Identification of Prorocentrum minimum and Takayama pulchella by fluorescence in situ hybridization through epifluorescence microscopy and flow cytometry

Partial rDNA sequences of Prorocentrum minimum and Takayama pulchella were amplified, cloned and sequenced. and these sequence data were deposited in the GenBank. Eight oligonucleotide probes (DNA probes) were designed based on the sequence analysis. The probes were employed to detect and identify P. minimum and T. pulchella in unialgal and mixed algal samples with a fluorescence in situ hybridization method using flow cytometry. Epifluorescence micrographs showed that these specific probes labeled with fluorescein isothiocyanate entered the algal cells and bound to target sequences, and the fluorescence signal resulting from whole-cell hybridization varied from probe to probe. These DNA probes and the hybridization protocol we developed were specific and effective for P. minimum and T. pulchella, without any specific binding to other algal species. The hybridization efficiency of different probes specific to P. minimum was in the order: PM18S02 > PM28S02 > PM28S01 > PM18S01, and that of the probes specific to T. pulchella was TP18S02 > TP28S01 > TP28S02 > TP18S01. The different hybridization efficiency of the DNA probes could also be shown in the fluorescent signals between the labeled and unlabeled cells demonstrated using flow cytometry. The DNA probes PM18S02, PM28S02; TP18S02 and TP28S01, and the protocol, were also useful for the detection of algae in natural samples.

环境因子对有毒赤潮生物微型原甲藻（Prorocentrum minimum）生长增殖的影响

本文研究了温度、盐度、PH、营养盐（N、P）；维生素（B_(12)、B_1）、微量元素（Fe、Mn）、底泥抽提液和赤潮生物骨条藻与微型原甲藻生长的关系。结果表明，微型原甲藻增殖生长的适温范围为20－30 ℃，28 ℃时有最大生长率；最适盐度为10－35‰，25‰盐度生长率最高；PH 7.18-8.72范围内生长良好，影响不明显。N、P营养盐能促进微型原甲藻增殖生长，N盐以硝态氮效果最好，容易被吸收利用；氨盐为唯一N源时效果较差，浓度超过100uM时则有抑制作用；亚硝酸盐作用不明显。微型原甲藻主要是利用无机磷；有机磷作唯一P源时不能有效地被吸收利用，对微型原甲藻生长不利；维生素B_(12)促进微型原甲藻增殖生长效果明显，B_(12)与B_1综合效果更好；底泥抽提液、微量元素Fe、Mn都能有效地促进微型原甲藻增殖生长；研究证实底泥中腐植酸（HFA）虽有促进生长的作用，但效果尚不及抽提液，推测可能还有其他有效成分。研究发现中肋骨条藻有刺激微型原甲藻生长的作用，但其具体作用成分还有待研究。所得这些结果对进一步研究阐明微型原甲藻赤潮成因和机理有重要科学意义。

Three-dimensional fluorescence characteristics of dissolved organic matter produced by Prorocentrum donghaiense Lu

Filtration and cross-flow ultrafiltration techniques were used to separate culture media of Prorocentrum donghaiense at the exponential growth, stationary and decline stages into < 0.45 mu m filtrate, 100 kDa-0.45 mu m, 10-100 kDa and 1-10 kDa retentate and < 1 kDa ultrafiltrate fractions. The fluorescence. properties of different molecular weights of dissolved organic matter (DOM) were measured by excitation-emission matrix spectra. Protein-like and humic-like fluorophores were observed in the DOM produced by P. donghaiense. The central positions of protein-like fluorophores showed a red shift with prolonged growth duration, shifting from tyrosine-like properties at the exponential growth stage to tryptophan-like properties at the stationary and decline stages. The excitation wavelengths of protein-like fluorophores exhibited some change in the exponential growth and stationary stages with increased molecular size, but showed little change in the decline stage. However, the emission wavelengths in the decline stage exhibited a blue shift. Very distinct C type and A type peaks in humic-like fluorophores were observed. With a prolonged culture time, the intensities of both of the peaks became strong and the excitation wavelengths of peak A showed a red shift, while the A:C ratios fell. More than 94% of fluorescent DOM was in the lower than 1 kDa molecular weight fraction.

Morphological and genetic study of Prorocentrum donghaiense Lu from the East China Sea, and comparison with some related Prorocentrum species

In this paper, the detailed morphology of Prorocentrum donghaiense Lu from both field samples and cultures was examined, and a taxonomic comparison was made between P donghaiense and some related Prorocentrum spp. using morphological and molecular data and other published information. There were distinct differences among these species in morphological characteristics that historically have been presented as conservative features. The discrepancies extended beyond that of individual variations within the same species due to environmental factors. Therefore, these morphological features may not be conservative but, rather, polymorphic depending on environmental conditions. Based on this analysis, we suggest that the high-biomass bloom-forming species in the East China Sea, previously reported as Prorocentrum dentatum Stein, is P donghaiense Lu. The species reported from the East China Sea and Japanese and Korean waters appear to be the same species. Molecular data also suggest that P. dentatum (CCMP1517) and P. donghaiense are genetically identical. Therefore, the geographic distribution of P. donghaiense may be much wider than expected. (C) 2004 Elsevier B.V. All rights reserved.

A comparison of future increased CO2 and temperature effects on sympatric Heterosigma akashiwo and Prorocentrum minimum

Very little is known about how global anthropogenic changes will affect major harmful algal bloom groups. Shifts in the growth and physiology of HAB species like the raphidophyte Heterosigma akashiwo and the dinoflagellate Prorocentrum minimum due to rising CO2 and temperature could alter their relative abundance and environmental impacts in estuaries where both form blooms, such as the Delaware Inland Bays (DIB). We grew semi-continuous cultures of sympatric DIB isolates of these two species under four conditions: (1) 20 degrees C and 375 ppm CO2 (ambient control), (2)20 degrees C and 750 ppm CO2 (high CO2),(3) 24 degrees C and 375 ppm CO2 (high temperature), and (4) 24 degrees C and 750 ppm CO2 (combined). Elevated CO2 alone or in concert with temperature stimulated Heterosigma growth, but had no significant effect on Prorocentrum growth. P-Bmax (the maximum biomass-normalized light-saturated carbon fixation rate) in Heterosigma was increased only by simultaneous CO2 and temperature increases, whereas P-Bmax in Prorocentrum responded significantly to CO2 enrichment, with or without increased temperature. CO2 and temperature affected photosynthetic parameters alpha, Phi(max), E-k, and Delta F/F'(m) in both species. Increased temperature decreased and increased the Chl a content of Heterosigma and M Prorocentrum, respectively. CO2 availability and temperature had pronounced effects on cellular quotas of C and N in Heterosigma, but not in Prorocentrum. Ratios of C:P and N:P increased with elevated carbon dioxide in Heterosigma but not in Prorocentrum. These changes in cellular nutrient quotas and ratios imply that Heterosigma could be more vulnerable to N limitation but less vulnerable to P-limitation than Prorocentrum under future environmental conditions. In general, Heterosigma growth and physiology showed a much greater positive response to elevated CO2 and temperature compared to Prorocentrum, consistent with what is known about their respective carbon acquisition mechanisms. Hence, rising temperature and CO2 either alone or in combination with other limiting factors could significantly alter the relative dominance of these two co-existing HAB species over the next century. (c) 2007 Elsevier B.V. All rights reserved.

Interactions between the bloom-forming dinoflagellates Prorocentrum donghaiense and Alexandrium tamarense in laboratory cultures

Interactions between Prorocentrum donghaiense and Alexandrium tamarens, two bloom-forming dinoflagellates, were investigated using bi-algal cultures. All R donghaiense died, but A. tamarense was hardly affected by the end of the experiment when the initial cell density was set at 1.0 X 10(4) cells mL(-1) for P. donghaiense and 0.28 x 10(4) cells mL(-1) for A. tamarense. However, significant growth suppression occurred in either species when the initial cell density of P donghaiense increased to I. 0 X 105 Cells mL(-1) in the bi-algal culture, but no out-competement was observed. The simultaneous assay on the culture filtrates showed that P donghaiense filtrate prepared at a lower initial density (1.0 X 10(4) cells mL(-1)) stimulated growth of the co-cultured A. tanzarense (0.28 x 10(4) cells mL(-1)), but filtrate at a higher initial density (1.0 x 10(5) cells mL(-1)) depressed its growth. The filtrate of A. tamarense at a density of 0.28 x 10(4) cells mL(-1) killed all R donghaiense at a lower density (1.0 x 10(4) cells mL(-1)), but only exhibited an inhibitory effect on it at a higher density (1.0 x 10(5) cells mL(-1)). It is likely that these two species of microalgae interfere with each other mainly by releasing allelochemical substance(s) into the culture medium, and a direct cell-to-cell contact was not necessary for their mutual interaction. The allelopathic test further proved that A. tamarense could affect the growth of co-cultured P. donghaiense by producing allelochemical(s); moreover, A. tamarense culture filtrate at the stationary growth phase (SP) had a strongly inhibitory effect on P donghaiense compared to that at the exponential phase (EP). Results also demonstrated a dose-dependent relationship between the microalgal initial cell density and the degree of the allelopathic effect. The growth of R donghaiense and A. tamarense in the bi-algal cultures was simulated using a mathematical model to quantify the interaction. The estimated parameters from the model showed that the inhibition exerted by A. tamarense on P. donghaiense was about 17 and 8 times stronger than the inhibition P. donghaiense exerted on A. tamarense, when the initial cell density was set at 1.0 X 10(4) and 1.0 X 10(5) cells mL(-1) for P donghaiense, respectively. and 0.28 x 10(4) cells mL(-1) for A. tamarense in the bi-algal cultures. A. tamarense seems to have a survival strategy that is superior to that of P. donghaiense in bi-algal cultures under controlled laboratory conditions. (c) 2006 Elsevier B.V. All rights reserved.

Cancro do tronco da lima ácida Tahiti.

Esta enfermidade afeta somente a lima ácida Tahiti não causando nenhum dano a outras variedades de citros, ainda que sejam cultivadas no mesmo pomar. Inicialmente registrada no Texas em 1987, foi relatada no Brasil em 1993 na comunidade de Lagoa do Cedro no estado da Bahia e o seu agente causal identificado como Lasiodiplodia theobromae. Atualmente já foi registrada em vários estados do Norte, do Nordeste e do Sudeste do Brasil

Manual prático para o monitoramento e controle das pragas da lima ácida Tahiti.

Para a execução do Manejo Integrado de Pragas é necessário se fazer inspeções (amostragem ou vistoria) das pragas, doenças e seus inimigos naturais, de modo a fornecer dados seguros para as decisões a serem tomadas, não só para o controle das pragas mas também para a preservação dos inimigos naturais. A identificação de pragas e doenças e a proposição do uso de técnicas de Manejo Integrado de Pragas (MIP) são fortes aliados da implantação da produção integrada no cultivo da lima ácida Tahiti. O maior aliado no controle otimizado nos pomares é o monitoramento da dinâmica populacional das pragas e seus inimigos naturais, e da evolução espacial e temporal das doenças, aliando conhecimentos epidemiológicos e climáticos.

A exocorte da lima ácida 'Tahiti'.

Os citros podem ser infectados por várias espécies de viróides, dentre elas destaca-se o Citrus exocotis viróide, CEVd como o agente causal da exocorte dos citros. Esta doença está presente em praticamente todos as regiões citrícolas do mundo, associada aos sintomas de descamação em porta-enxertos como o limão ´Cravo' (Citrus limonia Osbeck) e o Poncirus trifoliata e ao nanismo de plantas neles estabelecidas. Na América Latina a lima ácida ´Tahiti' (C. latifolia Tan) é também descrita como suscetível à exocorte.

Produção integrada viabiliza certificação de propriedades de lima ácida 'Tahiti' no norte de Minas Gerais.

A produção da lima ácida 'Tahiti' tem evoluído no país tanto na adoção de novas tecnologias como em maiores áreas plantadas pelos citricultores, fato esse diretamente ligado à crescente demanda de mercados externos por essa fruta. A região do Perímetro Irrigado de Jaíba (MG) é uma nova fronteira agrícola onde a lima ácida 'Tahiti' tem se tornado uma cultura de destaque em função das condições climáticas e pela presença de diversas instituições de apoio àagricultura, tanto públicas (Empresa de Assistência Técnica e Extensão Rural de Minas Gerais - Emater- MG, Embrapa, Serviço Nacional de Aprendizagem Rural - Senar-MG, Companhia de Desenvolvimento dos Vales do São Francisco e do Parnaíba - Codevasf, Distrito de Irrigação de Jaíba - DIJ, Empresa de Pesquisa Agropecuária de Minas Gerais - Epamig, Instituto Mineiro de Agropecuária - IMA, Federação da Agricultura e Pecuária do Estado de Minas Gerais - Faemg e Central Exportaminas) quanto privadas, a exemplo da Centraljai, importante central de associações de produtores de limão, que tem papel decisivo no beneficiamento e na comercialização da produção local, pois possui uma empacotadora sob sua responsabilidade. Entretanto, paralelamente à demanda crescente por essa fruteira, também crescem as exigências dos mercados importadores nos quesitos ambientais e sociais, em que a rastreabilidade da produção é fator preponderante para garantir sucesso na comercialização. Por isso, o sistema de Produção Integrada (PI) aparece como uma opção ideal à cadeia produtiva do limão 'Tahiti', pois é constituído por um conjunto de práticas agronômicas selecionadas a partir daquelas disponíveis, que asseguram a qualidade e produtividade da cultura e prioriza princípios baseados na sustentabilidade, em que se tem maior controle tanto na utilização dos recursos naturais como na regulação de insumos poluentes, a fim de possibilitar o correto monitoramento do processo e a verificação de todo o sistema.

Plan de marketing para la inserción de una bebida natural a base de yacón con piña, en el mercado de Lima, Perú

El consumo de productos naturales en el mundo es cada vez más creciente, y dentro de ello las bebidas funcionales que desde sus orígenes han tenido una performance relativamente satisfactoria. El yacón (Smallanthus sochifolius)es una planta perene, herbácea, que crece en toda la serranía peruana. Su demanda en el mercado exterior ha hecho que cada vez tenga mayor importancia. En el 2014 se exportó USD 2 899.398,00 en forma de polvo, jarabe, deshidratado; sin embargo su consumo en el mercado nacional es bajo, a pesar del gran potencial nutritivo que posee. El mercado de Lima moderna se presenta como un importante atractivo de mercado que podría consumir la bebida. Por ello el objetivo del presente trabajo es Realizar un Plan de Marketing para el mercado de Lima moderna a fin de evaluar la factibilidad de insertar una bebida nueva: yacón con piña, para fomentar el consumo interno de este producto. La metodología utilizada fue de carácter exploratorio, descriptivo cuantitativa y cualitativa mediante información primaria y secundaria que ayudó a su desarrollo. Dentro de la información primaria se utilizó como herramienta la encuesta y la información secundaria a través de informes y estudios que se hicieron para el sector de bebidas no alcohólicas. El sector de bebidas no alcohólicas en el Perú, muestra un importante crecimiento en lo referente a bebidas saludables. En el último año (2014) el crecimiento de este sector fue de 6 por ciento, motivados por la percepción del consumidor en los beneficios saludables de estas bebidas. La zona de Lima moderna acoge al 72,2 por ciento del nivel socio económico A y B, este segmento concentra el más alto promedio de ingresos. En el análisis de Porter, se describe a la empresa como una industria, el cual para este rubro las barreras de ingreso son altas por la inversión en activos físicos y la inversión en publicidad y promoción por ser un producto nuevo. A través del estudio realizado se encontró que existe demanda para este producto, el 31,1 por ciento de los encuestados compraría definitivamente, 25,5 por ciento una vez por semana y el 18,3 por ciento todos los días. La estrategia utilizada en el presente trabajo fue la diferenciación, por las características saludables de la bebida y única en el mercado. La bebida es un producto de introducción en el mercado, el precio fijado es alto porque se orienta a un segmento de mercado selectivo y además fijado en la imagen y calidad del producto. La distribución se realizará a través de los supermercados, minimarkets, hoteles categorizados. Y por último se concluye que la introducción de un producto novedoso, podría marcar estándares en el mercado de bebidas no alcohólicas y permitir la creación de una nueva categoría de productos Premium naturales y funcionales.

Vulnerability of coastal ecosystems to changes in harmful algal bloom distribution in response to climate change: projections based on model analysis

Harmful algal blooms (HABs), those proliferations of algae that can cause fish kills, contaminate seafood with toxins, form unsightly scums, or detrimentally alter ecosystem function have been increasing in frequency, magnitude, and duration worldwide. Here, using a global modeling approach, we show, for three regions of the globe, the potential effects of nutrient loading and climate change for two HAB genera, pelagic Prorocentrum and Karenia, each with differing physiological characteristics for growth. The projections (end of century, 2090-2100) are based on climate change resulting from the A1B scenario of the Intergovernmental Panel on Climate Change Institut Pierre Simon Laplace Climate Model (IPCC, IPSL-CM4), applied in a coupled oceanographic-biogeochemical model, combined with a suite of assumed physiological 'rules' for genera-specific bloom development. Based on these models, an expansion in area and/or number of months annually conducive to development of these HABs along the NW European Shelf-Baltic Sea system and NE Asia was projected for both HAB genera, but no expansion (Prorocentrum spp.), or actual contraction in area and months conducive for blooms (Karenia spp.), was projected in the SE Asian domain. The implications of these projections, especially for Northern Europe, are shifts in vulnerability of coastal systems to HAB events, increased regional HAB impacts to aquaculture, increased risks to human health and ecosystems, and economic consequences of these events due to losses to fisheries and ecosystem services.