Extracellular release of a heterologous phytase from roots of transgenic plants: does manipulation of rhizosphere biochemistry impact microbial community structure?

To maintain the sustainability of agriculture, it is imperative that the reliance of crops on inorganic phosphorus (P) fertilizers is reduced. One approach is to improve the ability of crop plants to acquire P from organic sources. Transgenic plants that produce microbial phytases have been suggested as a possible means to achieve this goal. However, neither the impact of heterologous expression of phytase on the ecology of microorganisms in the rhizosphere nor the impact of rhizosphere microorganisms on the efficacy of phytases in the rhizosphere of transgenic plants has been tested. In this paper, we demonstrate that the presence of rhizosphere microorganisms reduced the dependence of plants oil extracellular secretion of phytase from roots when grown in a P-deficient soil. Despite this, the expression of phytase in transgenic plants had little or no impact on the microbial community structure as compared with control plant lines, whereas soil treatments, such as the addition of inorganic P, had large effects. The results demonstrate that soil microorganisms are explicitly involved in the availability of P to plants and that the microbial community in the rhizosphere appears to be resistant to the impacts of single-gene changes in plants designed to alter rhizosphere biochemistry and nutrient cycling.

Characterization of the complex locus of bean encoding polygalacturonase-inhibiting proteins reveals subfunctionalization for defense against fungi and insects.

Polygalacturonase-inhibiting proteins (PGIPs) are extracellular plant inhibitors of fungal endopolygalacturonases (PGs) that belong to the superfamily of Leu-rich repeat proteins. We have characterized the full complement of pgip genes in the bean (Phaseolus vulgaris) genotype BAT93. This comprises four clustered members that span a 50-kb region and, based on their similarity, form two pairs (Pvpgip1/Pvpgip2 and Pvpgip3/Pvpgip4). Characterization of the encoded products revealed both partial redundancy and subfunctionalization against fungal-derived PGs. Notably, the pair PvPGIP3/PvPGIP4 also inhibited PGs of two mirid bugs (Lygus rugulipennis and Adelphocoris lineolatus). Characterization of Pvpgip genes of Pinto bean showed variations limited to single synonymous substitutions or small deletions. A three-amino acid deletion encompassing a residue previously identified as crucial for recognition of PG of Fusarium moniliforme was responsible for the inability of BAT93 PvPGIP2 to inhibit this enzyme. Consistent with the large variations observed in the promoter sequences, reverse transcription-PCR expression analysis revealed that the different family members differentially respond to elicitors, wounding, and salicylic acid. We conclude that both biochemical and regulatory redundancy and subfunctionalization of pgip genes are important for the adaptation of plants to pathogenic fungi and phytophagous insects.

Density of insect-pollinated grassland plants decreases with increasing surrounding land-use intensity

Pollinator declines have raised concerns about the persistence of plant species that depend on insect pollination, in particular by bees, for their reproduction. The impact of pollinator declines remains unknown for species-rich plant communities found in temperate seminatural grasslands. We investigated effects of land-use intensity in the surrounding landscape on the distribution of plant traits related to insect pollination in 239 European seminatural grasslands. Increasing arable land use in the surrounding landscape consistently reduced the density of plants depending on bee and insect pollination. Similarly, the relative abundance of bee-pollination-dependent plants increased with higher proportions of non-arable agricultural land (e.g. permanent grassland). This was paralleled by an overall increase in bee abundance and diversity. By isolating the impact of the surrounding landscape from effects of local habitat quality, we show for the first time that grassland plants dependent on insect pollination are particularly susceptible to increasing land-use intensity in the landscape.

Parasitoids select plants more heavily infested with their caterpillar hosts: a new approach to aid interpretation of plant headspace volatiles

Plants produce volatile organic compounds (VOCs) in response to herbivore attack, and these VOCs can be used by parasitoids of the herbivore as host location cues. We investigated the behavioural responses of the parasitoid Cotesia vestalis to VOCs from a plant–herbivore complex consisting of cabbage plants (Brassica oleracea) and the parasitoids host caterpillar, Plutella xylostella. A Y-tube olfactometer was used to compare the parasitoids' responses to VOCs produced as a result of different levels of attack by the caterpillar and equivalent levels of mechanical damage. Headspace VOC production by these plant treatments was examined using gas chromatography–mass spectrometry. Cotesia vestalis were able to exploit quantitative and qualitative differences in volatile emissions, from the plant–herbivore complex, produced as a result of different numbers of herbivores feeding. Cotesia vestalis showed a preference for plants with more herbivores and herbivore damage, but did not distinguish between different levels of mechanical damage. Volatile profiles of plants with different levels of herbivores/herbivore damage could also be separated by canonical discriminant analyses. Analyses revealed a number of compounds whose emission increased significantly with herbivore load, and these VOCs may be particularly good indicators of herbivore number, as the parasitoid processes cues from its external environment

Water, irrigation and plant diseases

Irrigation is a major husbandry tool, vital for world food production and security. The purpose of this review is twofold:- firstly drawing attention to the beneficial and deleterious aspects of irrigation resulting from interactions with the microbial world; secondly, forming a basis for encouraging further research and development. Irrigation is for example, a valuable component in the control of some soil borne pathogens such as Streptomyces scabies, the cause of potato common scab and Fusarium cubense, a cause of banana wilt. By contrast, applying irrigation encourages some foliar pathogens and factors such as splash dispersal of propagules and the retention of leaf wetness are important elements in the successful establishment of disease foci. Irrigation applied at low levels in the canopy directly towards the stem bases and root zones of plants also provides means encouraging disease development. Irrigation also offers means for the direct spread of microbes such as water borne moulds, Oomycetes, and plasmodial pathogens coming from populations present in the water supply. The presence of plant disease causing microbes in sources of irrigation has been associated with outbreaks of diseases such as clubroot (Plasmodiophora brassicae). Irrigation can be utilised as a means for applying agrochemicals, fungigation. The developing technologies of water restriction and root zone drying also have an impact on the success of disease causing organisms. This is an emerging technology and its interactions with benign and pathogenic microbes require consideration.

Ectomycorrhizal symbiosis can enhance plant nutrition through improved access to discrete organic nutrient patches of high resource quality

It is known that roots can respond to patches of fertility; however, root proliferation is often too slow to exploit resources fully, and organic nutrient patches may be broken down and leached, immobilized or chemically fixed before they are invaded by the root system. The ability of fungal hyphae to exploit resource patches is far greater than that of roots due to their innate physiological and morphological plasticity, which allows comprehensive exploration and rapid colonization of resource patches in soils. The fungal symbionts of ectomycorrhizal plants excrete significant quantities of enzymes such as chitinases, phosphatases and proteases. These might allow the organic residue to be tapped directly for nutrients such as N and P. Pot experiments conducted with nutrient-stressed ectomycorrhizal and control willow plants showed that when high quality organic nutrient patches were added, they were colonized rapidly by the ectomycorrhizal mycelium. These established willows (0.5 m tall) were colonized by Hebeloma syrjense P. Karst. for 1 year prior to nutrient patch addition. Within days after patch addition, colour changes in the leaves of the mycorrhizal plants (reflecting improved nutrition) were apparent, and after I month the concentration of N and P in the foliage of mycorrhizal plants was significantly greater than that in non-mycorrhizal plants subject to the same nutrient addition. It seems likely that the mycorrhizal plants were able to compete effectively with the wider soil microbiota and tap directly into the high quality organic resource patch via their extra-radical mycelium. We hypothesize that ectomycorrhizal plants may reclaim some of the N and P invested in seed production by direct recycling from failed seeds in the soil. The rapid exploitation of similar discrete, transient, high-quality nutrient patches may have led to underestimations when determining the nutritional benefits of ectomycorrhizal colonization.

Piriformospora indica reduces Fusarium head blight disease severity and mycotoxin DON contamination in wheat under UK weather conditions

Piriformospora indica (Sebacinaceae) is a cultivable root endophytic fungus. It colonises the roots of a wide range of host plants. In many settings colonisation promotes host growth, increases yield and protects the host from fungal diseases. We evaluated the effect of P. indica on Fusarium head blight (FHB) disease of winter (cv. Battalion) and spring (cv. Paragon, Mulika, Zircon, Granary, KWS Willow and KWS Kilburn) wheat and consequent contamination by the mycotoxin deoxynivalenol (DON) under UK weather conditions. Interactions of P. indica with an arbuscular mycorrhizal fungus (Funneliformis mosseae), fungicide application (Aviator Xpro) and low and high fertiliser levels were considered. P. indica application reduced FHB disease severity and incidence by 70%. It decreased mycotoxin DON concentration of winter and spring wheat samples by 70% and 80% respectively. P. indica also increased above ground biomass, 1000 grain weight and total grain weight. P. indica reduced disease severity and increased yield in both high and low fertiliser levels. The effect of P. indica was compatible with F. mosseae and foliar fungicide application. P. indica did not have any effects on plant tissue nutrients. These results suggest that P. indica might be useful in biological control of Fusarium diseases of wheat.

Cloning, expression, purification and characterization of recombinant glutathione-S-transferase from Xylella fastidiosa

Xylella fastidiosa is an important pathogen bacterium transmitted by xylem-feedings leafhoppers that colonizes the xylem of plants and causes diseases on several important crops including citrus variegated chlorosis (CVC) in orange and lime trees. Glutathione-S-transferases (GST) form a group of multifunctional isoenzymes that catalyzes both glutathione (GSH)-dependent conjugation and reduction reactions involved in the cellular detoxification of xenobiotic and endobiotic compounds. GSTs are the major detoxification enzymes found in the intracellular space and mainly in the cytosol from prokaryotes to mammals, and may be involved in the regulation of stress-activated signals by suppressing apoptosis signal-regulating kinase 1. In this study, we describe the cloning of the glutathione-S-transferase from X. fastidiosa into pET-28a(+) vector, its expression in Escherichia coli, purification and initial structural characterization. The purification of recombinant xfGST (rxfGST) to near homogeneity was achieved using affinity chromatography and size-exclusion chromatography (SEC). SEC demonstrated that rxfGST is a homodimer in solution. The secondary and tertiary structures of recombinant protein were analyzed by circular dichroism and fluorescence spectroscopy, respectively. The enzyme was assayed for activity and the results taken together indicated that rxfGST is a stable molecule, correctly folded, and highly active. Several members of the GST family have been extensively studied. However, xfGST is part of a less-studied subfamily which yet has not been structurally and biochemically characterized. In addition, these studies should provide a useful basis for future studies and biotechnological approaches of rxfGST. (C) 2008 Elsevier Inc. All rights reserved.

Chemical Composition and Antimicrobial Activities of the Essential Oil of Hypericum cordatum (Vell. Conc.) N. Robson (Hypericaceae)

The volatile Constituents of the fresh materials of Hypericum cordatum were isolated by hydrodistillation kind analyzed by CC and GC/MS. The leaves produced 0.04% of a yellowish essential oil and the flowers did not. The main components of the oil were myrcene (40.18%), alpha-pinene (16.40%), and limonene (12%). The antibacterial activities of the oil against Saccharomyces aureus and Escherichia coli and the anti-fungal activities of the oil against the fungi Cladosporium cladosporioides and C. sphaerospemum were evaluated. The oil showed an antibacterial activity against the bacteria S. aureus and anti-fungal activity against the two fungi.

Avaliação de fungicidas no controle de Asperisporium caricae na cultura do mamoeiro

Dentre as doenças fúngicas do mamoeiro, a varíola (Asperisporium caricae) é umas das doenças mais importantes. Os objetivos deste trabalho foram avaliar a eficácia de fungicidas no controle da varíola em folhas e frutos do mamoeiro. O experimento foi conduzido em área comercial, no município de Taquarintiga - SP, em delineamento de blocos casualizados, com cinco tratamentos e quatro repetições. Os tratamentos foram tebuconazol + trifloxistrobina (0,06 L.100L-1 de água), piraclostrobina (0,04 L.100L-1 de água), difenoconazol (0,03 L.100L-1 de água), azoxistrobina (128 g.ha-1) e a testemunha. Foram realizadas três avaliações, nas quais foram avaliadas a severidade da varíola através de escala diagramática, nas folhas e nos frutos de três plantas previamente marcadas. Os dados registrados foram submetidos à análise de variância pelo teste F, e as médias, comparadas pelo teste de Scott-Knott (p<0,05). Os fungicidas tebuconazol+trifloxistrobina, piraclostrobina, difenoconazol e azoxistrobina foram eficientes no controle da varíola.

Mite diversity on plants of different families found in the Brazilian Atlantic Forest

Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)

Differential gene expression analysis of Paracoccidioides brasiliensis during keratinocyte infection

Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)

Binding of extracellular matrix proteins to Paracoccidioides brasiliensis

Adhesion to extracellular matrix (ECM) proteins plays a crucial role in invasive fungal diseases. ECM proteins bind to the surface of Paracoccidioides brasiliensis yeast cells in distinct qualitative patterns. Extracts from Pb18 strain, before (18a) and after animal inoculation (18b), exhibited differential adhesion to ECM components. Pb18b extract had a higher capacity for binding to ECM components than Pb18a. Laminin was the most adherent component for both samples, followed by type I collagen, fibronectin, and type IV collagen for Pb18b. A remarkable difference was seen in the interaction of the two extracts with fibronectin and their fragments. Pb18b extract interacted significantly with the 120-kDa fragment. Ligand affinity binding assays showed that type I collagen recognized two components (47 and 80 kDa) and gp43 bound both fibronectin and laminin. The peptide 1 (NLGRDAKRHL) from gp43, with several positively charged amino acids, contributed most to the adhesion of P. brasiliensis to Vero cells. Synthetic peptides derived from peptide YIGRS of laminin or from RGD of both laminin and fibronectin showed the greatest inhibition of adhesion of gp43 to Vero cells. In conclusion, this work provided new molecular details on the interaction between P. brasiliensis and ECNI components. (c) 2006 Elsevier SAS. All rights reserved.

Clues to the presence of pathogenic fungi in certain environments

The presence of various pathogenic fungi in rather unsuspected hosts and environments has always attracted the attention of the scientific community. Reports on the putative role of animals in fungal infections of humans bear important consequences on public health as well as on the understanding of fungal ecology. Fungi are ubiquitous in nature and their great capacity for adaptation allows them to survive and indeed, to thrive, in plants, trees and other natural substrata. Nonetheless, we are just beginning to learn the significance that these diverse fungal habitats have on the increasing number of immunosuppressed individuals. The accidental or permanent presence of fungi in animals, plants, soils and watercourses should not be taken too lightly because they constitute the source where potential pathogens will be contracted. If those fungal habitats that carry the largest risks of exposure could be defined, if seasonal variations in the production of infectious propagules could be determined, and if their mode of transmission were to be assessed, it would be possible to develop protective measures in order to avoid human infection. Additionally, unsuspected avenues for the exploration of fungal survival strategies would be opened, thus enhancing our capacity to react properly to their advancing limits. This paper explores several ecological connections between human pathogenic fungi and certain animals, trees, waterways and degraded organic materials. The occurrence of such connections in highly endemic areas will hopefully furnish more precise clues to fungal habitats and allow the design of control programs aimed at avoiding human infection.

Fungos causadores de podridões pós-colheita em uvas apirênicas no pólo agrícola de Juazeiro - BA e Petrolina - PE

In Brazil, postharvest diseases caused by pathogenic microorganisms are a major problem that causes damage to around 80% of the total fruit production. In the lower middle Sao Francisco river valley numerous studies on identification and control of fungal diseases during postharvest of grapes are needed, in order to minimize losses in this step. In this context, bunches of seedless varieties 'Crimson', 'Sonaka'; 'Superior' and 'Thompson' were collected from July to November 2009, in order to identify and quantify the incidence of pathogenic fungi. The grapes were collected on five farms which specialize in the production of table grapes for export, all located in Juazeiro - BA and Petrolina - PE. During this period, 10 samples were taken. In the fruit farm five plants were used for sampling, and removal of two bunches of grapes per plant, totaling 10 bunches per variety. Subsequently, they were sent to the laboratory of Plant Pathology at UNEB/DTCS where they were placed separately in a moist chamber for 48 hours at an average temperature of 23 degrees C. After this period, isolations of berries and stems in Petri plates containing PDA - potato-dextrose-agar were carried out with 10 repetitions, which were placed on benches under laboratory conditions. From the 8th day on, the presence of Aspergillus niger, Alternaria alternata, Cladosporium herbarum, Lasiodiploidia theobromae was observed, which presented the highest incidence, as well as Rhizopus stolonifer, Penicillium expansum.