Epicoccum nigrum P16, a Sugarcane Endophyte, Produces Antifungal Compounds and Induces Root Growth

Background: Sugarcane is one of the most important crops in Brazil, mainly because of its use in biofuel production. Recent studies have sought to determine the role of sugarcane endophytic microbial diversity in microorganism-plant interactions, and their biotechnological potential. Epicoccum nigrum is an important sugarcane endophytic fungus that has been associated with the biological control of phytopathogens, and the production of secondary metabolites. In spite of several studies carried out to define the better conditions to use E. nigrum in different crops, little is known about the establishment of an endophytic interaction, and its potential effects on plant physiology. Methodology/Principal Findings: We report an approach based on inoculation followed by re-isolation, molecular monitoring, microscopic analysis, plant growth responses to fungal colonization, and antimicrobial activity tests to study the basic aspects of the E. nigrum endophytic interaction with sugarcane, and the effects of colonization on plant physiology. The results indicate that E. nigrum was capable of increasing the root system biomass and producing compounds that inhibit the in vitro growth of sugarcane pathogens Fusarium verticillioides, Colletotrichum falcatum, Ceratocystis paradoxa, and Xanthomomas albilineans. In addition, E. nigrum preferentially colonizes the sugarcane surface and, occasionally, the endophytic environment. Conclusions/Significance: Our work demonstrates that E. nigrum has great potential for sugarcane crop application because it is capable of increasing the root system biomass and controlling pathogens. The study of the basic aspects of the interaction of E. nigrum with sugarcane demonstrated the facultative endophytism of E. nigrum and its preference for the phylloplane environment, which should be considered in future studies of biocontrol using this species. In addition, this work contributes to the knowledge of the interaction of this ubiquitous endophyte with the host plant, and also to a better use of microbial endophytes in agriculture.

Changes in spatial and temporal gene expression during incompatible interaction between common bean and anthracnose pathogen

Common bean, one of the most important legumes for human consumption, may have drastic reduction in yield due to anthracnose, a disease caused by the fungus Colletotrichum lindemuthianum. Rapid induction of the plant defense mechanisms is essential to establish an incompatible interaction with this pathogenic fungus. In this study, we evaluated spatial (leaves, epicotyls and hypocotyls) and temporal (24, 48, 72 and 96 hours after inoculation [HAI]) relative expression (RE) of 12 defense-related transcripts selected from previously developed ESTs libraries, during incompatible interaction between the resistant common bean genotype SEL 1308 and the avirulent anthracnose pathogen race 73, using real time quantitative RT-PCR (RT-qPCR) analysis. All selected transcripts, including the ones coding for pathogenesis-related (PR) proteins (PR1a, PR1b, PR2, and PR16a and PR16b) were differentially regulated upon pathogen inoculation. The expression levels of these transcripts were dependent on the tissue and time post inoculation. This study contributes to a better understanding of the kinetics of induced defenses against a fungal pathogen of common bean and may be used as a base line to study defenses against a broad range of pathogens including bacteria as well as non-host resistance. (C) 2012 Elsevier GmbH. All rights reserved.

Validation of reference genes for RT-qPCR normalization in common bean during biotic and abiotic stresses

Selection of reference genes is an essential consideration to increase the precision and quality of relative expression analysis by the quantitative RT-PCR method. The stability of eight expressed sequence tags was evaluated to define potential reference genes to study the differential expression of common bean target genes under biotic (incompatible interaction between common bean and fungus Colletotrichum lindemuthianum) and abiotic (drought; salinity; cold temperature) stresses. The efficiency of amplification curves and quantification cycle (C (q)) were determined using LinRegPCR software. The stability of the candidate reference genes was obtained using geNorm and NormFinder software, whereas the normalization of differential expression of target genes [beta-1,3-glucanase 1 (BG1) gene for biotic stress and dehydration responsive element binding (DREB) gene for abiotic stress] was defined by REST software. High stability was obtained for insulin degrading enzyme (IDE), actin-11 (Act11), unknown 1 (Ukn1) and unknown 2 (Ukn2) genes during biotic stress, and for SKP1/ASK-interacting protein 16 (Skip16), Act11, Tubulin beta-8 (beta-Tub8) and Unk1 genes under abiotic stresses. However, IDE and Act11 were indicated as the best combination of reference genes for biotic stress analysis, whereas the Skip16 and Act11 genes were the best combination to study abiotic stress. These genes should be useful in the normalization of gene expression by RT-PCR analysis in common bean, the most important edible legume.

Endophytic fungi from Vitis labrusca L. ('Niagara Rosada') and its potential for the biological control of Fusarium oxysporum

We investigated the diversity of endophytic fungi found on grape (Vitis labrusca cv. Niagara Rosada) leaves collected from Salesopolis, SP, Brazil. The fungi were isolated and characterized by amplified ribosomal DNA restriction analysis, followed by sequencing of the ITS1-5.8S-ITS2 rDNA. In addition, the ability of these endophytic fungi to inhibit the grapevine pathogen Fusarium oxysporum f. sp herbemontis was determined in vitro. We also observed that the climatic factors, such as temperature and rainfall, have no effect on the frequency of infection by endophytic fungi. The endophytic fungal community that was identified included Aporospora terricola, Aureobasidium pullulans, Bjerkandera adusta, Colletotrichum boninense, C. gloeosporioides, Diaporthe helianthi, D. phaseolorum, Epicoccum nigrum, Flavodon flavus, Fusarium subglutinans, F. sacchari, Guignardia mangiferae, Lenzites elegans, Paraphaeosphaeria pilleata, Phanerochaete sordida, Phyllosticta sp, Pleurotus nebrodensis, Preussia africana, Tinctoporellus epiniltinus, and Xylaria berteri. Among these isolates, two, C. gloeosporioides and F. flavus, showed potential antagonistic activity against F. oxysporum f. sp herbemontis. We suggest the involvement of the fungal endophyte community of V. labrusca in protecting the host plant against pathogenic Fusarium species. Possibly, some endophytic isolates could be selected for the development of biological control agents for grape fungal disease; alternatively, management strategies could be tailored to increase these beneficial fungi.

Potencial do flavedo (epicarpo) de Citrus aurantifolia cv. Tahiti no controle do bolor verde e da antracnose em citros

O Brasil é considerado o maior produtor de citros e o maior exportador de suco de laranja. Doenças de pós-colheita representam uma grande perda para a citricultura, sendo que para a exportação de frutos são rígidas as exigências com relação a isenção de resíduos químicos nos mesmos. Patógenos de importância em pós-colheita de citros incluem o Penicillium digitatum, agente causal do bolor-verde e o Colletotrichum gloeosporioides, agente causal da antracnose. Dada a importância econômica que representam estas doenças dos frutos cítricos, tanto em termos de comprometimento da qualidade e dificuldade de controle, a busca de alternativas adicionais que possam viabilizar a capacidade produtiva e garantir a obtenção de frutos com excelentes padrões de qualidade torna-se imprescindível. Portanto, estudou-se os efeitos dos extratos aquosos do flavedo de Citrus aurantifolia var. Tahiti, Lentinula edodes, Agaricus subrufescens (syn. Agaricus brasiliensis), albedo de Citrus sinensis var. Valência e do ácido jasmônico no controle póscolheita do bolor verde e da antracnose e na indução de resistência em frutos de laranjeira Valência (Citrus sinensis). Foi possível observar que o extrato aquoso do flavedo (C. aurantifolia) apresentou efeito inibitório sobre os patógenos, quando tratados em pós-colheita, em função da redução dos sintomas e esporulação. Porém, os extratos de albedo (C. sinensis), L. edodes, A. subrufescens e o ácido jasmônico não apresentaram efeitos sobre P. digitatum e C. gloeosporioides.

Study of thiabendazole resistance and volatile organic compounds production of Penicillium expansum strains

The years of excessive use of thiabendazole to control Penicillium expansum has induced the development of resistance. Sensitivity of fourty eight strains collected from orchards and packinghouses in Emilia Romagna to pure and commercial TBZ was determined in vitro on TBZ amended medium (400μg/mL). Out of 48 strains, 35 were thiabendazole-sensitive (S) and 13 were thiabendazole-resistant (R). Microtiter assay adapted to P. expansum, showed EC50 values ranging from 54 to 320 μg/mL for ten TBZ-resistant strains. At the highest dose (50 μg/mL), resistant strains growth was not inhibited and the reported MICs value were >1000 μg/mL. Therefore, preliminary screening combined with microtiter assay, can be a good strategy to test susceptibility to TBZ. Mutations in the β-tubulin gene were studied on amino acid sequences from residue 167 to residue 357 of 10 P. expansum strains. Mutation at codon 198 was associated with TBZ-resistance. However, its absence in 3 resistant strains can be explained by the involvement of other mechanisms. Moreover, a P. expansum strain LB8/99 showed good antifungal effect against some fungal pathogens through double petri dish assay. It inhibited both mycelium growth and conidia germination of B. cinerea, C. acutatum, and M. laxa, and reduced significantly by 53% and 18% respectively P. expansum. Three major VOCS: geosmin, phenethyl alcolhol (PEA) and an unknown substance were identified by GC-MS analysis. Consistent fumigation of fungal pathogens with PEA (1230 mg/mL), inhibited both conidia germination and mycelium growth of all pathogens, except conidia germination of P. expansum that was reduced by 90% with respect to control. While, the concentration of PEA produced naturally by LB8/99 was ineffective in controlling the pathogens and seemed to have a synergic or additive effect with the other VOCS. Investigations to study the biofumigant effect of LB8/99 on other commodities like seeds and seedlings are in progress.

Trips (Frankliniella brevicaulis Hood) de las erupciones del fruto de banano (Musa acuminata Colla) en la provincia de Formosa, Argentina

El banano en la provincia de Formosa (Argentina) es uno de los principales cultivos de renta y generador de mano de obra para el productor por lo que representa un importante lugar en lo social y económico. La sanidad de los cultivos es fundamental para lograr una mayor rentabilidad. Se han observado en los frutos daños atribuibles a trips. Los objetivos de este trabajo fueron identificar la/s especie/s de trips causal de las erupciones del fruto de banano en la provincia de Formosa, realizar una descripción detallada de su morfología y caracterizar y evaluar los daños producidos. La zona de trabajo correspondió al Área-Laguna Naineck en el Departamento Pilcomayo de la provincia de Formosa considerando un total de 1.103,74 ha pertenecientes a 233 productores. Para la determinación del estado sanitario de las plantaciones con respecto a la presencia del insecto se realizó un diseño de muestreo estadístico. Se recolectaron muestras del insecto y se identificaron con ayuda de claves. La única especie de trips encontrada fue Frankliniella brevicaulis. Los daños producidos por esta especie serían debidos a las heridas causadas por la oviposición, siendo éstas posibles fuentes de entrada para el hongo Colletotrichum musae. Se observaron daños en el 100% de los cachos de banana en todas las plantaciones evaluadas.

Comparative Biochemistry of the Oxidative Burst Produced by Rose and French Bean Cells Reveals Two Distinct Mechanisms1

Cultured cells of rose (Rosa damascena) treated with an elicitor derived from Phytophthora spp. and suspension-cultured cells of French bean (Phaseolus vulgaris) treated with an elicitor derived from the cell walls of Colletotrichum lindemuthianum both produced H2O2. It has been hypothesized that in rose cells H2O2 is produced by a plasma membrane NAD(P)H oxidase (superoxide synthase), whereas in bean cells H2O2 is derived directly from cell wall peroxidases following extracellular alkalinization and the appearance of a reductant. In the rose/Phytophthora spp. system treated with N,N-diethyldithiocarbamate, superoxide was detected by a N,N′-dimethyl-9,9′-biacridium dinitrate-dependent chemiluminescence; in contrast, in the bean/C. lindemuthianum system, no superoxide was detected, with or without N,N-diethyldithiocarbamate. When rose cells were washed free of medium (containing cell wall peroxidase) and then treated with Phytophthora spp. elicitor, they accumulated a higher maximum concentration of H2O2 than when treated without the washing procedure. In contrast, a washing treatment reduced the H2O2 accumulated by French bean cells treated with C. lindemuthianum elicitor. Rose cells produced reductant capable of stimulating horseradish (Armoracia lapathifolia) peroxidase to form H2O2 but did not have a peroxidase capable of forming H2O2 in the presence of reductant. Rose and French bean cells thus appear to be responding by different mechanisms to generate the oxidative burst.

Surface signaling in pathogenesis.

Surface signaling plays a major role in fungal infection. Topographical features of the plant surface and chemicals on the surface can trigger germination of fungal spores and differentiation of the germ tubes into appressoria. Ethylene, the fruit-ripening hormone, triggers germination of conidia, branching of hyphae, and multiple appressoria formation in Colletotrichum, thus allowing fungi to time their infection to coincide with ripening of the host. Genes uniquely expressed during appressoria formation induced by topography and surface chemicals have been isolated. Disruption of some of them has been shown to decrease virulence on the hosts. Penetration of the cuticle by the fungus is assisted by fungal cutinase secreted at the penetration structure of the fungus. Disruption of cutinase gene in Fusarium solani pisi drastically decreased its virulence. Small amounts of cutinase carried by spores of virulent pathogens, upon contact with plant surface, release small amounts of cutin monomers that trigger cutinase gene expression. The promoter elements involved in this process in F. solani pisi were identified, and transcription factors that bind these elements were cloned. One of them, cutinase transcription factor 1, expressed in Escherichia coli, is phosphorylated. Several protein kinases from F. solani pisi were cloned. The kinase involved in phosphorylation of specific transcription factors and the precise role of phosphorylation in regulating cutinase gene transcription remain to be elucidated.

Uso de Metarhizium spp. na produção de mudas pré-brotadas de cana-de-açúcar e seus efeitos na planta, em pragas e doenças

Os fungos do gênero Metarhizium são entomopatogênicos e ainda apresentam relações endofíticas e podem viver saprofiticamente no solo. A associação desses microrganismos com insetos é bem conhecida, mas as interações diretas com as plantas ainda são incipientes. Objetivou-se com este estudo, determinar a capacidade de colonização endofítica em raízes de cana-de-açúcar (Saccharum spp.) de M. robertsii, M. anisopliae e três linhagens brasileiras recém-descobertas, bem como revelar o potencial destes fungos como antagonistas a dois fungos fitopatogênicos responsáveis pela podridão-vermelha (Fusarium moniliforme e Colletotrichum falcatum) e no controle de duas importantes pragas, a broca da cana-de-açúcar, Diatraea saccharalis e o nematóide-das-galhas, Meloidogyne javanica. Foram utilizados 24 isolados de Metarhizium spp. nos experimentos de promoção de crescimento de plantas após a inoculação em gemas de cana-de-açúcar em condições de casa-de-vegetação e campo. O efeito da inoculação do fungo em mudas de cana-de-açúcar foi investigado quanto a mortalidade e redução no desenvolvimento de D. saccharalis e redução nas populações de M. javanica. Experimentos de inibição in vitro em cultivo pareado foram conduzidos com os fungos F. moniliforme e C. falcatum e Trichoderma harzianum. Além disto, foram realizados testes qualitativos \"in vitro\" para avaliar a capacidade desses fungos em produzir β-1,3-Glucanase e Sideróforos. A inoculação de Metarhizium de todas as espécies testadas promoveu o crescimento de parte aérea, peso úmido e seco de raízes em relação ao controle (água). Enquanto o tratamento de gemas rotineiramente usado na usina onde se realizou a produção de mudas pré-brotadas com aplicação do fungicida Comet® + o fertilizante Biozyme TF resultou em pegamento de 32% e 59,6%, apenas com o uso de isolados de Metarhizium spp. estes valores foram de até 50% e 86,4%, nos dois experimentos em campo, respectivamente. As mortalidades observadas em lagartas de D. saccharalis que se alimentaram das plantas inoculadas com isolados de Metarhizium spp. atingiram valores de até 80%, sendo a mortalidade confirmada pela esporulação do fungo de até 55%. As lagartas sobreviventes apresentaram menor peso do que aquelas do controle não tratado. A inoculação de Metarhizium spp. em mudas de cana-de-açúcar conferiu proteção a M. javanica, resultando em uma densidade de massa de ovos por grama de raiz nas mudas inoculadas com o fungo até 59,8% menor no primeiro experimento e 64,1% menor no segundo em relação as mudas sem inoculação. Os ensaios de antagonismo mostraram que, todos os isolados de Metarhizium spp. apresentaram alguma forma de inibição tanto para F. moniliforme como para C. falcatum. Dos 24 isolados testados, 20,8% produziram a enzima hidrolítica, β-1,3-glucanase, o que pode estar associado a capacidade de inibição dos fungos fitopatogênicos. Constatou-se que 8 (33,3%) dos isolados produziram sideróforos e sugere que esses fungos apresentam mecanismos de disponibilização do ferro. O conhecimento gerado com este estudo poderá subsidiar novas estratégias de utilização de Metarhizium spp. em campo na produção de MPB de cana-de-açúcar.

Using molecular markers to assess the effect of introgression on quantitative attributes of common bean in the Andean gene pool

Progress in bean breeding programs requires the exploitation of genetic variation that is present among races or through introgression across gene pools of Phaseolus vulgaris L. Of the two major common bean gene pools, the Andean gene pool seems to have a narrow genetic base, with about 10% of the accessions in the CIAT core collection presenting evidence of introgression. The objective of this study was to quantify the degree of spontaneous introgression in a sample of common bean landraces from the Andean gene pool. The effects of introgression on morphological, economic and nutritional attributes were also investigated. Homogeneity analysis was performed on molecular marker data from 426 Andean-type accessions from the primary centres of origin of the CIAT common bean core collection and two check varieties. Quantitative attribute diversity for 15 traits was studied based on the groups found from the cluster analysis of marker prevalence indices computed for each accession. The two-group summary consisted of one group of 58 accessions (14%) with low prevalence indices and another group of 370 accessions (86%) with high prevalence indices. The smaller group occupied the outlying area of points displayed from homogeneity analysis, yet their geographic origin was widely distributed over the Andean region. This group was regarded as introgressed, since its accessions displayed traits that are associated with the Middle American gene pool: high resistance to Andean disease isolates but low resistance to Middle American disease isolates, low seed weight and high scores for all nutrient elements. Genotypes generated by spontaneous introgression can be helpful for breeders to overcome the difficulties in transferring traits between gene pools.

Transcriptional response of sugarcane roots to methyl jasmonate

The effect of methyl jasmonate treatment on gene expression in sugarcane roots signalling between roots and shoots was studied. A collection of 829 ESTs were obtained from sugarcane roots treated with the defence-regulator methyl jasmonate (MJ) treatment. A subset of 747 of these were combined with 4793 sugarcane ESTs obtained from stem tissues in a cDNA microarray and experiments undertaken to identify genes that were induced in roots 24-120 h following treatment with MJ. Two data analysis systems (t-statistic and tRMA) were used to analyse the microarray results and these methods identified a common set of 21 ESTs corresponding to transcripts significantly induced by MJ in roots and 23 that were reduced in expression following MJ treatment. The induction of six transcripts identified in the microarray analysis was tested and confirmed using northern blotting. Homologues of genes encoding lipoxygenase and PR-10 proteins were induced 824 It after MJ treatment while the other four selected transcripts were induced at later time points. Following treatment of roots with MJ, the lipoxygenase homologue, but not the PR-10 homologue, was induced in untreated stem and leaf tissues. The PR-10 homologue and a PR-1 homologue, but not the lipoxygenase homologue, were induced in untreated tissues after the application of SA to roots. Repeated foliar application of MJ had no apparent effects on plant growth and was demonstrated to increase lipoxygenase transcripts in roots, but did not increase transcript levels-of other genes tested. These results lay a foundation for further studies of induced pest and disease resistance in sugarcane roots. (C) 2004 Elsevier Ireland Ltd. All rights reserved.

Manipulating avocado fruit ripening with 1-methylcyclopropene

Previous investigations with 1-methylcyclopropene (1-MCP) on avocado (Persea americana Mill.) fruit have focussed mainly on improving storage life by reducing the severity of disorders causing discolouration of the flesh. Development of 1-MCP and ethylene treatments, which also help control the time to reach the eating ripe stage, may confer additional practical benefits. In this context, the current study investigated the potential of 1-MCP to accurately manipulate ripening of non-stored 'Hass' avocado fruit by treatment before or after ethylene and at different times during ripening. To investigate this, 500 nL L-1 1-MCP was applied within 1 day after harvest, followed by ethylene 0-14 days after 1-MCP. In addition, fruit were treated with ethylene, then 1-MCP 0-8 days after ethylene. Treatment of fruit with 500 nL L-1 1-MCP for 18 h at 20 degreesC provided the maximum effect by increasing the days from harvest to ripe (DTR) from 8 (with no 1-MCP) to 20. Fruit treated with 500 nL L-1 1-MCP for 18 h at 20 degreesC remained insensitive to 100 muL L-1 ethylene applied between 0 and 14 days after 1-MCP for 24 h at 20 degreesC. Ripening of fruit exposed to 100 muL L-1 ethylene for 24 h at 20 degreesC could be delayed by up to 3.3 days by applying 500 nL L-1 1-MCP for 18 h at 20 degreesC up to 2 days after ethylene treatment. However, once the fruit started to soften (sprung) there was little effect of 1-MCP on DTR, compared with no 1-MCP. 1-MCP treatment was associated with increased severity of body rots (caused mainly by Colletotrichum spp.) and stem-end rots (caused mainly by Dothiorella spp.), which was likely due to the increased DTR in these treatments. Significant differences in disease severity were found between orchards (replications), with replicates with low disease severity being less affected by 1-MCP treatment. These results indicate that 1-MCP can delay ripening, but careful sourcing of fruit is required to reduce the risk of diseases in ripe fruit. There is some capacity to delay ripening using 1-MCP after ethylene. There is little potential to control ripening using ethylene after treatment with 500 nL L-1 1-1-MCP, but lower concentrations may be more effective. (C) 2004 Elsevier B.V. All rights reserved.

Developing a commercial hot water treatment to control post-harvest rots on ‘Fiji Red’ papaya

Fiji exports approximately 800 t year-1 of 'Solo Sunrise' papaya marketed as 'Fiji Red' to international markets which include New Zealand, Australia and Japan. The wet weather conditions from November to April each year result in a significant increase in fungal diseases present in Fiji papaya orchards. The two major pathogens that are causing significant post-harvest losses are: stem end rot (Phytophthora palmivora) and anthracnose (Colletotrichum spp.). The high incidence of post-harvest rots has led to increased rejection rates all along the supply chain, causing a reduction in income to farmers, exporters, importers and retailers of Fiji papaya. It has also undermined the superior quality reputation on the market. In response to this issue, the Fiji Papaya industry led by Nature's Way Cooperative, embarked on series of trials supported by the Australian Centre for International Agricultural Research (ACIAR) to determine the most effective and economical post-harvest control in Fiji papaya. Of all the treatments that were examined, a hot water dip treatment was selected by the industry as the most appropriate technology given the level of control that it provide, the cost effectiveness of the treatment and the fact that it was non-chemical. A commercial hot water unit that fits with the existing quarantine treatment and packing facilities has been designed and a cost benefit analysis for the investment carried out. This paper explores the research findings as well as the industry process that has led to the commercial uptake of this important technology.