High-throughput sequencing of black pepper root transcriptome

Background: Black pepper (Piper nigrum L.) is one of the most popular spices in the world. It is used in cooking and the preservation of food and even has medicinal properties. Losses in production from disease are a major limitation in the culture of this crop. The major diseases are root rot and foot rot, which are results of root infection by Fusarium solani and Phytophtora capsici, respectively. Understanding the molecular interaction between the pathogens and the host's root region is important for obtaining resistant cultivars by biotechnological breeding. Genetic and molecular data for this species, though, are limited. In this paper, RNA-Seq technology has been employed, for the first time, to describe the root transcriptome of black pepper. Results: The root transcriptome of black pepper was sequenced by the NGS SOLiD platform and assembled using the multiple-k method. Blast2Go and orthoMCL methods were used to annotate 10338 unigenes. The 4472 predicted proteins showed about 52% homology with the Arabidopsis proteome. Two root proteomes identified 615 proteins, which seem to define the plant's root pattern. Simple-sequence repeats were identified that may be useful in studies of genetic diversity and may have applications in biotechnology and ecology. Conclusions: This dataset of 10338 unigenes is crucially important for the biotechnological breeding of black pepper and the ecogenomics of the Magnoliids, a major group of basal angiosperms.

Two novel MvaT-like global regulators control exoproduct formation and biocontrol activity in root-associated Pseudomonas fluorescens CHA0.

Pseudomonas fluorescens CHA0 protects various crop plants against root diseases caused by pathogenic fungi. Among a range of exoproducts excreted by strain CHA0, the antifungal compounds 2,4-diacetylphloroglucinol (DAPG) and pyoluteorin (PLT) are particularly relevant to the strain's biocontrol potential. Here, we report on the characterization of MvaT and MvaV as novel regulators of biocontrol activity in strain CHA0. We establish the two proteins as further members of an emerging family of MvaT-like regulators in pseudomonads that are structurally and functionally related to the DNA-binding protein H-NS. In mvaT and mvaV in frame-deletion mutants of strain CHA0, PLT production was enhanced about four- and 1.5-fold, respectively, whereas DAPG production remained at wild-type levels. Remarkably, PLT production was increased up to 20-fold in an mvaT mvaV double mutant. DAPG biosynthesis was almost completely repressed in this mutant. The effects on antibiotic production could be confirmed by following expression of gfp-based reporter fusions to the corresponding biosynthetic genes. MvaT and MvaV also influenced levels of other exoproducts, motility, and physicochemical cell-surface properties to various extents. Compared with the wild type, mvaT and mvaV mutants had an about 20% reduced capacity (in terms of plant fresh weight) to protect cucumber from a root rot caused by Pythium ultimum. Biocontrol activity was nearly completely abolished in the double mutant Our findings indicate that MvaT and MvaV act together as further global regulatory elements in the complex network controlling expression of biocontrol traits in plant-beneficial pseudomonads.

In vitro toxin production by Fusarium solani f. sp. piperis

Fusarium solani f. sp. piperis (teleomorph: Nectria haematococca f. sp. piperis), causal agent of root rot and stem blight on black pepper (Piper nigrum), produces secondary metabolites with toxigenic properties, capable of inducing vein discoloration in detached leaves and wilting in transpiring microcuttings. Production of F. solani f. sp. piperis (Fsp) toxic metabolites reached a peak after 25 days of static incubation on potato sucrose broth at 25 ºC under illumination. Changes in the pH of the culture filtrate did not alter the effect of toxic metabolites. However, when the pH was changed before the medium had been autoclaved, a more intense biological response was observed, with an optimum at pH 6.0. Isolates that produced red pigments in liquid cultures were more efficient in producing biologically active culture filtrates than those which produced pink coloured or clear filtrates suggesting that these pigments could be related to toxigenic activity. Detached leaves of seven black pepper cultivars and Piper betle showed symptoms of vein discoloration after immersion in autoclaved and non-autoclaved Fsp culture filtrates indicating the thermostable nature of these toxic metabolites.

Macrophomina phaseolina: density and longevity of microsclerotia in soybean root tissues and free on the soil, and competitive saprophytic ability

In field experiments, the density of Macrophomina phaseolina microsclerotia in root tissues of naturally colonized soybean cultivars was quantified. The density of free sclerotia on the soil was determined for plots of crop rotation (soybean-corn) and soybean monoculture soon after soybean harvest. M. phaseolina natural infection was also determined for the roots of weeds grown in the experimental area. To verify the ability of M. phaseolina to colonize dead substrates, senesced stem segments from the main plant species representing the agricultural system of southern Brazil were exposed on naturally infested soil for 30 and 60 days. To quantify the sclerotia, the methodology of Cloud and Rupe (1991) and Mengistu et al. (2007) was employed. Sclerotium density, assessed based on colony forming units (CFU), ranged from 156 to 1,108/g root tissue. Sclerotium longevity, also assessed according to CFU, was 157 days for the rotation and 163 days for the monoculture system. M. phaseolina did not colonize saprophytically any dead stem segment of Avena strigosa,Avena sativa,Hordeum vulgare,Brassica napus,Gossypium hirsutum,Secale cereale,Helianthus annus,Triticosecalerimpaui, and Triticum aestivum. Mp was isolated from infected root tissues of Amaranthus viridis,Bidens pilosa,Cardiospermum halicacabum,Euphorbia heterophylla,Ipomoea sp., and Richardia brasiliensis. The survival mechanisms of M. phaseolina studied in this paper met the microsclerotium longevity in soybean root tissues, free on the soil, as well as asymptomatic colonization of weeds.

The analysis of Metarhizium robertsii potential as endophytic plant root coloniser, plant growth enhancer and antagonist to bean root pathogen Fusarium solani f. sp. phaseolis.

The soil-inhabiting insect-pathogenic fungus Metarhizium robertsii also colonizes plant roots endophytically, thus showing potential as a plant symbiont. M robertsii is not randomly distributed in soils but preferentially associates with the plant rhizosphere when applied in agricultural settings. Root surface and endophytic colonization of switchgrass (Panicum virgatum) and haricot beans (Phaseolus vulgaris) by M robertsii were examined after inoculation with fungal conidia. Light and confocal microscopies were used to ascertain this rhizosphere association. Root lengths, root hair density and emergence of lateral roots were also measured. Initially, M robertsii conidia adhered to, germinated on, and colonized, roots. Furthermore, plant roots treated with Metarhizium grew faster and the density of plant root hairs increased when compared with control plants. The onset of plant root hair proliferation was initiated before germination of M robertsii on the root (within 1-2 days). Plants inoculated with M robertsii AMAD2 (plant adhesin gene) took significantly longer to show root hair proliferation than the wild type. Cell free extracts of M robertsii did not stimulate root hair proliferation. Longer term (60 days) associations showed that M robertsii endophytically colonized individual cortical cells within bean roots. Metarhizium appeared as an amorphous mycelial aggregate within root cortical cells as well as between the intercellular spaces with no apparent damage to the plant. These results suggested that not only is M robertsii rhizosphere competent but displays a beneficial endophytic association with plant roots that results in the proliferation of root hairs. The biocontrol of bean (Phaseolis vulgaris) root rot fungus Fusarium solani f. sp. phaseolis by Metarhizium robertsii was investigated in vitro and in vivo. Dual cultures on Petri dishes showed antagonism of M robertsii against F. solani. A relative inhibition of ca. 60% of F. solani growth was observed in these assays. Cell free culture filtrates of M robertsii inhibited the germination of F. solani conidia by 83% and the inhibitory metabolite was heat stable. Beans plants colonized by M robertsii then exposed to F. solani showed healthier plant profiles and lower disease indices compared to plants not colonized by M robertsii. These results suggested that the insect pathogenic/endophytic fungus M robertsii could also be utilized as a biocontrol agent against certain plant pathogens occurring in the rhizosphere.

Pathogens occuring in the winter pea-maize-winter wheat rotation, their host specificity and the potential of compost in suppressing foot and root disease of peas

The overall aim of the work presented was to evaluate soil health management with a specific focus on soil borne diseases of peas. For that purpose field experiments were carried out from 2009 until 2013 to assess crop performance and pathogen occurrence in the rotation winter pea-maize-winter wheat and if the application of composts can improve system performance. The winter peas were left untreated or inoculated with Phoma medicaginis, in the presence or absence of yard waste compost at rate of 5 t dry matter ha-1. A second application of compost was made to the winter wheat. Fusarium ssp. were isolated and identified from the roots of all three crops and the Ascochyta complex pathogens on peas. Bioassays were conducted under controlled conditions to assess susceptibility of two peas to Fusarium avenaceum, F. solani, P. medicaginis and Didymella pinodes and of nine plant species to F. avenaceum. Also, effects of compost applications and temperature on pea diseases were assessed. Application of composts overall stabilized crop performance but it did not lead to significant yield increases nor did it affect pathogen composition and occurrence. Phoma medicaginis was dominating the pathogen complex on peas. F. graminearum, F. culmorum, F. proliferatum, Microdochium nivale, F. crookwellense, F. sambucinum, F. oxysporum, F. avenaceum and F. equiseti were frequently isolated species from maize and winter wheat with no obvious influence of the pre-crop on the Fusarium species composition. The spring pea Santana was considerably more susceptible to the pathogens tested than the winter pea EFB33 in both sterile sand and non-sterilized field soil. F. avenaceum was the most aggressive pathogen, followed by P. medicaginis, D. pinodes, and F. solani. Aggressiveness of all pathogens was greatly reduced in non-sterile field soil. F. avenaceum caused severe symptoms on roots of all nine plant species tested. Especially susceptible were Trifolium repens, T. subterraneum, Brassica juncea and Sinapis alba in addition to peas. Reduction of growing temperatures from 19/16°C day/night to 16/12°C and 13/10°C did not affect the efficacy of compost. It reduced plant growth and slightly increased disease on EFB33 whereas the highest disease severity on Santana was observed at the highest temperature, 19/16°C. Application of 20% v/v of compost reduced disease on peas due to all four pathogens depending on pea variety, pathogen and growing media used. Suppression was also achieved with lower application rate of 3.5% v/v. Tests with γ sterilized compost suggest that the suppression of disease caused by Fusarium spp. is biological in origin, whereas chemical and physical properties of compost are playing an additional role in the suppression of disease caused by D. pinodes and P. medicaginis.

Rhizoctonia solani GA-3PT es el principal patógeno asociado con el chancro del tallo y la sarna negra de la papa en Colombia

Stem canker and black scurf diseases of potatoes are caused by the basidiomycetous fungus Tanatephorus cucumeris (ana-morphic species complex Rhizoctonia solani). Tese diseases have worldwide distribution wherever potato is grown but their etiology varies depending on the predominance of distinct R. solani anastomosis groups (AGs) in a particular area. Within the species complex, several AGs have been associated with stem canker or black scurf diseases, including AG-1, AG-2-1, AG-2-2, AG-3, AG-4, AG-5 and AG-9. Tis article reports on the most comprehensive population-based study, providing evidence on the distribution of R. solani AGs in Colombian potato fields. A total of 433 isolates were sampled from the main potato cropping areas in Colombia from 2005 to 2009. Isolates were assigned to AGs by conventional PCR assays using specific primers for AG-3, sequencing of the ITS-rDNA and hyphal interactions. Most of the isolates evaluated were assigned to AG-3PT (88.45%), and a few to AG-2-1 (2.54%). Te remaining isolates were binucleate Rhizoctonia (AG-A, E, and I). Pathogenicity tests on the stems and roots of different plant species, including the potato, showed that AG-3PT affects the stems of solanaceous plants. In other plant species, damage was severe in the roots, but not the stems. AG-2-1 caused stem canker of Solanum tuberosum cv. Capiro and in R. raphanistrum and B. campestris subsp. Rapa plantlets and root rot in other plants. Te results of our study indicated that R. solani AG-3PT was the principal pathogen associated with potato stem canker and black scurf diseases of potatoes in Colombia.

Extração e análise eletroforética em gel de poliacrilamida (SDS-PAGE) de proteínas totais de folhas e raízes de Piper tuberculatum

O Estado do Pará é o principal produtor brasileiro de pimenta-do-reino (Piper nigrum Link), entretanto a sua produção tem sido bastante afetada pela doença conhecida como fusariose. O Fusarium solani f. sp. piperis é o agente causador desta doença que afeta o sistema radicular da planta, causando o apodrecimento das raízes e a queda das folhas levando à morte da planta. Algumas piperáceas nativas da região amazônica, entre elas a espécie Piper tuberculatum Jacq., têm se mostrado resistentes à infecção pelo F. solani f. sp. piperis, e desta forma têm sido utilizadas em estudos de interação planta-patógeno. Neste trabalho foram avaliadas cinco condições de extração de proteínas com o objetivo de selecionar tampões adequados para a extração de proteínas totais de folhas e raízes de P. tuberculatum. Os tampões utilizados para a extração de proteínas de raízes e folhas foram: tampão salino, tampão sacarose, tampão glicerol, tampão uréia e tampão fosfato de sódio. As análises quantitativas mostraram que os tampões sacarose, glicerol e uréia foram mais eficientes na extração de proteínas de folhas e raízes. Análises de SDS-PAGE mostraram padrões diferenciados de bandas em extratos protéicos de folhas e raízes obtidos com os diferentes tampões. Os resultados obtidos neste trabalho contribuem para a identificação de tampões de extração adequados para a obtenção de amostras de proteínas totais em estudos de interação P. tuberculatum - F. solani f. sp. piperis.

Isolamento, caracterização e expressão em sistema bacteriano de um gene que codifica uma proteína transportadora de lipídeos de Piper nigrum

A pimenteira-do-reino (Piper nigrum L.) constitui uma das espécies de pimenta mais amplamente utilizadas no mundo, pertencendo à família Piperaceae, a qual compreende cerca de 1400 espécies distribuídas principalmente no continente americano e sudeste da Ásia, onde esta cultura originou. A pimenteira-do-reino foi introduzida no Brasil no século XVII, e tornou-se uma cultura de importância econômica desde 1933. O Estado do Pará é o principal produto brasileiro de pimenta-do-reino, contudo sua produção vem sendo afetada pela doença fusariose causada pelo fungo Fusarium solani f. sp. piperis. Estudos prévios revelaram a identificação de sequencias de cDNA diferencialmente expressas durante a interação da pimenteira-do-reino com o F. solani f. sp. piperis. Entre elas, uma sequencia de cDNA parcial que codifica para uma proteína transportadora de lipídeos (LTP), a qual é conhecida por seu importante papel na defesa de plantas contra patógenos e insetos. Desta forma, o objetivo principal deste trabalho foi isolar e caracterizar as sequencias de cDNA e genômica de uma LTP de pimenteira-do-reino, denominada PnLTP. O cDNA completo da PnLTP isolado por meio de experimentos de RACE apresentou 621 bp com 32 pb and 235 bp nas regiões não traduzidas 5‘ e 3‘, respectivamente. Este cDNA contem uma ORF de 354 bp codificando uma proteína deduzida de 117 resíduos de aminoácidos que apresentou alta identidade com LTPs de outras espécies vegetais. Análises das sequencias revelou que a PnLTP contem um potencial peptídeo sinal na extremidade amino-terminal e oito resíduos de cisteína preditos por formar quatro pontes de dissulfeto, as quais poderiam contribuir para a estabilidade desta proteína. O alinhamento entre as sequencias de cDNA e genômica revelou a ausência de introns na região codificante do gene PnLTP, o que está de acordo ao encontrado em outros genes de LTPs de plantas. Por último, a PnLTP madura foi expressa em sistema bacteriano. Experimentos adicionais serão realizados com o objetivo de avaliar a habilidade da PnLTP recombinante em inibir o crescimento do F. solani f. sp. piperis.

Armillaria root disease /

Includes index.

Evaluation of citrus somatic hybrids for tolerance to Phytophthora nicotianae and citrus tristeza virus

Somatic hybridization is a biotechnology tool that can be used in citrus breeding programs to produce somatic hybrids with the complete genetic combination of both parents. The goal of this work was to test the reaction of citrus somatic hybrids that may be useful as rootstocks to trunk and root infections caused by Phytophthora nicotianae van Breda de Haan (P parasitica Dastur) and to citrus tristeza virus (CTV). The somatic hybrids evaluated were `Caipira` sweet orange (Citrus sinensis L. Osbeck) + `Rangpur` lime (C. limonia Osbeck), `Caipira` sweet orange + `Cleopatra` mandarin (C. reshni hort. ex Tanaka), `Caipira` sweet orange + `Volkamer` lemon (C. volkameriana V Ten. & Pasq.), `Caipira` sweet orange + rough lemon (C. jambhiri Lush.), `Cleopatra` mandarin + `Volkamer` lemon, `Cleopatra` mandarin + sour orange (C. aurantium L.), `Rangpur` lime + `Sunki` mandarin (C. sunki (Hayata) hort. ex Tanaka), `Ruby Blood` sweet orange (C. sinensis L. Osbeck) + `Volkamer` lemon, `Rohde Red` sweet orange (C. sinensis L. Osbeck) + `Volkamer` lemon, and `Valencia` sweet orange + Fortunella obovata hort. ex Tanaka. For P. nicotianae trunk and root infection assays, plants of the somatic hybrids, obtained from 9-month semi-hardwood cuttings, were evaluated and compared with diploid citrus rootstock cultivars after mycelia inoculation in the trunk or spore infestation in the substrate, respectively. `Cleopatra` mandarin + sour orange, `Rangpur` lime + `Sunki` mandarin, `Cleopatra` mandarin + `Volkamer` lemon, `Ruby Blood` sweet orange + `Volkamer` lemon, `Rohde Red` sweet orange + `Volkamer` lemon, and `Caipira` sweet orange + `Volkamer` lemon had less trunk rot occurrence, whereas the somatic hybrids `Cleopatra` mandarin + `Volkamer` lemon, `Cleopatra` mandarin + sour orange, `Caipira` sweet orange + `Volkamer` lemon, and `Caipira` sweet orange + `Rangpur` lime were tolerant to root rot. For CTV assays, plants of the somatic hybrids along with tolerant and intolerant rootstocks were budded with a mild strain CTV-infected or healthy `Valencia` sweet orange budwood. Differences in average scion shoot length indicated that the hybrids `Cleopatra` mandarin + sour orange and `Valencia` sweet orange + Fortunella obovata were intolerant to CTV (c) 2007 Elsevier B.V. All rights reserved.

The proportion of individual lucerne plants resistant to Phytophthora medicaginis and Colletotrichum trifolii in Australian cultivars

Phytophthora root rot (Phytophthora medicaginis) and colletotrichum crown rot (Colletotrichum trifoli) are the 2 most serious pathogens of lucerne in eastern Australia. Work reported in this paper shows that in glasshouse tests of the 11 most commonly grown Australian lucerne cultivars, the proportion of individual plants with resistance to both pathogens ranges from 0 (Hunter River and Aurora) through to a maximum of 19.8% (Sequel HR). Within 9 of the cultivars, the proportion of individual plants resistant to the 2 pathogens was <7%. Since these 2 diseases are known to cause serious losses in eastern Australia, the results indicate further improvement in lucerne production can be obtained by increasing the proportion of individual plants in a cultivar resistant to both pathogens. This would be best achieved by identifying dominant sources of resistance and incorporating this into on-going lucerne breeding programs.

Gene and genotypic diversity of Phytophthora cinnamomi in South Africa and Australia revealed by DNA polymorphisms

Phytophthora cinnamomi isolates from South Africa and Australia were compared to assess genetic differentiation between the two populations. These two populations were analysed for levels of phenotypic diversity using random amplified polymorphic DNAs (RAPDs) and gene and genotypic diversity using restriction fragment length polymorphisms (RFLPs). Sixteen RAPD markers from four decanucleotide Operon primers and 34 RFLP alleles from 15 putative loci were used. A few isolates from Papua New Guinea known to posses alleles different from Australian isolates were also included for comparative purposes. South African and Australian P. cinnamomi populations were almost identical with an extremely low level of genetic distance between them (D-m = 0.003). Common features for the two populations include shared alleles, low levels of phenotypic/genotypic diversity, high clonality, and low observed and expected levels of heterozygosity. Furthermore, relatively high levels of genetic differentiation between mating type populations (D-m South Africa = 0.020 and D-m Australia = 0.025 respectively), negative fixation indices, and significant deviations from Hardy-Weinberg equilibrium, all provided evidence for the lack of frequent sexual reproduction in both populations. The data strongly suggest that both the South African and Australian P. cinnamomi populations are introduced.

Expression of resistance by tetraploid and diploid lucerne genotypes to Phytophthora medicaginis is not influenced by inoculum level or by temperature

Phytophthora-resistant lucerne cultivars do not always perform well under conditions of high disease pressure in the field. To determine whether resistance expression remains stable under different infection intensities, tetraploid and diploid lucerne genotypes, genotypically defined for their reactions to Phytophthora medicaginis, were clonally propagated, and the influence of different reproducible inoculum levels (0 . 5 and 5 . 0 g dry weight mycelium/kg dry weight potting mix), the period of exposure to these levels (10-60 days), and temperature (16/22 degrees C and 24/30 degrees C) on disease expression was determined in controlled environments. Generally, expression of resistance by resistant genotypes, remained stable under these conditions. Biotic (e.g. Aphanomyces eutiches) or abiotic factors other than P. medicaginis may be responsible for the poorer than expected performance under field conditions in some instances, or the percentage of resistant plants in some cultivars currently classified as resistant is insufficient to provide buffering against productivity reductions under severe epidemics. Further research is needed to clarify the situation.

Differential disease reactions on lucerne genotypes inoculated with Phytophthora medicaginis isolates from lucerne and chickpea

Stem inoculation of clonally propagated lucerne genotypes was used to assess levels of host species and genotype specialisation in Phytophthora medicaginis. A quantitative assessment of pathogenic aggressiveness of 29 P. medicaginis isolates (from lucerne and chickpea) on 9 different clonally propagated lucerne genotypes revealed no significant difference in aggressiveness between isolates from lucerne and those from chickpea on all of the lucerne genotypes. This supported previous studies which showed that P. medicaginis isolates from lucerne and chickpea were indistinguishable using random amplified polymorphic DNA (RAPD) analysis. Analysis of pathogenic aggressiveness towards individual lucerne genotypes revealed, for the first time, specificity of individual P. medicaginis isolates. This has implications for breeding for resistance to P. medicaginis in lucerne, where screening should be done using the widest range of pathogen specificity obtainable.