Food preservation using antifungal lactic acid bacteria

Fungal spoilage of food and feed prevails as a major problem for the food industry. The use antifungal-producing lactic acid bacteria (LAB) may represent a safer, natural alternative to the use of chemical preservatives in foods. A large scale screen was undertaken to identify a variety of LAB with antifungal properties from plant, animal and human sources. A total of 6,720 LAB colonies were isolated and screened for antifungal activity against the indicator Penicillium expansum. 94 broad-spectrum producers were identified through 16S rRNA sequencing with the majority of the population comprising Lactobacillus plantarum isolates. Six broad-spectrum isolates were consequently characterised. Pedicococcus pentosaceous 54 displayed potent anti-mould capabilities in pear, plum and grape models and may represent an ideal candidate for use in the beverage industry. Two antifungal Lb. plantarum isolates were assessed for their technological robustness and potential as biopreservatives in refrigerated foods. Lb. plantarum 16 and 62 displayed high levels of tolerance to freeze-drying, low temperature exposure and high salt concentrations. Both lactobacilli were introduced as supplements into orange juice to retard the growth of the spoilage yeast Rhodotorula mucilaginosa. Furthermore the isolates were applied as adjuncts in yoghurt production to successfully reduce yeast growth. Lb. plantarum 16 proved to be the optimal inhibitor of yeast growth in both food matrices. To date there is limited information available describing the mechanisms behind fungal inhibition by LAB. The effects of concentrated cell-free supernatant (cCFS), derived from Lb. plantarum 16, on the growth of two food-associated moulds was assessed microscopically. cCFS completely inhibited spore, germ tube and hyphal development. A transcriptomic approach was undertaken to determine the impact of antifungal activity on Aspergillus fumigatus Af293. A variety of genes, most notably those involved in cellular metabolism, were found to have their transcription modulated in response to cCFS which is indicative of global cellular shutdown. This study provides the first insights into the molecular targets of antifungal compounds produced by LAB. The genome sequence of the steep water isolate Lb. plantarum 16 was determined. The complete genome of Lb. plantarum16 consists of a single circular chromosome of 3,044,738 base pairs with an average G+C content of 44.74 % in addition to eight plasmids. The genome represents the smallest of this species to date while harbouring the largest plasmid complement. Some features of particular interest include the presence of two prophages, an interrupted plantaricin cluster and a chromosomal and plasmid encoded polysaccharide cluster. The sequence presented here provides a suitable platform for future studies elucidating the mechanisms governing antifungal production.

Arsenic accumulation and metabolism in rice (Oryza sativa L.)

The use of arsenic (As) contaminated groundwater for irrigation of crops has resulted in elevated concentrations of arsenic in agricultural soils in Bangladesh, West Bengal (India), and elsewhere. Paddy rice (Oryza sativa L.) is the main agricultural crop grown in the arsenic-affected areas of Bangladesh. There is, therefore, concern regarding accumulation of arsenic in rice grown those soils. A greenhouse study was conducted to examine the effects of arsenic-contaminated irrigation water on the growth of rice and uptake and speciation of arsenic. Treatments of the greenhouse experiment consisted of two phosphate doses and seven different arsenate concentrations ranging from 0 to 8 mg of As L(-1) applied regularly throughout the 170-day post-transplantation growing period until plants were ready for harvesting. Increasing the concentration of arsenate in irrigation water significantly decreased plant height, grain yield, the number of filled grains, grain weight, and root biomass, while the arsenic concentrations in root, straw, and rice husk increased significantly. Concentrations of arsenic in rice grain did not exceed the food hygiene concentration limit (1.0 mg of As kg(-1) dry weight). The concentrations of arsenic in rice straw (up to 91.8 mg kg(-1) for the highest As treatment) were of the same order of magnitude as root arsenic concentrations (up to 107.5 mg kg(-1)), suggesting that arsenic can be readily translocated to the shoot. While not covered by food hygiene regulations, rice straw is used as cattle feed in many countries including Bangladesh. The high arsenic concentrations may have the potential for adverse health effects on the cattle and an increase of arsenic exposure in humans via the plant-animal-human pathway. Arsenic concentrations in rice plant parts except husk were not affected by application of phosphate. As the concentration of arsenic in the rice grain was low, arsenic speciation was performed only on rice straw to predict the risk associated with feeding contaminated straw to the cattle. Speciation of arsenic in tissues (using HPLC-ICP-MS) revealed that the predominant species present in straw was arsenate followed by arsenite and dimethylarsinic acid (DMAA). As DMAA is only present at low concentrations, it is unlikely this will greatly alter the toxicity of arsenic present in rice.

Do leaves of plants on phosphorus-impoverished soils contain high concentrations of phenolic defence compounds?

Prominent theories of plant defence have predicted that plants growing on nutrient-poor soils produce more phenolic defence compounds than those on richer soils. Only recently has the Protein Competition Model (PCM) of phenolic allocation suggested that N and P limitation could have different effects because the nutrients are involved in different cellular metabolic processes. 2. We extend the prediction of the PCM and hypothesize that N will have a greater influence on the production of phenolic defensive compounds than P availability, because N limitation reduces protein production and thus competition for phenylalanine, a precursor of many phenolic compounds. In contrast, P acts as a recyclable cofactor in these reactions, allowing protein and hence phenolic production to continue under low P conditions. 3. We test this hypothesis by comparing the foliar concentrations of phenolic compounds in (i) phenotypes of 21 species growing on P-rich alluvial terraces and P-depleted marine terraces in southern New Zealand, and (ii) 87 species growing under similar climates on comparatively P-rich soils in New Zealand vs. P-depleted soils in Tasmania. 4. Foliar P concentrations of plants from the marine terraces were about half those of plants from alluvial soils, and much lower in Tasmania than in New Zealand. However, foliar concentrations of N and phenolic compounds were similar across sites in both comparisons, supporting the hypothesis that N availability is a more important determinant of plant investment in phenolic defensive compounds than P availability. We found no indication that reduced soil P levels influenced plant concentrations of phenolic compounds. There was wide variation in the foliar N and P concentrations among species, and those with low foliar nutrient concentrations produced more phenolics (including condensed tannins). 5. Our study is the first trait comparison extending beyond standard leaf economics to include secondary metabolites related to defence in forest plants, and emphasizes that N and P have different influences on the production of phenolic defence compounds. © 2009 British Ecological Society.

Impacts of culling and exclusion of browsers on vegetation recovery across New Zealand forests

Introduced browsing animals negatively impact New Zealand's indigenous ecosystems. Eradicating introduced browsers is currently unfeasible at large scales, but culling since the 1960s has successfully reduced populations to a fraction of their earlier sizes. Here we ask whether culling of ungulates has allowed populations of woody plant species to recover across New Zealand forests. Using 73 pairs of permanent fenced exclosure and unfenced control plots, we found rapid increases in sapling densities within exclosures located in disturbed forests, particularly if a seedling bank was already present. Recovery was slower in thinning stands and hampered by dense fern cover. We inferred ungulate diet preference from species recovery rates inside exclosures to test whether culling increased abundance of preferred species across a national network of 574 unfenced permanent forest plots. Across this network, saplings were observed irrespective of their preference to ungulates in the 1970s, but preferred species were rarer within disturbed sites in the 1990s after long-term culling and despite nationwide increases in sapling densities. This indicates that preferred species are relatively heavily affected by browsing after culling, presumably because remaining animals will increase consumption of preferred species as competition is reduced. Our results clearly suggest that culling will not return preferred plants to the landscape immediately, even given suitable conditions for regeneration. Complete removal of ungulates rather than simply reducing their densities may be required for recovery in heavily browsed temperate forests, but since this is only feasible at small spatial scales, management efforts must target sites of high conservation value. © 2012 Elsevier Ltd.

Microbe-mediated recovery of salt marshes contaminated with oil hydrocarbons

Salt marshes are highly productive intertidal habitats that serve as nursery grounds for many commercially and economically important species. Because of their location and physical and biological characteristics, salt marshes are considered to be particularly vulnerable to anthropogenic inputs of oil hydrocarbons. Sediment contamination with oil is especially dangerous for salt marsh vegetation, since low molecular weight aromatic hydrocarbons can affect plants at all stages of development. However, the use of vegetation for bioremediation (phytoremediation), by removal or sequestration of contaminants, has been intensively studied. Phytoremediation is an efficient, inexpensive and environmental friendly approach for the removal of aromatic hydrocarbons, through direct incorporation by the plant and by the intervention of degrading microbial populations in the rhizosphere (microbe-assisted phytoremediation). Rhizosphere microbial communities are enriched in important catabolic genotypes for degradation of oil hydrocarbons (OH) which may have a potential for detoxification of the sediment surrounding the roots. In addition, since rhizosphere bacterial populations may also internalize into plant tissues (endophytes), rhizocompetent AH degrading populations may be important for in planta AH degradation and detoxification. The present study involved field work and microcosms experiments aiming the characterization of relevant plant-microbe interactions in oilimpacted salt marshes and the understanding of the effect of rhizosphere and endosphere bacteria in the role of salt marsh plants as potential phytoremediation agents. In the field approach, molecular tools were used to assess how plant species- and OH pollution affect sediment bacterial composition [bulk sediment and sediment surrounding the roots (rhizosphere) of Halimione portulacoides and Sarcocornia perennis subsp. perennis] in a temperate estuary (Ria de Aveiro, Portugal) chronically exposed to OH pollution. In addition, the 16S rRNA gene sequences retrieved in this study were used to generate in silico metagenomes and to evaluate the distribution of potential bacterial traits in different microhabitats. Moreover, a combination of culture-dependent and -independent approaches was used to investigate the effect of oil hydrocarbons contamination on the structure and function of endophytic bacterial communities of salt marsh plants.Root systems of H. portulacoides and S. perennis subsp. perennis appear to be able to exert a strong influence on bacterial composition and in silico metagenome analysis showed enrichment of genes involved in the process of polycyclic aromatic hydrocarbon (PAH) degradation in the rhizosphere of halophyte plants. The culturable fraction of endophytic degraders was essentially closely related to known OH-degrading Pseudomonas species and endophytic communities revealed sitespecific effects related to the level of OH contamination in the sediment. In order to determine the effects of oil contamination on plant condition and on the responses in terms of structure and function of the bacterial community associated with plant roots (rhizosphere, endosphere), a microcosms approach was set up. The salt marsh plant Halimione portulacoides was inoculated with a previous isolated Pseudomonas sp. endophytic degrader and the 2-methylnaphthalene was used as model PAH contaminant. The results showed that H. portulacoides health and growth were not affected by the contamination with the tested concentration. Moreover, the decrease of 2-methylnaphthalene at the end of experiment, can suggest that H. portulacoides can be considered as a potential plant for future uses in phytoremedition approaches of contaminated salt marsh. The acceleration of hydrocarbon degradation by inoculation of the plants with the hydrocarbon-degrading Pseudomonas sp. could not, however, be demonstrated, although the effects of inoculation on the structure of the endophytic community observed at the end of the experiment indicate that the strain may be an efficient colonizer of H. portulacoides roots. The results obtained in this work suggest that H. portulacoides tolerates moderate concentrations of 2-methylnaphthalene and can be regarded as a promising agent for phytoremedition approaches in salt marshes contaminated with oil hydrocarbons. Plant/microbe interactions may have an important role in the degradation process, as plants support a diverse endophytic bacterial community, enriched in genetic factors (genes and plasmids) for hydrocarbon degradation.

Genome-wide association studies reveal genomic windows and candidate genes related to fat deposition in chickens.

2016

Identification of CNVs on the genome of Brazilian chicken lines.

2016

Pretreatment Of Agricultural Waste With Pleurotus Sp. For Ethanol Production

Bioethanol is a liquid fuel obtained from fermentation of sugar/starch crops. Lignocellulosic biomass being less expensive is considered a future alternative for the food crops. One of the main challenges for the use of lignocellulosics is the development of an efficient pre-treatment process. Pretreatments are classified into three - physical, chemical, and biological pretreatment. Chemical process has not been proven suitable so far, due to high costs and production of undesired by-products. Biologically, hydrolysis can be enhanced by microbial or enzymatic pretreatment. Studies show that the edible mushrooms of Pleurotus sp. produce several extracellular enzymes which reduce the structural and chemical complexity of fibre. In the present study, P. ostreatus and P. eous were cultivated on paddy straw. Spent substrate left after mushroom cultivation was powdered and used for ethanol production. Saccharomyces sp. was used for fermentation studies. Untreated paddy straw was used as control. Production of ethanol from P. ostreatus substrate was 5.5 times more when compared to untreated paddy straw, while the spent substrate of P. eous gave 5 times increase in ethanol yield. Assays showed the presence of several extracellular enzymes in the spent substrate of both species, which together contributed to the increase in ethanol yield

Hyperspectral field measurements to determine dry matter yield and nutritive value of legume-grass swards

Summary: Productivity, botanical composition and forage quality of legume-grass swards are important factors for successful arable farming in both organic and conventional farming systems. As these attributes can vary considerably within a field, a non-destructive method of detection while doing other tasks would facilitate a more targeted management of crops, forage and nutrients in the soil-plant-animal system. This study was undertaken to explore the potential of field spectral measurements for a non destructive prediction of dry matter (DM) yield, legume proportion in the sward, metabolizable energy (ME), ash content, crude protein (CP) and acid detergent fiber (ADF) of legume-grass mixtures. Two experiments were conducted in a greenhouse under controlled conditions which allowed collecting spectral measurements which were free from interferences such as wind, passing clouds and changing angles of solar irradiation. In a second step this initial investigation was evaluated in the field by a two year experiment with the same legume-grass swards. Several techniques for analysis of the hyperspectral data set were examined in this study: four vegetation indices (VIs): simple ratio (SR), normalized difference vegetation index (NDVI), enhanced vegetation index (EVI) and red edge position (REP), two-waveband reflectance ratios, modified partial least squares (MPLS) regression and stepwise multiple linear regression (SMLR). The results showed the potential of field spectroscopy and proved its usefulness for the prediction of DM yield, ash content and CP across a wide range of legume proportion and growth stage. In all investigations prediction accuracy of DM yield, ash content and CP could be improved by legume-specific calibrations which included mixtures and pure swards of perennial ryegrass and of the respective legume species. The comparison between the greenhouse and the field experiments showed that the interaction between spectral reflectance and weather conditions as well as incidence angle of light interfered with an accurate determination of DM yield. Further research is hence needed to improve the validity of spectral measurements in the field. Furthermore, the developed models should be tested on varying sites and vegetation periods to enhance the robustness and portability of the models to other environmental conditions.

The fate and toxicity of the flavonoids naringenin and formononetin in soil

The flavonoid class of plant secondary metabolites play a multifunctional role in below-ground plant-microbe interactions with their best known function as signals in the nitrogen fixing legume-rhizobia symbiosis. Flavonoids enter rhizosphere soil as a result of root exudation and senescence but little is known about their subsequent fate or impacts on microbial activity. Therefore, the present study examined the sorptive behaviour, biodegradation and impact on dehydrogenase activity (as determined by iodonitrotetrazolium chloride reduction) of the flavonoids naringenin and formononetin in soil. Organic carbon normalised partition coefficients, log K-oc, of 3.12 (formononetin) and 3.19 (naringenin) were estimated from sorption isotherms and, after comparison with literature log K-oc values for compounds whose soil behaviour is better characterised, the test flavonoids were deemed to be moderately sorbed. Naringenin (spiked at 50 mu g g(-1)) was biodegraded without a detectable lag phase with concentrations reduced to 0.13 +/- 0.01 mu g g(-1) at the end of the 96 h time course. Biodegradation of formononetin proceeded after a lag phase of similar to 24 with concentrations reduced to 4.5 +/- 1% of the sterile control after 72 h. Most probable number (MPN) analysis revealed that prior to the addition of flavonoids, the soil contained 5.4 x 10(6) MPNg(-1) (naringenin) and 7.9 x 10(5) MPNg(-1) (formononetin) catabolic microbes. Formononetin concentration had no significant (p > 0.05) effect on soil dehydrogenase activity, whereas naringenin concentration had an overall but non-systematic impact (p = 0.045). These results are discussed with reference to likely total and bioavailable concentrations of flavonoids experienced by microbes in the rhizosphere. (c) 2007 Elsevier Ltd. All rights reserved.

Role of polyhydroxybutyrate and glycogen as carbon storage compounds in pea and bean bacteroids

Rhizobium leguminosarum synthesizes polyhydroxybutyrate and glycogen as its main carbon storage compounds. To examine the role of these compounds in bacteroid development and in symbiotic efficiency, single and double mutants of R. legumosarum bv. viciae were made which lack polyhydroxybutyrate synthase (phaC), glycogen synthase (glgA), or both. For comparison, a single phaC mutant also was isolated in a bean-nodulating strain of R. leguminosarum bv. phaseoli. In one large glasshouse trial, the growth of pea plants inoculated with the R. leguminosarum bv. viciae phaC mutant were significantly reduced compared with wild-type-inoculated plants. However, in subsequent glasshouse and growth-room studies, the growth of pea plants inoculated with the mutant were similar to wildtype-inoculated plants. Bean plants were unaffected by the loss of polyhydroxybutyrate biosynthesis in bacteroids. Pea plants nodulated by a glycogen synthase mutants or the glgA/phaC double mutant, grew as well as the wild type in growth-room experiments. Light and electron micrographs revealed that pea nodules infected with the glgA mutant accumulated large amounts of starch in the II/III interzone. This suggests that glycogen may be the dominant carbon storage compound in pea bacteroids. Polyhydroxybutyrate was present in bacteria in the infection thread of pea plants but was broken down during bacteroid formation. In nodules infected with a phaC mutant of R. leguminosarum bv. viciae, there was a drop in the amount of starch in the II/III interzone, where bacteroids form. Therefore, we propose a carbon burst hypothesis for bacteroid formation, where polyhydroxybutyrate accumulated by bacteria is degraded to fuel bacteroid differentiation.

Role of polyhydroxybutyrate and glycogen as carbon storage compounds in pea and bean bacteroids

Rhizobium leguminosarum synthesizes polyhydroxybutyrate and glycogen as its main carbon storage compounds. To examine the role of these compounds in bacteroid development and in symbiotic efficiency, single and double mutants of R. legumosarum bv. viciae were made which lack polyhydroxybutyrate synthase (phaC), glycogen synthase (glgA), or both. For comparison, a single phaC mutant also was isolated in a bean-nodulating strain of R. leguminosarum bv. phaseoli. In one large glasshouse trial, the growth of pea plants inoculated with the R. leguminosarum bv. viciae phaC mutant were significantly reduced compared with wild-type-inoculated plants. However, in subsequent glasshouse and growth-room studies, the growth of pea plants inoculated with the mutant were similar to wildtype-inoculated plants. Bean plants were unaffected by the loss of polyhydroxybutyrate biosynthesis in bacteroids. Pea plants nodulated by a glycogen synthase mutants or the glgA/phaC double mutant, grew as well as the wild type in growth-room experiments. Light and electron micrographs revealed that pea nodules infected with the glgA mutant accumulated large amounts of starch in the II/III interzone. This suggests that glycogen may be the dominant carbon storage compound in pea bacteroids. Polyhydroxybutyrate was present in bacteria in the infection thread of pea plants but was broken down during bacteroid formation. In nodules infected with a phaC mutant of R. leguminosarum bv. viciae, there was a drop in the amount of starch in the II/III interzone, where bacteroids form. Therefore, we propose a carbon burst hypothesis for bacteroid formation, where polyhydroxybutyrate accumulated by bacteria is degraded to fuel bacteroid differentiation.

Confocal imaging of pseudomonas syringae pv. phaseolicola colony development in bean reveals reduced multiplication of strains containing the genomic island PPHGI-1.

Pseudomonas syringae pv. phaseolicola is the seed borne causative agent of halo blight in the common bean Phaseolus vulgaris. Pseudomonas syringae pv. phaseolicola race 4 strain 1302A contains the avirulence gene hopAR1 (located on a 106-kb genomic island, PPHGI-1, and earlier named avrPphB), which matches resistance gene R3 in P. vulgaris cultivar Tendergreen (TG) and causes a rapid hypersensitive reaction (HR). Here, we have fluorescently labeled selected Pseudomonas syringae pv. phaseolicola 1302A and 1448A strains (with and without PPHGI-1) to enable confocal imaging of in-planta colony formation within the apoplast of resistant (TG) and susceptible (Canadian Wonder [CW]) P. vulgaris leaves. Temporal quantification of fluorescent Pseudomonas syringae pv. phaseolicola colony development correlated with in-planta bacterial multiplication (measured as CFU/ml) and is, therefore, an effective means of monitoring Pseudomonas syringae pv. phaseolicola endophytic colonization and survival in P. vulgaris. We present advances in the application of confocal microscopy for in-planta visualization of Pseudomonas syringae pv. phaseolicola colony development in the leaf mesophyll to show how the HR defense response greatly affects colony morphology and bacterial survival. Unexpectedly, the presence of PPHGI-1 was found to cause a reduction of colony development in susceptible P. vulgaris CW leaf tissue. We discuss the evolutionary consequences that the acquisition and retention of PPHGI-1 brings to Pseudomonas syringae pv. phaseolicola in planta.

Pseudomonas syringae pv. phaseolicola: from ‘has bean’ to supermodel

Pseudomonas syringae pv. phaseolicola causes halo blight of the common bean, Phaseolus vulgaris, worldwide and remains difficult to control. Races of the pathogen cause either disease symptoms or a resistant hypersensitive response on a series of differentially reacting bean cultivars. The molecular genetics of the interaction between P. syringae pv. phaseolicola and bean, and the evolution of bacterial virulence, have been investigated in depth and this research has led to important discoveries in the field of plant-microbe interactions. In this review, we discuss several of the areas of study that chart the rise of P. syringae pv. phaseolicola from a common pathogen of bean plants to a molecular plant-pathogen supermodel bacterium. Taxonomy: Bacteria; Proteobacteria, gamma subdivision; order Pseudomonadales; family Pseudomonadaceae; genus Pseudomonas; species Pseudomonas syringae; Genomospecies 2; pathogenic variety phaseolicola. Microbiological properties: Gram-negative, aerobic, motile, rod-shaped, 1.5 µm long, 0.7-1.2 µm in diameter, at least one polar flagellum, optimal temperatures for growth of 25-30 °C, oxidase negative, arginine dihydrolase negative, levan positive and elicits the hypersensitive response on tobacco. Host range: Major bacterial disease of common bean (Phaseolus vulgaris) in temperate regions and above medium altitudes in the tropics. Natural infections have been recorded on several other legume species, including all members of the tribe Phaseoleae with the exception of Desmodium spp. and Pisum sativum. Disease symptoms: Water-soaked lesions on leaves, pods, stems or petioles, that quickly develop greenish-yellow haloes on leaves at temperatures of less than 23 °C. Infected seeds may be symptomless, or have wrinkled or buttery-yellow patches on the seed coat. Seedling infection is recognized by general chlorosis, stunting and distortion of growth. Epidemiology: Seed borne and disseminated from exudation by water-splash and wind occurring during rainfall. Bacteria invade through wounds and natural openings (notably stomata). Weedy and cultivated alternative hosts may also harbour the bacterium. Disease control: Some measure of control is achieved with copper formulations and streptomycin. Pathogen-free seed and resistant cultivars are recommended. Useful websites: Pseudomonas-plant interaction http://www.pseudomonas-syringae.org/; PseudoDB http://xbase.bham.ac.uk/pseudodb/; Plant Associated and Environmental Microbes Database (PAMDB) http://genome.ppws.vt.edu/cgi-bin/MLST/home.pl; PseudoMLSA Database http://www.uib.es/microbiologiaBD/Welcome.html.