Polinização de Cacauí (Theobroma speciosum Willd. ex Spreng. - Sterculiaceae): implicações para conservação

O cacauí (Theobroma speciosum) é uma fruteira típica da Região Norte, cujas flores sapromiofílicas atraem dípteros. Os recursos que os insetos utilizam são muito variados e entre eles estão as flores, as quais podem ser utilizadas como alimento e local para reprodução. Este trabalho visou fazer uma comparação do sistema de polinização de T. speciosum em duas áreas de preservação distintas, contribuindo para a avaliação das estratégias de conservação da espécie. O trabalho foi realizado nos anos de 2000 a 2002, no Centro de Pesquisa do Trópico Úmido da Empresa Brasileira de Pesquisa Agropecuária (EMBRAPA/CPATU) e na Estação Científica Ferreira Penna (ECFPn/MPEG). Foram realizados estudos sobre estrutura populacional e fenologia de T. speciosum nas duas áreas de preservação, observação comportamental dos insetos no campo, coletas de insetos visitantes das flores de cacauí, análise de carreamento de pólen pelos insetos e verificação dos insetos emergentes no laboratório. As árvores de cacauí, em ambos os locais, florescem em períodos mais secos e frutificam em períodos mais úmidos. As populações de cacauí nas duas áreas diferem pela distribuição espacial e etária dos seus indivíduos. A falta de jovens e subadultos na área de preservação ex situ, mostra falha no "tum over' populacional, demonstrando fragilidade no sistema estabelecido. As inflorescências de T. speciosum são utilizadas por uma grande diversidade de invertebrados. Nove ordens de insetos, e pelo menos duas ordens de outros invertebrados, visitaram as flores de cacau'. As ordens com maior quantidade de indivíduos foram Hymenoptera, Thysanoptera, Diptera e Coleoptera. Os visitantes das flores de cacauí foram classificados como: oportunistas, predadores e polinizadores. Drosofilideos foram indicados como polinizadores de T speciosum, com a possibilidade da participação de alguns forídeos. As guildas dos drosofilídeos foram distintas nas duas áreas de preservação e mais diversa na área de preservação in situ. Quatro espécies foram comuns as duas áreas. A perda de diversidade e alteração na composição na guilda de polinizadores parece não ter afetado a população de T. speciosum na área de preservação ex situ. No caso em estudo o sistema de polinização apesar de profundamente alterado, ainda apresenta sinais de viabilidade.

Atividade antioxidante do extrato seco de cacau orgânico (Theobroma cacao): estudo de estabilidade e teste de aceitação de cremes acrescidos deste extrato

Conselho Nacional de Desevolvimento Científico e Tecnológico (CNPq)

EVALUATION OF IN VITRO AND IN VIVO EFFECTS OF SEMIPURIFIED PROTEINASE INHIBITORS FROM THEOBROMA SEEDS ON MIDGUT PROTEASE ACTIVITY OF LEPIDOPTERAN PEST INSECTS

We have characterized in vitro and in vivo effects of trypsin inhibitors from Theobroma seeds on the activity of trypsin- and chymotrypsin-like proteins from Lepidopteran pest insects. The action of semipurified trypsin inhibitors from Theobroma was evaluated by the inhibition of bovine trypsin and chymotrypsin activities determined by the hydrolysis of N-Benzoyl-DL-Arginine-p-Nitroanilide (BAPA) and N-Succinyl-Ala-Ala-Pho-Phe p-Nitroanilide (S-(Ala)2ProPhe-pNA). Proteinase inhibitor activities from Theobroma cacao and T. obovatum seeds were the most effective in inhibiting trypsin-like proteins, whereas those from T. obovatum and T. sylvestre were the most efficient against chymotrypsin-like proteins. All larvae midgut extracts showed trypsin-like proteolytic activities, and the putative trypsin inhibitors from Theobroma seeds significantly inhibited purified bovine trypsin. With respect to the influence of Theobroma trypsin inhibitors on intact insects, the inclusion of T. cacao extracts in artificial diets of velvet bean caterpillars (Anticarsia gemmatalis) and sugarcane borer (Diatraea saccharalis) produced a significant increase in the percentage of adult deformation, which is directly related to both the survival rate of the insects and oviposition.

Effects of Cor15a-IPT Gene Expression on Leaf Senescence in Transgenic Petunia x hybrida and Dendranthema x grandiflorum.

To prevent leaf senescence of young transplants or excised shoots during storage under dark and cold conditions, the cytokinin biosynthetic gene isopentenyl transferase (ipt) was placed under the control of a cold-inducible promoter cor15a from Arabidopsis thaliana and introduced into Petunia x hybrida 'Marco Polo Odyssey' and Dendranthema x grandiflorum (chrysanthemum) 'Iridon'. Transgenic cor15a-ipt petunia and chrysanthemum plants and excised leaves remained green and healthy during prolonged dark storage (4 weeks at 25 degrees C) after an initial exposure to a brief cold-induction period (4 degrees C for 72 h). However, cor15a-ipt chrysanthemum plants and excised leaves that were not exposed to a cold-induction period, senesced under the same dark storage conditions. Regardless of cold-induction treatment, leaves and plants of non-transformed plants senesced under prolonged dark storage. Analysis of ipt expression indicated a marked increase in gene expression in intact transgenic plants as well as in isolated transgenic leaves exposed to a short cold-induction treatment prior to dark storage. These changes correlated with elevated concentrations of cytokinins in transgenic leaves after cold treatment. Cor15a-ipt transgenic plants showed a normal phenotype when grown at 25 degrees C.

Einiges über die Taqanot Schum

Ferdinand Rosenthal

Evaluación in vitro de hongos nativos antagonistas de Moniliophthora rereri (Cif. & Par., Evans et al.,) en el cultivo de cacao (Theobroma cacao L.)

El cacao es afectado por una diversidad de microorganismos patógenos, entre ellos el hongo Moniliophthora roreri (Cif. & Par., Evans et al.,) causante de la moniliasis, que es una de las enfermedades de mayor importancia de este cultivo. El uso irracional de plaguicidas sintéticos para el manejo de esta enfermedad ha generado problemas al agroecosistema y la salud humana, por lo que se está demandando el uso de alternativas agroecológicas. En este contexto, el objetivo principal de esta investigación fue generar información acerca de la capacidad antagónica in vitro mostrada por hongos nativos contra la moniliasis del cacao. Se recolectaron muestras de suelo, hojas, corteza, frutos sanos y enfermos en tres zonas cacaoteras de Nicaragua para la caracterización morfológica del patógeno y de los potenciales microorganismos antagonistas. Las pruebas de antagonismo “in vitro” se realizaron a través de la técnica del cultivo dual. Se evaluó el crecimiento radial del patógeno y de los antagonistas, el porcentaje de inhibición del crecimiento radial (PICR) del patógeno y la capacidad de control biológico de los hongos antagonistas. Se probaron cuatro técnicas para la conservación de los hongos antagonistas. Se obtuvieron cuatro aislados del patógeno M. roreri y 17 aislados de hongos antagonistas. El PICR del patógeno ejercida por aislados del antagonista Trichoderma varió de 40.13% a 46.77%, en los aislados del antagonista Paecilomyces el PICR osciló entre 59.38% y 67.43%, en el único aislado del antagonista Clonostachys el PICR fue de 62.33%-67.22%. Los 17 aislados se ubicaron en las clases 1, 2 y 3 de la escala de valoración de antagonismo. La mejor técnica de conservación de hongos antagonistas y de M. roreri fue la de conservación con glicerol. Los resultados de este estudio indican que en el agroecosistema de cacao existen microorganismos nativos que tienen potencial para ser usados como agentes de control biológico del patógeno M. roreri.

Effect of Cocoa ( Theobroma cacao ) Plantation Types on the Distribution of Anuran Communities in Ore, Ondo State, Nigeria

Anuran species diversity and abundance were evaluated in different farming status of cocoa plantation in Ore, Ondo State, Nigeria. Applying the combination of visual encounter survey (VES) and acoustical survey (AES), the different farms surveyed were categorized as; 1) pure cocoa farms with pesticides applied (PCWP); 2) pure cocoa farms without pesticides application; 3) intercropped cocoa farms with pesticides applied (ICWP); and 4) intercropped cocoa farms without pesticides application (ICNP). The surrounding primary/secondary forest (PSFV) was sampled applying the transect method. A mean total of 690±2.6 anurans belonging to 28 species, 14 genera and 9 families were recorded during the study. Out of these, 10, 19, 17, 22 and 26 species were recorded respectively from PCWP, PCNP, ICWP, ICNP and PSFV. The anuran species richness was significantly different between the different cocoa plantation status and the forest sites (F4, 10 = 20.55, P< 0.01). The highest mean number of individuals (190±9.5) was observed at ICNP followed by PSFV (183±5.7), while the least was at PCWP (77±8.0). There was also significant difference (F4, 10 = 150.48, P< 0.01) between the abundance of anuran species at the various sites. Diversity indices (Shannon and Margalef) showed that the forest sites had the greatest (3.204 and 4.799) respectively while the pesticide using pure cocoa farms had the least (1.853 and 2.072). Generally, the result of the study clearly indicated that pesticide use cocoa farms were significantly lower compared to other sites. Farmers are encouraged to engage in intercropping (polyculture) especially with food and economic crops in a biodiversity friendly manner which could have similar characteristics of a natural ecosystem, thereby enhancing the biological diversity of agro-ecosystems.

Adubação de estabelecimento de capim elefante (Pennisetum purpureum, Schum) na região bragantina.

Foi realizado na Estação Experimental de Tracuateua, um experimento objetivando determinar a melhor forma de adubação de estabelecimento em Capim Elefante. Foram comparadas várias maneiras de aplicação de adubo mineral, em dois sistemas de utilização: com e sem corte intermediário da gramínea, aos 45 dias do plantio. Os efeitos dos tratamentos foram semelhantes quando não houve corte intermediário. Os resultados demonstram haver viabilidade de utilização de capineiras, mesmo ainda não estabelecidas, apesar de se obter menores produções nos primeiros cortes.

Analysis Of The Ergosterol Biosynthesis Pathway Cloning, Molecular Characterization And Phylogeny Of Lanosterol 14 α-demethylase (erg11) Gene Of Moniliophthora Perniciosa.

The phytopathogenic fungus Moniliophthora perniciosa (Stahel) Aime & Philips-Mora, causal agent of witches' broom disease of cocoa, causes countless damage to cocoa production in Brazil. Molecular studies have attempted to identify genes that play important roles in fungal survival and virulence. In this study, sequences deposited in the M. perniciosa Genome Sequencing Project database were analyzed to identify potential biological targets. For the first time, the ergosterol biosynthetic pathway in M. perniciosa was studied and the lanosterol 14α-demethylase gene (ERG11) that encodes the main enzyme of this pathway and is a target for fungicides was cloned, characterized molecularly and its phylogeny analyzed. ERG11 genomic DNA and cDNA were characterized and sequence analysis of the ERG11 protein identified highly conserved domains typical of this enzyme, such as SRS1, SRS4, EXXR and the heme-binding region (HBR). Comparison of the protein sequences and phylogenetic analysis revealed that the M. perniciosa enzyme was most closely related to that of Coprinopsis cinerea.

Apoplastic And Intracellular Plant Sugars Regulate Developmental Transitions In Witches' Broom Disease Of Cacao.

Witches' broom disease (WBD) of cacao differs from other typical hemibiotrophic plant diseases by its unusually long biotrophic phase. Plant carbon sources have been proposed to regulate WBD developmental transitions; however, nothing is known about their availability at the plant-fungus interface, the apoplastic fluid of cacao. Data are provided supporting a role for the dynamics of soluble carbon in the apoplastic fluid in prompting the end of the biotrophic phase of infection. Carbon depletion and the consequent fungal sensing of starvation were identified as key signalling factors at the apoplast. MpNEP2, a fungal effector of host necrosis, was found to be up-regulated in an autophagic-like response to carbon starvation in vitro. In addition, the in vivo artificial manipulation of carbon availability in the apoplastic fluid considerably modulated both its expression and plant necrosis rate. Strikingly, infected cacao tissues accumulated intracellular hexoses, and showed stunted photosynthesis and the up-regulation of senescence markers immediately prior to the transition to the necrotrophic phase. These opposite findings of carbon depletion and accumulation in different host cell compartments are discussed within the frame of WBD development. A model is suggested to explain phase transition as a synergic outcome of fungal-related factors released upon sensing of extracellular carbon starvation, and an early senescence of infected tissues probably triggered by intracellular sugar accumulation.

Endophytic and pathogenic isolates of the cacao fungal pathogen Moniliophthora perniciosa (Tricholomataceae) are indistinguishable based on genetic and physiological analysis

We evaluated the genetic and physiological variability of Moniliophthora perniciosa obtained from healthy and diseased branches of cacao (Theobroma cacao) plants. The diversity of the isolates was evaluated by RAPD technique and by studies of virulence and exoenzyme production. The genetic variability of endophytic and pathogenic M. perniciosa was evaluated in association with pathogenicity assays. RAPD analysis showed eight genetic groups, which were not related to plant disease status (healthy versus diseased branches). Isolates from cacao were included in three groups, excluding isolates from other host plants. Pathogenicity and enzyme analysis showed that the virulence of the isolates is not related to exoenzyme production. This is the first evidence that M. perniciosa colonizes healthy parenchymatic tissues, showing that endophytic behavior may occur in this species.

Searching for Moniliophthora perniciosa pathogenicity genes

The basidiomycete Moniliophthora perniciosa is the causal agent of witches` broom disease of Theobroma cacao (cacao). Pathogenesis mechanisms of this hemibiotrophic fungus are largely unknown. An approach to identify putative pathogenicity genes is searching for sequences induced in mycelia grown under in vitro conditions. Using this approach, genes from M. perniciosa induced under limiting nitrogen and light were identified from a cDNA library enriched by suppression subtractive hybridization as potential putative pathogenicity genes. From the 159 identified unique sequences, 59 were annotated and classified by gene ontology. Two sequences were categorized as ""Defence genes, Virulence, and Cell response"" presumably coding for allergenic proteins, whose homologues from other fungi are inducers of animal or plant defences. Differential gene expression was evaluated by quantitative amplification of reversed transcripts (RT-qPCR) of the putative identified genes coding for the two allergenic proteins (Aspf13 and 88KD), and for the enzymes Arylsulfatase (AS); Aryl-Alcohol Oxidase; Aldo-Keto Reductase (AK); Cytochrome P450 (P450); Phenylalanine Ammonia-Lyase; and Peroxidase from mycelia grown under contrasting N concentrations. All genes were validated for differential expression, except for the putative Peroxidase. The same eight genes were analysed for expression in susceptible plants inoculated with M. perniciosa, and six were induced during the early asymptomatic stage of the disease. In infected host tissues, transcripts of 88KD and AS were found more abundant at the biotrophic phase, while those from Aspf13, AK, PAL, and P450 accumulated at the necrotrophic phase, enabling to suggest that mycelia transition from biotrophic to necrotrophic might occur earlier than currently considered. These sequences appeared to be virulence life-style genes, which encode factors or enzymes that enable invasion, colonization or intracellular survival, or manipulate host factors to benefit the pathogen`s own survival in the hostile environment. (C) 2010 The British Mycological Society. Published by Elsevier Ltd. All rights reserved.

The mitochondrial genome of the phytopathogenic basidiomycete Moniliophthora perniciosa is 109 kb in size and contains a stable integrated plasmid

We present here the sequence of the mitochondrial genome of the basidiomycete phytopathogenic hemibiotrophic fungus Moniliophthora perniciosa, causal agent of the Witches` Broom Disease in Theobroma cacao. The DNA is a circular molecule of 109103 base pairs, with 31.9 % GC, and is the largest sequenced so far. This size is due essentially to the presence of numerous non-conserved hypothetical ORFs. It contains the 14 genes coding for proteins involved in the oxidative phosphorylation, the two rRNA genes, one ORF coding for a ribosomal protein (rps3), and a set of 26 tRNA genes that recognize codons for all amino acids. Seven homing endonucleases are located inside introns. Except atp8, all conserved known genes are in the same orientation. Phylogenetic analysis based on the cox genes agrees with the commonly accepted fungal taxonomy. An uncommon feature of this mitochondrial genome is the presence of a region that contains a set of four, relatively small, nested, inverted repeats enclosing two genes coding for polymerases with an invertron-type structure and three conserved hypothetical genes interpreted as the stable integration of a mitochondrial linear plasmid. The integration of this plasmid seems to be a recent evolutionary event that could have implications in fungal biology. This sequence is available under GenBank accession number AY376688. (c) 2008 The British Mycological Society. Published by Elsevier Ltd. All rights reserved.

Differential gene expression between the biotrophic-like and saprotrophic mycelia of the witches` broom pathogen Moniliophthora perniciosa

Moniliophthora perniciosa is a hemibiotrophic fungus that causes witches` broom disease (WBD) in cacao. Marked dimorphism characterizes this fungus, showing a monokaryotic or biotrophic phase that causes disease symptoms and a later dikaryotic or saprotrophic phase. A combined strategy of DNA microarray, expressed sequence tag, and real-time reverse-transcriptase polymerase chain reaction analyses was employed to analyze differences between these two fungal stages in vitro. In all, 1,131 putative genes were hybridized with cDNA from different phases, resulting in 189 differentially expressed genes, and 4,595 reads were clusterized, producing 1,534 unigenes. The analysis of these genes, which represent approximately 21% of the total genes, indicates that the biotrophic-like phase undergoes carbon and nitrogen catabollite repression that correlates to the expression of phytopathogenicity genes. Moreover, downregulation of mitochondrial oxidative phosphorylation and the presence of a putative ngr1 of Saccharomyces cerevisiae could help explain its lower growth rate. In contrast, the saprotrophic mycelium expresses genes related to the metabolism of hexoses, ammonia, and oxidative phosphorylation, which could explain its faster growth. Antifungal toxins were upregulated and could prevent the colonization by competing fungi. This work significantly contributes to our understanding of the molecular mechanisms of WBD and, to our knowledge, is the first to analyze differential gene expression of the different phases of a hemibiotrophic fungus.

Alterações fisiológicas após aplicação de silício em cacau e sua influência na preferência por pulgões

Os aspectos fisiológicos e bioquímicos influenciados pelo silício e que condicionam reações de resistência a pragas no cacaueiro ainda são pouco conhecidos. O objetivo deste experimento foi avaliar os efeitos da aplicação de silício sobre a fotossíntese, sobre o teor foliar de fenóis solúveis totais e a sua influência na preferência de Toxoptera aurantii em genótipos de cacau. O experimento foi conduzido em casa de vegetação, no delineamento de blocos casualizados, com quatro repetições, em arranjo fatorial 3 x 3, constituído por três genótipos (TSH 1188, CCN 51 e Catongo), duas doses de silicato de potássio (3 e 6 mL L-1), aplicadas por via foliar e um controle pulverizado somente com água. Foram realizadas avaliações das trocas gasosas, da fluorescência da clorofila a e do teor de compostos fenólicos foliar, além de um teste de preferência com chance de livre escolha com T. aurantii. Os genótipos TSH 1188 e CCN 51 apresentam maior eficiência fotoquímica e fotossintética, comparados com o Catongo. Independentemente do genótipo, a aplicação de silicato de potássio não alterou o índice de desempenho das plantas (PI ABS e PI TOTAL); no entanto, a dose 3 mL L-1 aumentou a fotossíntese líquida e o teor de fenóis solúveis totais. A dose 6 mL L-1 reduziu a preferência de T. aurantii pelas folhas do genótipo TSH 1188. A aplicação de silicato de potássio é promissora no aumento da resistência do cacaueiro a T. aurantii.