Secreção de enzimas mediada pelo pH do ambiente em isolados patogênicos e endofíticos do fungo Colletotrichum

Em fungos, um sistema de regulação gênica garante que enzimas sejam secretadas predominantemente em valores de pH do ambiente próximos aos pH ótimos de atividade correspondentes. Embora muita informação tenha sido acumulada sobre essa resposta adaptativa, não existem estudos envolvendo fungos fitopatogênicos, endofíticos e entomopatogênicos, bem como sobre outros aspectos relacionados às interações fungo-hospedeiro. No presente trabalho foi comparado, em meio sólido, o efeito do pH do ambiente na secreção das enzimas amilase, celulase, lipase, pectinase e protease por isolados endofíticos, fitopatogênico e entomopatogênicos pertencentes a diferentes espécies de Colletotrichum. Para todas as enzimas e em todos os isolados, observou-se um padrão de secreção dependente dos valores do pH do ambiente. Isolados endofíticos e patogênicos apresentaram padrões distintos de secreção de protease, com ótimos em pH de crescimento alcalino e ácido, respectivamente. Em meio líquido, uma fosfatase ácida Pi-repressível, secretada por um isolado endofítico, respondeu ao pH do ambiente, apresentando um aumento de 14 vezes na sua atividade específica durante o crescimento do fungo em meio ácido, quando comparado a meio alcalino. Além disso, foi clonada parte do gene pacC de Colletotrichum, o qual codifica um fator de transcrição responsável pela regulação dependente do pH do ambiente. É plausível a hipótese de que o pH ambiente é um fator de amplo espectro controlando a secreção enzimática durante as interações fungo-hospedeiro por meio de um circuito genético conservado.In fungi a genetic system ensures that enzymes are secreted mainly at ambient pH values corresponding to their optima of activity. Although a great deal of information has been obtained concerning this environmental response, there is a lack of studies involving phytopathogenic, endophytic and entomopathogenic fungi as well as different aspects of fungus-host interactions. This study compares in a plate-clearing assays, the effect of ambient pH in the secretion of amylase, cellulase, lipase, pectinase and protease by endophytic, phytopathogenic, and entomopathogenic isolates belonging to several species of Colletotrichum. All enzymes were secreted in a pH-dependent manner by all isolates. Endophytes and pathogens showed distinct patterns of protease secretion, with optima at alkaline and acid growth conditions, respectively. In liquid medium, a Pi-repressible acid phosphatase of an endophytic isolate responded to ambient pH, having a 14-fold increase in secreted specific activity at acid pH, as compared to alkaline pH. Furthermore, part of a Colletotrichum pacC homologue gene, coding for a transcriptional factor responsible for pH-regulated gene expression, was cloned. Ambient pH seems to be a general factor controlling enzyme secretion in fungus-host interactions through a conserved genetic circuit

A novel linkage map of sugarcane with evidence for clustering of retrotransposon-based markers

The development of sugarcane as a sustainable crop has unlimited applications. The crop is one of the most economically viable for renewable energy production, and CO2 balance. Linkage maps are valuable tools for understanding genetic and genomic organization, particularly in sugarcane due to its complex polyploid genome of multispecific origins. The overall objective of our study was to construct a novel sugarcane linkage map, compiling AFLP and EST-SSR markers, and to generate data on the distribution of markers anchored to sequences of scIvana_1, a complete sugarcane transposable element, and member of the Copia superfamily. The mapping population parents (‘IAC66-6’ and ‘TUC71-7’) contributed equally to polymorphisms, independent of marker type, and generated markers that were distributed into nearly the same number of co-segregation groups (or CGs). Bi-parentally inherited alleles provided the integration of 19 CGs. The marker number per CG ranged from two to 39. The total map length was 4,843.19 cM, with a marker density of 8.87 cM. Markers were assembled into 92 CGs that ranged in length from 1.14 to 404.72 cM, with an estimated average length of 52.64 cM. The greatest distance between two adjacent markers was 48.25 cM. The scIvana_1-based markers (56) were positioned on 21 CGs, but were not regularly distributed. Interestingly, the distance between adjacent scIvana_1-based markers was less than 5 cM, and was observed on five CGs, suggesting a clustered organization. Results indicated the use of a NBS-profiling technique was efficient to develop retrotransposon-based markers in sugarcane. The simultaneous maximum-likelihood estimates of linkage and linkage phase based strategies confirmed the suitability of its approach to estimate linkage, and construct the linkage map. Interestingly, using our genetic data it was possible to calculate the number of retrotransposon scIvana_1 (~60) copies in the sugarcane genome, confirming previously reported molecular results. In addition, this research possibly will have indirect implications in crop economics e.g., productivity enhancement via QTL studies, as the mapping population parents differ in response to an important fungal disease13CONSELHO NACIONAL DE DESENVOLVIMENTO CIENTÍFICO E TECNOLÓGICO - CNPQCOORDENAÇÃO DE APERFEIÇOAMENTO DE PESSOAL DE NÍVEL SUPERIOR - CAPESFUNDAÇÃO DE AMPARO À PESQUISA DO ESTADO DE SÃO PAULO - FAPESPnão temnão tem2010/51708-

Ambient pH-regulated enzime secretion in endophytic and pathogenic isolates of the fungal genus Colletotrichum Secreção de enzimas mediada pelo pH do ambiente em isolados patogênicos e endofíticos do fungo Colletotrichum

In fungi a genetic system ensures that enzymes are secreted mainly at ambient pH values corresponding to their optima of activity. Although a great deal of information has been obtained concerning this environmental response, there is a lack of studies involving phytopathogenic, endophytic and entomopathogenic fungi as well as different aspects of fungus-host interactions. This study compares in a plate-clearing assays, the effect of ambient pH in the secretion of amylase, cellulase, lipase, pectinase and protease by endophytic, phytopathogenic, and entomopathogenic isolates belonging to several species of Colletotrichum. All enzymes were secreted in a pH-dependent manner by all isolates. Endophytes and pathogens showed distinct patterns of protease secretion, with optima at alkaline and acid growth conditions, respectively. In liquid medium, a Pi-repressible acid phosphatase of an endophytic isolate responded to ambient pH, having a 14-fold increase in secreted specific activity at acid pH, as compared to alkaline pH. Furthermore, part of a Colletotrichum pacC homologue gene, coding for a transcriptional factor responsible for pH-regulated gene expression, was cloned. Ambient pH seems to be a general factor controlling enzyme secretion in fungus-host interactions through a conserved genetic circuit.<br>Em fungos, um sistema de regulação gênica garante que enzimas sejam secretadas predominantemente em valores de pH do ambiente próximos aos pH ótimos de atividade correspondentes. Embora muita informação tenha sido acumulada sobre essa resposta adaptativa, não existem estudos envolvendo fungos fitopatogênicos, endofíticos e entomopatogênicos, bem como sobre outros aspectos relacionados às interações fungo-hospedeiro. No presente trabalho foi comparado, em meio sólido, o efeito do pH do ambiente na secreção das enzimas amilase, celulase, lipase, pectinase e protease por isolados endofíticos, fitopatogênico e entomopatogênicos pertencentes a diferentes espécies de Colletotrichum. Para todas as enzimas e em todos os isolados, observou-se um padrão de secreção dependente dos valores do pH do ambiente. Isolados endofíticos e patogênicos apresentaram padrões distintos de secreção de protease, com ótimos em pH de crescimento alcalino e ácido, respectivamente. Em meio líquido, uma fosfatase ácida Pi-repressível, secretada por um isolado endofítico, respondeu ao pH do ambiente, apresentando um aumento de 14 vezes na sua atividade específica durante o crescimento do fungo em meio ácido, quando comparado a meio alcalino. Além disso, foi clonada parte do gene pacC de Colletotrichum, o qual codifica um fator de transcrição responsável pela regulação dependente do pH do ambiente. É plausível a hipótese de que o pH ambiente é um fator de amplo espectro controlando a secreção enzimática durante as interações fungo-hospedeiro por meio de um circuito genético conservado

Endophytic microorganisms: a review on insect control and recent advances on tropical plants

In the past two decades, a great deal of information on the role of endophytic microorganisms in nature has been collected. The capability of colonizing internal host tissues has made endophytes valuable for agriculture as a tool to improve crop performance. In this review, we addressed the major topics concerning the control of insects-pests by endophytic microorganisms. Several examples of insect control are described, notably those involving the interactions between fungi and grazing grasses from temperate countries. The mechanisms by which endophytic fungi control insect attacks are listed and include toxin production as well as the influence of these compounds on plant and livestock and how their production may be affected by genetic and environmental conditions. The importance of endophytic entomopathogenic fungi for insect control is also addressed. As the literature has shown, there is a lack of information on endophytes from tropical hosts, which are more severely affected by pests and diseases. Having this in mind, we have included an updated and extensive literature in this review, concerning new findings from tropical plants, including the characterization of endophytic fungi and bacteria microbiota from several Amazon trees, citrus and medicinal plants among others

High molecular diversity of the fungus Guignardia citricarpa and Guignardia mangiferae and new primers for the diagnosis of the citrus black spot

RAPD markers were used to investigate the distribution of genetic variability among a group of Guignardia citricarpa, G. mangiferae, and Phyllosticta spinarum isolates obtained from several hosts in Brazil, Argentina, Mexico, Costa Rica, Thailand, Japan, United States and South Africa. Pathogenic isolates G. citricarpa Kiely (anamorph form P. citricarpa McAlp Van Der Aa) are the etiological agent of the Citrus Black Spot (CBS), a disease that affects several citric plants and causes substantial injuries to the appearance of their fruits, thus preventing their export. Several previous studies have demonstrated the existence of an endophytic species with high morphological similarity to the causal agent of CBS that could remain latent in the same hosts. Consequently, the identification of the plants and fruits free from the causal agent of the disease is severely hampered. The RAPD analysis showed a clear discrimination among the pathogenic isolates of G. citricarpa and endophytic isolates (G. mangiferae and P. spinarum). In addition, a Principal Coordinate Analysis (PCO) based on a matrix of genetic similarity estimated by the RAPD markers showed four clusters, irrespective of their host or geographical origin. An Analysis of Molecular Variance (AMOVA) indicated that 62.8% of the genetic variation was found between the populations (G. citricarpa, G. mangiferae, P. spinarum and Phyllosticta sp.). Substantial variation was found in the populations (37.2%). Exclusive RAPD markers of isolates of G. citricarpa were cloned, sequenced and used to obtain SCARS (Sequence Characterized Amplified Regions), which allowed the development of new specific primers for the identification of G. citricarpa PCR (Polymerase Chain Reaction) analysis using a pair of primers specific to pathogenic isolates corroborating the groupings obtained by the RAPD markers, underscoring its efficiency in the identification of the causal agent of CBS.Marcadores de RAPD foram utilizados para investigar a distribuição da variabilidade genética de linhagens de Guignardia citricarpa, G. mangiferae, e Phyllosticta spinarum isolados em diversos hospedeiros no Brasil, Argentina, México, Costa Rica, Tailândia, Japão, EUA e África do Sul. O fungo Guignardia citricarpa Kiely (Phyllosticta citricarpa McAlp Van Der Aa) é o agente causal da Mancha Preta dos Citros (CBS), uma doença que afeta diversas plantas cítricas, causando dano a aparência dos frutos, prejudicando a exportação. Diversos estudos têm demonstrado a existência de uma espécie endofítica muito semelhante morfologicamente a G. citricarpa, e que permanece de forma endofítica no mesmo hospedeiro. Dificultando assim, a identificação de plantas e frutos livres do agente causa da CBS. A análise do perfil de RAPD revelou uma clara discriminação entre isolados patogênicos de G. citricarpa e isolados endofíticos (G. mangiferae e P. spinarum). A Análise de Coordenadas Principais (PCO) baseada na matriz de similaridade genética dos marcadores RAPD, demonstrou a formação de quatro grupos, sem relação com origem geográfica ou com hospedeiros utilizados. A análise de Variância de Marcadores Moleculares (AMOVA) indicou que 62,8% da variação genética é encontrada entre as populações (G. citricarpa, G. mangiferae, P. spinarum and Phyllosticta sp.). Entretanto, variação substancial foi encontrada dentro destas populações (37,2%). Bandas de RAPD exclusivas de isolados de G. citricarpa foram clonadas, sequenciadas e utilizadas na obtenção de SCARS (Sequence Characterized Amplified Regions), que permitiram o desenvolvimento de novos primers específicos para a identificação de G. citricarpa. Reações de PCR (Polymerase Chain Reaction) utilizando este par de primers corroboraram os agrupamentos obtidos pelos marcadores de RAPD, revelando sua eficiência na identificação do agente causal da CBS

BRAZILIAN ARCHIVES OF BIOLOGY AND TECHNOLOGY A N I N T E R N A T I O N A L J O U R N A L High Molecular Diversity of the Fungus Guignardia citricarpa and Guignardia mangiferae and New Primers for the Diagnosis of the Citrus Black Spot

ABSTRACT RAPD markers were used to investigate the distribution of genetic variability among a group o