoeiros com mosaico mostrou-se infectada com esse vírus. O PRSV-P foi recuperado da abobrinha de moita em proporções variáveis em 2 dos 3 testes realizados em Rinópolis, e em 3 dos 4 testes realizados em Piracicaba. Nenhuma planta de melancia e pepino cultivada entre mamoeiros com mosaico foi infectada com o PRSV-P. Não foi possível recuperar o PRSV-P de nenhuma planta de abobrinha cultivada entre 5 e 80 metros de distância dos mamoeiros com mosaico em Piracicaba. Foram capturados afídeos vetores do PRSV-P e foi possível detectar plantas infectadas com os potyvirus PRSV-W e ZYMV, o que demonstra a presença e atividade dos vetores de vírus. Os resultados confirmaram a suscetibilidade variável das espécies de cucurbitáceas ao PRSV-P. Embora a abobrinha de moita fosse a única espécie encontrada naturalmente infectada pelo PRSV-P quando cultivada entre linhas de mamoeiro com mosaico, a presença de cucurbitáceas nos campos de produção de mamoeiro, especialmente quando o controle do mosaico do mamoeiro é feito através do roguing, não é recomendada. / Besides Carica papaya, Papaya ringspot virus type P (PRSV-P) infects systemically only species belonging to the family Cucurbitaceae. Their susceptibility varies according to the species/cultivar, virus isolate and the method of inoculation. Attempts to recovery PRSV-P from naturally infected cucurbit plants grown near to or among diseased papaya trees have shown distinct results worldwide. This study aimed to evaluate the susceptibility of Cucurbita pepo cv. Caserta, Cucurbita maxima cv. Exposiçao, Cucumis sativus hybrid Primepack Plus, and Citrullus lanatus cv. Crimson Sweet to five isolates of PRSV-P obtained from different regions of Brazil. It was also evaluated the natural infection of cucurbit plants grown between rows and in the vicinity of papaya trees infected with PRSV-P. The five PRSV-P isolates were maintained in papaya plants. Cucurbit plants grown in pots under greenhouse conditions were mechanically inoculated with each isolate at the cotiledonary stage. The plants were assessed based on symptoms and infection was confirmed by PTA-ELISA using extracts from the inoculated cotyledons and upper leaves. The same extracts were also mechanically inoculated on papaya plants in order to recover the virus isolate. Inoculated papaya plants were also tested by PTA-ELISA. Zucchini squash was the most susceptible species to PRSV-P, followed by watermelon and cucumber. Pumpkin cv. Exposição was not infected. To study the natural infection of zucchini squash cv. Caserta by PRSV-P, a trial was carried out in Linhares, State of Espírito Santo; three independent trials were carried out in Rinópolis; and four trials were carried out in Piracicaba, both regions located in the State of São Paulo. Watermelon and cucurbit were also included in some trials in Piracicaba. After approximately 40 to 70 days, leaf samples were collected and tested individually or in groups of three to five plants for the presence of PRSV-P by mechanical inoculation on papaya plants under greenhouse conditions. None of the zucchini squash plants grown between rows of infected papaya trees in Linhares was found infected by PRSV-P based on the virus recovery test to papaya plants. The virus was also not recovered from watermelon and cucurbit plants grown between rows of infected papaya trees in Piracicaba. On the other hand, PRSV-P was recovery from zucchini squash plants grown intercalated with diseased papayas in Rinópolis and Piracicaba. The number of infected plants varied among the trial. Several attempts to recover PRSV-P from innumerous zucchini squash plants grown approximately five to 80 meters from diseased papaya trees in Piracicaba failed. Alates of several species of aphids were captured in the field at Piracicaba. Also, innumerous cucurbit plants were found infected by the potyviruses Papaya ringspot virus type W and Zucchini yellow mosaic virus, suggesting aphids activity in the area. The results confirmed the variable susceptibility of cucurbit species to infection with PRSV-P. Although natural infection with PRSV-P was restricted to zucchini squash cv. Caserta grown among infected papaya trees, the presence of cucurbit plants in the vicinity of papaya orchards, especially where disease control is done by systematic rouging of diseased plants, should not be allowed.

O presente trabalho teve como objetivo avaliar a eficiência do sistema de análise de imagens de plântulas (Seedling Vigor Imaging System® - SVIS®) na determinação do potencial fisiológico de sementes de pepino, em comparação com informações fornecidas por testes de vigor recomendados para essa espécie. O trabalho foi desenvolvido em três épocas de avaliação com intervalos aproximados de três meses. Em cada época, a avaliação do potencial fisiológico foi efetuada usando-se os testes de germinação, primeira contagem de germinação, envelhecimento acelerado tradicional e com solução saturada de NaCl, comprimento de plântulas e emergência de plântulas. Os resultados desses testes foram comparados com os dados obtidos com o uso do software Seedling Vigor Imaging System (SVIS®), que avaliou o desenvolvimento de plântulas escaneadas, mediante a obtenção de valores do comprimento da raiz primária, do hipocótilo e das plântulas, do índice de vigor e de uniformidade de desenvolvimento. Além disso, foram estudadas variações na proporção da participação do crescimento e da uniformidade de desenvolvimento das plântulas para o cálculo do índice de vigor no \"software SVIS®, na tentativa de verificar possíveis variações na sensibilidade do sistema para detectar diferenças no potencial fisiológico dos lotes avaliados. Concluiu-se que a análise computadorizada de imagens de plântulas é uma alternativa consistente e promissora para avaliação do potencial fisiológico de sementes de pepino, com eficiência comprovada equivalente à verificada em testes tradicionais para determinação do vigor. As análises pelo SVIS® em plântulas com quatro dias de idade são mais precisas e a proporção crescimento/uniformidade para a composição do cálculo do índice de vigor não afeta a precisão da determinação do potencial fisiológico de sementes de pepino pela análise SVIS®. / This study aimed to evaluate the efficiency of seedling digital image analysis (Seedling Vigor Imaging System® - SVIS®) to determine the physiological quality of cucumber seed lots, compared to information provided by vigor tests recommended for this species. Research comprised three evaluation times with a threemonth interval. Physiological quality determination comprised germination, germination first count, traditional and saturated salt accelerated aging, seedling length and seedling emergence tests. Results obtained were compared to data by the evaluation using the software Seedling Vigor Imaging System (SVIS®); in this system, seedlings were scanned and the image analysis made possible to obtain values of the primary root, hypocotyl and seedling lengths in addition to a vigor index and uniformity of seedling of development. It also studied the importance of the contribution of growth intensity and uniformity to compose the value of the vigor index calculated by the software. It was concluded that digital image analysis of seedlings is a consistent and promising alternative to provide reliable information of cucumber seed physiological quality, comparable to those from traditional tests. Analyses by SVIS ® of four-day old seedlings are more accurate, and the proportion growth/uniformity in the composition of the vigor index does not affect the accuracy of determining the physiological quality of cucumber seeds by analyses SVIS®.

Anbauverfahren von Gurken am »Hohen Draht« mit einer Spanndrahthöhe von 3,40 m werden hinsichtlich Ertragsleistung, Arbeitszeitaufwand und Wirtschaftlichkeit mit Standardanbauverfahren (Spanndrahthöhe 2,20 m) verglichen. Die Ergebnisse des vierjährigen Forschungsprojektes zeigen, dass Gurken im Gewächshaus auf Substrat am »Hohen Draht« mit 200 bis 220 Gurken/m² deutlich höhere Erträge als die Standardverfahren erzielen. Pflege und Arbeitszeitaufwand sind allerdings eindeutig höher. Der Anbau von Gurken am »Hohen Draht« führte dennoch zu einer Verbesserung der Deckungsbeiträge um fast ein Drittel. Die Investitionskosten für die Umstellung von 1 ha Standardverfahren auf den Gurkenanbau am »Hohen Draht« betragen 70.000 bis 80.000 Euro. Bei steigenden Energiekosten ist das Verfahren am »Hohen Draht« eine wirtschaftliche Alternative zu den gegenwärtigen Standardverfahren. Allerdings mangelt es an guten Spezialsorten. Problematisch ist das Risiko von Ertragsausfällen durch Stängelbotrytis.

The mode of feeding exhibited by different organisms may influence the form or quantity of copper that is bioaccumulated. This hypothesis has been tested by exposing 2 species of sea cucumbers, Pentacta anceps and Stichopus chloronotus, which possess different feeding modes, to varying concentrations of copper. The digestive tract and body wall were dissected and analyzed for copper concentration using atomic absorption spectroscopy. A trend was present, exhibiting a small dose dependent curve. The results did not show a significant difference between species or treatments. This study indicates that feeding mode may influence the amount of copper accumulated but it could not be concluded because of a small sample size. The model used in this study demonstrated that copper was being actively precipitated out of the water and deposited into sediment. Future studies focusing on pollutant uptake may find this useful when evaluating the role of feeding mode or habitat.

京都大学 / 新制・論文博士 / 博士(農学) / 乙第13412号 / 論農博第2895号 / 新制||農||1085(附属図書館) / 学位論文||R3||N5322(農学部図書室) / 京都大学大学院農学研究科農学専攻 / (主査)教授 冨永 達, 教授 土井 元章, 教授 那須田 周平 / 学位規則第4条第2項該当 / Doctor of Agricultural Science / Kyoto University / DFAM

<p>Systemic Acquired Resistance (SAR) is a plant defense response induced by an initial infection in one part of the plant that leads to broad-spectrum resistance to normally virulent pathogens in distant naïve leaves. As part of the Cameron research team, I contributed to demonstrating that the lipid transfer protein, DIR1 is required for SAR long distance signaling in <em>Arabidopsis</em> and travels from induced to distant tissues during SAR. A highly similar<em> Arabidopsis</em> protein DIR1-like was identified and is thought to be responsible for the occasional SAR-competent phenotype observed in the <em>dir1-1</em> mutant. This work provides evidence for the idea that DIR1 and DIR1-like are paralogs created by a recent duplication event and that similar to DIR1, DIR1-like may travel to distant tissues during SAR. To better understand DIR1 and DIR1-like contribution during SAR, <em>dir1-1dir1-like</em> double mutant transgenic plants were created as well as transgenic plants expressing epitope- (HA and FLAG) and fluorescent- (iLOV and phiLOV) tagged DIR1 and DIR1-like to facilitate visualization of movement during SAR. Several putative DIR1 orthologs were identified in crop plants and cucumber CucDIR1 was shown to be functionally equivalent to AtDIR1 in <em>dir1-1</em> complementation studies providing further evidence that DIR1 plays an important role in SAR across plant species. By analyzing conservation between DIR1, DIR1-like and the putative DIR1 orthologs, several protein residues were identified that may be important for DIR1 function during SAR. DIR1 proteins were modified at these sites and the importance of these residues was supported by the reduced binding of the TNS hydrophobic probe in these DIR1 variants. Taken together, this thesis suggests that DIR1 and DIR1-like both participate in SAR in <em>Arabidopsis</em>, that DIR1 crop orthologs are also important for the SAR response and that DIR1 possesses several sites that are critical for its function in long distance SAR signaling.</p> / Doctor of Philosophy (PhD)

Greenhouse environments often promote bacterial and fungal infections in important crop plants. Exogenous application of chemical inducers could help reduce the severity of infection, or even prevent infection. Small molecules such as glycerol, azelaic acid and pipecolic acid have been implicated as being important signaling molecules during Systemic Acquired Resistance (SAR). To examine if these small molecules could be used to induce resistance in crop plants, exogenous treatment assays were developed in cucumber. Glycerol spray and azelaic acid infiltration induced modest resistance at locally treated leaves. Pipecolic acid soil treatment induced modest resistance in aerial tissue of cucumber plants, and strong resistance when plants were treated weekly. This knowledge may be useful in promoting the commercialization of SAR-associated compounds to protect important crop plants against disease. Plants possess multiple defense pathways that include an SA signaling component to initiate resistance to microbial pathogens. However, during Age-Related Resistance (ARR) in Arabidopsis, a number of studies support that SA acts as an anti-microbial and anti-biofilm agent against Pseudomonas syringae pv. tomato (Pst) in the plant intercellular space. Little is known about the role of Pst biofilm formation during infection of young plants or if other defense responses act to suppress bacterial biofilm formation. Therefore Pst biofilm formation and the effect of PAMP Triggered Immunity (PTI) on bacterial biofilm formation was examined. PTI was induced with flg22 in wild-type Col-0, fls2, bak1-3 (PTI mutants) and sid2-2 (SA biosynthesis mutant). In vivo bacterial biofilm-like aggregate formation was monitored using Pst DC3000 PDSK-GFPuv and epifluorescence microscopy. Pst aggregate occurrence and size were positively correlated with bacterial success in susceptible plants (wild-type Col-0, fls2, bak1-3, sid2-2), while fewer and smaller bacterial aggregates were observed in Col-0 undergoing PTI. To determine if the extracellular polysaccharide, alginate was a major contributor to biofilm formation, in vivo bacterial aggregate formation was monitored using alginate deficient Pst-GFP. Alginate deficient Pst-GFP and wild-type Pst grew to similar levels in wild-type plants suggesting that the ability to produce alginate was not necessary for Pst pathogenicity and success in planta. Fewer alginate-deficient Pst aggregates were observed compared to wild-type Pst in inoculated plants, suggesting that the ability to produce alginate was modestly important for aggregate formation. These data provide novel insights into how biofilms form in planta, the association between pathogen virulence and biofilm formation, and how plant defense responses such as PTI not only reduce bacterial growth, but also target biofilms. / Thesis / Master of Science (MSc)

The purpose of this research carried out in 1998 and 1999 was to develop methods for strip cropping of cucumbers with rye/vetch and black plastic mulch. Effects of planting methods, weed control measures, and cover crop management techniques on pest and beneficial insects, petiole sap nitrate-nitrogen, soil moisture, yields and economic viability were assessed. Four treatments, namely cucumber direct seeded in black plastic mulch on tilled bare ground (conventional); cucumber direct seeded in black plastic mulch laid over incorporated rye/vetch residue; cucumber direct seeded into no-till rolled rye/vetch; and seedlings transplanted into no- till rolled rye/vetch, were evaluated in a randomized complete block design. Weeds were controlled in half the plot by critical manual weeding and pre-emergence herbicides applied in the other half in all treatments except the conventional system. Three-week interval staggered plantings of buckwheat to provide flowers for adult beneficial insects were also evaluated. Results obtained indicate that rye/vetch habitat is more attractive to beneficial insects than cucumber beetles before rolling or flail mowing. There were higher densities of adult Diptera (an indicator for Tachinid parasitoids) in both years and of Pennsylvania leatherwings in 1999 in plots with rye/vetch than in plots with rye only before planting. Preferential attraction to adult Diptera was not found after planting with no differences in cumulative densities between no-till and black plastic mulch plots after rolling or flail mowing. However, adult Diptera densities were positively correlated with cucumber yield, higher densities of cucumber beetles occurred in black plastic mulch than in no-till plots in both years, and bacterial wilt, transmitted by cucumber beetles, was reduced in no-till in 1999. Similarly, rye/vetch habitat plots had a higher diversity of Carabidae species (in both habitat areas and crop rows) before rolling or flail mowing, and higher densities of Carabidae (in habitat areas), Staphylinidae (in both habitat areas and crop rows) and spiders (in crop rows) after rolling or flail mowing. Black plastic mulch plots with flail mowed, incorporated rye/vetch residues had higher petiole sap nitrate-nitrogen and higher early season cucumber plant dry weights than in conventional plots; later in the season, the highest petiole sap nitrate-nitrogen occurred in no-till plots. No-till had higher marketable cucumber yield than plastic systems. The profitability of these production systems depended more on differences in marketable yield than on cost differences. The plastic with incorporated rye/vetch and no-till transplant systems were more profitable during early harvests in late July, while no- till direct seeded and transplant systems were more profitable from early August onwards. The three-week interval staggered planting of buckwheat led to conservation of beneficial insects, and the second and third seeded buckwheat flowering periods coincided with the period vetch had lost flowers. / Ph. D.

Pseudoperonospora cubensis (Bert. et Curt) Rost. is the causal agent of cucurbit downy mildew (CDM). It is the most damaging cucumber pathogen on the Eastern Shore of Virginia and eastern parts of the United States. Pseudoperonospora cubensis is an obligate oomycete pathogen, infecting crops within the Cucurbitaceae family. The disease is characterized by angular chlorotic lesions and a downy or felt-like appearance on the abaxial side of the leaf. Control of this pathogen includes use of resistant cucumber cultivars and costly fungicide programs. Continuous use has led to resistance to commonly used fungicides. This has become a major concern and in response, seed companies have developed cucumber cultivars which claim downy mildew resistance.  This study evaluates different cucumber cultivars and assesses their level of resistance to CDM. The results indicate that an integrated management approach of reduced fungicide application and the use of resistant cultivars can suppress levels of CDM and yield a cucumber crop. Additionally, a molecular study was conducted, comparing the relative expression of genes encoding a basic PR-1 protein, a cytosolic ascorbate peroxidase protein and three resistance (R) gene proteins, in nineteen cultivars. All of the selected genes were analyzed using real-time PCR. The relative expression levels of the R-genes varied between cultivars. The basic PR-1 protein decreased expression in the majority of the cultivars, suggesting no involvement in the first twenty-four hours. Cytosolic ascorbate peroxidase relative expression levels suggest an increase in susceptible cultivars and a decrease in tolerant cultivars. / Master of Science

Fresh produce may be contaminated by bacterial pathogens including Listeria monocytogenes during harvesting, packaging, or transporting. Consumers may be at risk of foodborne illness if produce become contaminated. In this project, a cavitation process (formation of bubbles in water) was studied to determine the efficacy of microbubbles at inactivating the pathogen L. monocytogenes on stainless steel and the surface of fresh cucumber and avocado. Stainless steel coupons (1"×2''), cucumber, and avocado surfaces were inoculated with L. monocytogenes (LCDC strain). After 1, 24 or 48 h, loosely attached cells were washed off, and inoculated areas were targeted by microbubbles (~0.5 mm dia.) through an air stone (1.0 L air/min) for 1, 2, 5, or 10 min. After treatment, samples were transferred to sterile containers and serial diluted in peptone water and plated on Oxford agar. Plates were incubated for 48 h at 35℃. For stainless steel, the mean log reduction of L. monocytogenes (48 h drying) peaked at 2.95 after 10 min of microbubbles when compared to a no bubble treatment. After 48 h pathogen drying, cucumbers treated for 10 min resulted in a 1.78 mean log reduction of L. monocytogenes. For avocados, the mean log reduction of L. monocytogenes (24 hr drying) peaked at 1.65 after 10 min of microbubbles. This cavitation treatment (10 mins) reduced over 95% of L. monocytogenes on the surface of stainless steel, cucumber, and avocado. Microbubble applications may be an effective, economical, and environmental-friendly way to remove L. monocytogenes and possibly other bacterial pathogens from food impact surfaces and the surface of whole, intact fresh produce. / Master of Science in Life Sciences / Fresh produce may be contaminated by bacterial pathogens like Listeria monocytogenes during the process of harvesting, packaging, or transport. Consuming contaminated fresh produce without enough and proper decontaminated measures, consumers are in risk of being infected with foodborne illness. A cavitation process (formation of bubbles in water) was conducted to determine the efficacy of microbubbles at inactivating the pathogen Listeria monocytogenes on the impact surface of fresh produce (stainless steel), fresh cucumber, and fresh avocado. Stainless steel coupons (1"×2''), fresh cucumbers, and fresh avocados were inoculated respectively with Listeria monocytogenes (LCDC strain). After removing loosely attached pathogen cells on the surface of inoculated steel coupons, cucumbers, and avocados, their inoculated areas were targeted by microbubbles (~0.5 mm dia.) for 1, 2, or 10 minutes, with a constant air flow rate of 1.0 L/min through an air stone. The L. monocytogenes on stainless steel and cucumber, and avocado surfaces was reduced by 95% to 99%. This study suggests that microbubbles may remove, and possibly inactivate, L. monocytogenes effectively from the surface of fresh produce and their impact surfaces. Microbubbles thus could be an effective, economical, and environmental-friendly tool for minimizing produce contamination.