Diversity, distribution and multi-functional attributes of bacterial communities associated with the rhizosphere and endosphere of timothy (Phleum pratense L.).

AIMS: To characterize the bacterial communities of the rhizosphere and endosphere of the forage grass timothy (Phleum pratense L.) and evaluate the functional attributes with respect to growth promotion properties, antimicrobial and biosurfactant capacities. METHODS AND RESULTS: A total of 254 culturable bacteria were identified using 16S rRNA sequencing and grouped into 16 taxa that shared high homology of 98-99% with other known sequences. A majority of the isolates were recovered from the rhizosphere soil fraction and leaf and crown tissues. Bacillus genus was the most abundant in the bulk and rhizosphere soil fractions. Isolates belonging to the Methylobacterium genus were exclusively found in leaves making them tissue-specific. A majority of the bacterial isolates exhibited multi-functional growth promotion attributes and plant stress improvement related to the production of indole 3-acetic acid, VOC and siderophores and polymer-degrading enzymes and 1-aminocyclopropane-1-carboxylate (ACC) deaminase activities. Some demonstrated antimicrobial properties such as hydrogen cyanide and biosurfactant production and activities of fungal cell wall degrading enzymes. The internalization and spread of selected bacterial isolates in timothy seedlings under gnotobiotic conditions was confirmed using the culture-dependent method and SEM microscopy in proof-of-concept experiments. CONCLUSIONS: The attributes of some isolates with respect to growth promotion abilities, biocontrol potential and efficient colonization of timothy make them desirable for future development as potential biofertilizer tools. SIGNIFICANCE AND IMPACT OF THE STUDY: This study provides the first evidence of bacterial endophytes that have the necessary functional attributes to protect cool-season forage grasses against abiotic stress.

Soil microarthropods alter the outcome of plant-soil feedback experiments.

Plant-soil feedback (PSF) effects are studied as plant growth responses to soil previously conditioned by another plant. These studies usually exclude effects of soil fauna, such as nematodes, soil arthropods, and earthworms, although these organisms are known to influence plant performance. Here, we aimed to explore effects of a model microarthropod community on PSFs. We performed a PSF experiment in microcosms with two plant species, Phleum pratense and Poa pratensis. We added a model microarthropod community consisting of three fungivorous springtail species (Proisotoma minuta, Folsomia candida, and Sinella curviseta) and a predatory mite (Hypoaspis aculeifer) to half of the microcosms. We measured seedling establishment and plant biomass, nematode and microbial community composition, microbial biomass, and mycorrhizal colonization of roots. Microarthropods caused changes in the composition of nematode and microbial communities. Their effect was particularly strong in Phleum plants where they altered the composition of bacterial communities. Microarthropods also generally influenced plant performance, and their effects depended on previous soil conditioning and the identity of plant species. Microarthropods did not affect soil microbial biomass and mycorrhizal colonization of roots. We conclude that the role of soil microarthropods should be considered in future PSF experiments, especially as their effects are plant species-specific.

Inoculation of Brassica oxyrrhina with plant growth promoting bacteria for the improvement of heavy metal phytoremediation under drought conditions.

The aim of this study was to investigate the effects of drought resistant serpentine rhizobacteria on plant growth and metal uptake by Brassica oxyrrhina under drought stress (DS) condition. Two drought resistant serpentine rhizobacterial strains namely Pseudomonas libanensis TR1 and Pseudomonas reactans Ph3R3 were selected based on their ability to stimulate seedling growth in roll towel assay. Further assessment on plant growth promoting (PGP) parameters revealed their ability to produce indole-3-acetic acid, siderophore and 1-aminocyclopropane-1-carboxylate deaminase. Moreover, both strains exhibited high resistance to various heavy metals, antibiotics, salinity and extreme temperature. Inoculation of TR1 and Ph3R3 significantly increased plant growth, leaf relative water and pigment content of B. oxyrrhina, whereas decreased concentrations of proline and malondialdehyde in leaves under metal stress in the absence and presence of DS. Regardless of soil water conditions, TR1 and Ph3R3 greatly improved organ metal concentrations, translocation and bioconcentration factors of Cu and Zn. The successful colonization and metabolic activities of P. libanensis TR1 and P. reactans Ph3R3 represented positive effects on plant development and metal phytoremediation under DS. These results indicate that these strains could be used as bio-inoculants for the improvement of phytoremediation of metal polluted soils under semiarid conditions.

Pollen-Associated Microbiome Correlates with Pollution Parameters and the Allergenicity of Pollen.

Pollen allergies have been rapidly increasing over the last decades. Many allergenic proteins and non-allergenic adjuvant compounds of pollen are involved in the plant defense against environmental or microbial stress. The first aim of this study was to analyze and compare the colonizing microbes on allergenic pollen. The second aim was to investigate detectable correlations between pollen microbiota and parameters of air pollution or pollen allergenicity. To reach these aims, bacterial and fungal DNA was isolated from pollen samples of timothy grass (Phleum pratense, n = 20) and birch trees (Betula pendula, n = 55). With this isolated DNA, a terminal restriction fragment length polymorphism analysis was performed. One result was that the microbial diversity on birch tree and timothy grass pollen samples (Shannon/Simpson diversity indices) was partly significantly correlated to allergenicity parameters (Bet v 1/Phl p 5, pollen-associated lipid mediators). Furthermore, the microbial diversity on birch pollen samples was correlated to on-site air pollution (nitrogen dioxide (NO2), ammonia (NH3), and ozone (O3)). What is more, a significant negative correlation was observed between the microbial diversity on birch pollen and the measured NO2 concentrations on the corresponding trees. Our results showed that the microbial composition of pollen was correlated to environmental exposure parameters alongside with a differential expression of allergen and pollen-associated lipid mediators. This might translate into altered allergenicity of pollen due to environmental and microbial stress.

Lactobacillus hokkaidonensis sp. nov., isolated from subarctic timothy grass (Phleum pratense L.) silage.

Four strains of Gram-positive, non-spore-forming, rod-shaped, catalase-negative and non-motile lactic acid bacteria, LOOC260(T), LOOC253, LOOC273 and LOOC279, were isolated from timothy grass (Phleum pratense L.) silage produced in Hokkaido, a subarctic region of Japan. These isolates grew at 4-37 °C, indicating the psychrotolerant nature of these strains. Phylogenetic analysis on the basis of 16S rRNA and pheS gene sequences, as well as biochemical and physiological characteristics, indicated that these four strains were members of the genus Lactobacillus. 16S rRNA gene sequence analysis of strain LOOC260(T) demonstrated that the closest neighbours were the type strains of Lactobacillus suebicus (97.7 %), Lactobacillus oligofermentans (96.7 %) and Lactobacillus vaccinostercus (96.7 %). Strain LOOC260(T) showed low levels of DNA-DNA association with Lactobacillus suebicus JCM 9504(T) (14.7 ± 3.5 %), Lactobacillus oligofermentans JCM 16175(T) (15.1 ± 4.8 %) and Lactobacillus vaccinostercus JCM 1716(T) (10.7 ± 3.0 %). The cell wall contained meso-diaminopimelic acid and the major fatty acids were C18 : 1ω9c and C19 : 1 cyclo 9,10. On the basis of phenotypic, physiological and phylogenetic evidence, these isolates represent a novel species of the genus Lactobacillus, for which the name Lactobacillus hokkaidonensis sp. nov. is proposed. The type strain is LOOC260(T) ( = JCM 18461(T) = DSM 26202(T)).

Identification and characterization of lactic acid bacteria isolated from mixed pasture of timothy and orchardgrass, and its badly preserved silage.

In order to understand the relationship between lactic acid bacteria (LAB) species and silage fermentation, a total of 65 LAB strains isolated from mixed pasture of timothy (Phleum pratense L.) and orchardgrass (Dactylis glomerata L.), and its badly preserved silages were subjected to phenotypic and genetic analysis. According to these analyses, the isolates were divided into 13 groups, including Enterococcus gallinarum, Lactobacillus acidipiscis, L. coryniformis subsp. coryniformis, L. coryniformis subsp. torquens, L. curvatus, L. paraplantarum, L. plantarum subsp. argentoratensis, L. plantarum subsp. plantarum, L. sakei subsp. carnosus, Lactococcus garvieae, Lactococcus lactis subsp. cremoris, Leuconostoc pseudomesenteroides, Pediococcus acidilactici, Pediococcus pentosaceus, Weissella hellenica, Weissella paramesenteroides and Carnobacterium divergens. This is the first report to document that C. divergens, L. acidipiscis, L. sakei subsp. carnosus, L. garvieae, phenotypically novel L. lactis subsp. cremoris, E. gallinarum and W. hellenica are present in vegetative forage crops. L. plantarum group strains were most frequently isolated from the badly preserved silages. Some isolates showed a wide range of growth preferences for carbohydrate utilization, optimal growth pH and temperature in vitro, indicating that they have a high growth potential. These results are useful in understanding the diversity of LAB associated with decayed silage of timothy and orchardgrass.

Gram-positive bacteria on grass pollen exhibit adjuvant activity inducing inflammatory T cell responses.

BACKGROUND: Recently, it has been established that pollen grains contain Th2-enhancing activities besides allergens. OBJECTIVE: The aim of this study was to analyse whether pollen carry additional adjuvant factors like microbes and what immunological effects they may exert. METHODS: Timothy pollen grains were collected and disseminated on agar plates, and the growing microorganisms were cultivated and defined. Furthermore, the immunologic effects of microbial products on DC and T cell responses were analysed. RESULTS: A complex mixture of bacteria and moulds was detected on grass pollen. Besides Gram-negative bacteria that are known to favour Th1-directed immune responses, moulds were identified as being sources of allergens themselves. Herein, we focused on Gram-positive bacteria that were found in high numbers, e.g. Bacillus cereus and Bacillus subtilis. Contact of immature dendritic cells (DC) from grass pollen allergic donors with supernatants of homogenized Gram-positive bacteria induced maturation of DC as measured by up-regulation of CD80, CD83 and CD86 and by enhanced production of IL-6, IL-12p40 and TNF-α, which was less pronounced compared with effects induced by lipopolysaccharide (LPS). Consequently, stimulation of autologous CD4(+) T cells with supernatants of homogenized Gram-positive bacteria plus grass pollen allergen-pulsed DC led to an enhanced proliferation and production of IL-4, IL-13, IL-10, IL-17, IL-22 and IFN-γ production compared with T cells that were stimulated with allergen-pulsed immature DC alone, whereas production of the transcription factor for regulatory T cells FoxP3 was not significantly affected. CONCLUSIONS AND CLINICAL RELEVANCE: These data indicate that grass pollen is colonized by several microorganisms that influence the immune response differently. Similar to LPS, supernatants of homogenized Gram-positive bacteria may serve as adjuvants by augmenting DC maturation and inflammatory Th1, Th2 and Th17 responses helping to initiate allergic immune responses.

Detrimental and neutral effects of a wild grass-fungal endophyte symbiotum on insect preference and performance.

Seed-borne Epichloë/Neotyphodium Glenn, Bacon, Hanlin (Ascomycota: Hypocreales: Clavicipitaceae) fungal endophytes in temperate grasses can provide protection against insect attack with the degree of host resistance related to the grass-endophyte symbiotum and the insect species involved in an interaction. Few experimental studies with wild grass-endophyte symbiota, compared to endophyte-infected agricultural grasses, have tested for anti-insect benefits, let alone for resistance against more than one insect species. This study quantified the preference and performance of the bird cherry oat-aphid, Rhopalosiphum padi (L.) (Hemiptera: Aphididae) and the cereal leaf beetle, Oulema melanopus (L.) (Coleoptera: Chrysomelidae), two important pests of forage and cereal grasses, on Neotyphodium-infected (E+) and uninfected (E-) plants of the wild grass Alpine timothy, Phleum alpinum L. (Poales: Poaceae). The experiments tested for both constitutive and wound-induced resistance in E+ plants to characterize possible plasticity of defense responses by a wild E+ grass. The aphid, R. padi preferred E- over E+ test plants in choice experiments and E+ undamaged test plants constitutively expressed antibiosis resistance to this aphid by suppressing population growth. Prior damage of E+ test plants did not induce higher levels of resistance to R. padi. By contrast, the beetle, O. melanopus showed no preference for E+ or E- test plants and endophyte infection did not adversely affect the survival and development of larvae. These results extend the phenomenon of variable effects of E+ wild grasses on the preference and performance of phytophagous insects. The wild grass- Neotyphodium symbiotum in this study broadens the number of wild E+ grasses available for expanded explorations into the effects of endophyte metabolites on insect herbivory.

Elevated atmospheric carbon dioxide concentrations amplify Alternaria alternata sporulation and total antigen production.

BACKGROUND: Although the effect of elevated carbon dioxide (CO2) concentration on pollen production has been established in some plant species, impacts on fungal sporulation and antigen production have not been elucidated. OBJECTIVE: Our purpose was to examine the effects of rising atmospheric CO2 concentrations on the quantity and quality of fungal spores produced on timothy (Phleum pratense) leaves. METHODS: Timothy plants were grown at four CO2 concentrations (300, 400, 500, and 600 micromol/mol). Leaves were used as growth substrate for Alternaria alternata and Cladosporium phlei. The spore abundance produced by both fungi, as well as the size (microscopy) and antigenic protein content (ELISA) of A. alternata, were quantified. RESULTS: Leaf carbon-to-nitrogen ratio was greater at 500 and 600 micromol/mol, and leaf biomass was greater at 600 micromol/mol than at the lower CO2 concentrations. Leaf carbon-to-nitrogen ratio was positively correlated with A. alternata spore production per gram of leaf but negatively correlated with antigenic protein content per spore. At 500 and 600 micromol/mol CO2 concentrations, A. alternata produced nearly three times the number of spores and more than twice the total antigenic protein per plant than at lower concentrations. C. phlei spore production was positively correlated with leaf carbon-to-nitrogen ratio, but overall spore production was much lower than in A. alternata, and total per-plant production did not vary among CO2 concentrations. CONCLUSIONS: Elevated CO2 concentrations often increase plant leaf biomass and carbon-to-nitrogen ratio. Here we demonstrate for the first time that these leaf changes are associated with increased spore production by A. alternata, a ubiquitous allergenic fungus. This response may contribute to the increasing prevalence of allergies and asthma.

Specific bottom-up effects of arbuscular mycorrhizal fungi across a plant-herbivore-parasitoid system.

The majority of plants are involved in symbioses with arbuscular mycorrhizal fungi (AMF), and these associations are known to have a strong influence on the performance of both plants and insect herbivores. Little is known about the impact of AMF on complex trophic chains, although such effects are conceivable. In a greenhouse study we examined the effects of two AMF species, Glomus intraradices and G. mosseae on trophic interactions between the grass Phleum pratense, the aphid Rhopalosiphum padi, and the parasitic wasp Aphidius rhopalosiphi. Inoculation with AMF in our study system generally enhanced plant biomass (+5.2%) and decreased aphid population growth (-47%), but there were no fungal species-specific effects. When plants were infested with G. intraradices, the rate of parasitism in aphids increased by 140% relative to the G. mosseae and control treatment. When plants were associated with AMF, the developmental time of the parasitoids decreased by 4.3% and weight at eclosion increased by 23.8%. There were no clear effects of AMF on the concentration of nitrogen and phosphorus in plant foliage. Our study demonstrates that the effects of AMF go beyond a simple amelioration of the plants' nutritional status and involve rather more complex species-specific cascading effects of AMF in the food chain that have a strong impact not only on the performance of plants but also on higher trophic levels, such as herbivores and parasitoids.

Improvement of plant growth and nickel uptake by nickel resistant-plant-growth promoting bacteria.

In this study, among a collection of Ni-resistant bacterial strains isolated from the rhizosphere of Alyssum serpyllifolium and Phleum phleoides grown on serpentine soil, five plant growth-promoting bacteria (PGPB) were selected based on their ability to utilize 1-aminocyclopropane-1-carboxylate (ACC) as the sole N source and promote seedling growth. All of the strains tested positive for indole-3-acetic acid (IAA) production and phosphate solubilization. In addition, four of the strains exhibited significant levels of siderophores production. Further, the efficiency of PGPB in enhancing Ni solubilization in soils was analyzed. Compared with control treatment, inoculation of PGPB strains significantly increased the concentrations of bioavailable Ni. Furthermore, a pot experiment was conducted to elucidate the effects of inoculating Ni-resistant PGPB on the plant growth and the uptake of Ni by Brassica juncea and B. oxyrrhina in soil contaminated with 450 mg kg(-1) Ni. Psychrobacter sp. SRA2 significantly increased the fresh (351%) and dry biomass (285%) of the B. juncea test plants (p<0.05), whereas Psychrobacter sp. SRA1 and Bacillus cereus SRA10 significantly increased the accumulation of Ni in the root and shoot tissues of B. juncea compared with non-inoculated controls. This result indicates that the strains SRA1 and SRA10 facilitated the release of Ni from the non-soluble phases in the soil, thus enhancing the availability of Ni to plants. A significant increase, greater than that of the control, was also noted for growth parameters of the B. oxyrrhina test plants when the seeds were treated with strain SRA2. This effect can be attributed to the utilization of ACC, solubilization of phosphate and production of IAA. The results of the study revealed that the inoculation of Ni mobilizing strains Psychrobacter sp. SRA1 and B. cereus SRA10 increases the efficiency of phytoextraction directly by enhancing the metal accumulation in plant tissues and the efficient PGPB, Psychrobacter sp. SRA2 increases indirectly by promoting the growth of B. juncea and B. oxyrrhina.

Bacteriological and chemical changes occurring in Bunker-stored silage covered with biodegradable coating.

AIMS: To evaluate the efficacy of a biodegradable silage coating for the ability to protect timothy (Phleum pratensa) type silage against spoilage and its quality under natural conditions. METHODS AND RESULTS: Triplicate mini-silos of silage were prepared for three treatments (1: uncoated; 2: coated with biodegradable coating and 3: sealed with plastic), two types of storage (unprotected or protected from rain) and 10 sampling times (0, 7, 14, 21, 28, 35, 42, 56, 63 and 70 days postensiling). Triplicate mini-silos were opened at each sampling time for microbiological (total aerobic bacteria, lactic acid bacteria, moulds and yeasts) and biochemical analyses [pH, dry matter (DM), water-soluble sugars (WSC), lactic (LA), acetic, propionic and butyric acids content]. The study showed that at day 70, counts of moulds and yeasts in silages protected against rain and coated with biodegradable coating were 5.98 log CFU g(-1) when compared with 5.92 and 3.62 log CFU g(-1) in samples from plastic-sealed silage and uncoated silage, respectively. The pH was low and stable pH (4.34) when compared with uncoated (7.17) and plastic sealed (8.34) silages (P < or = 0.05). A DM, WSC and LA content of 421.7, 13.4 and 20.9 g kg(-1) was, respectively, observed. For silage stored outdoors, a level of moulds and yeasts of 3.77 log CFU g(-1) of silage was also observed in silages coated with biodegradable coating after 28 days of storage. A stable pH showing a mean value of 4 was also observed. The pH, DM, WSC and LA content were, respectively, 4.18, 341.1, 13.34 and 31.8 g kg(-1) in these samples. After 70 days of storage, the level of moulds and yeasts on silage sealed with biodegradable coating was 7.73 log CFU g(-1). A DM, WSC and LA content of 291.9, 5.56 and 10.0 g kg(-1) was, respectively, observed. CONCLUSIONS: When compared with uncoated silage, the application of biodegradable coating can preserve the quality of silage for up to a month when exposed to rain and up to 70 days when protected from rain. SIGNIFICANCE AND IMPACT OF THE STUDY: Results emphasize the possibility of the use of a biodegradable coating as an alternative to plastic film for sealing horizontal bunker silos.

Novel cyclic peptide, epichlicin, from the endophytic fungus, Epichloe typhina.

The novel cyclic peptide, epichlicin, was isolated from Epichloe typhina, an endophytic fungus of the timothy plant (Phleum pretense L.). Its structure was determined by NMR studies and by mass spectrometry. Enantiomers of 3-amino tetradecanoic acid, a constituent amino acid of epichlicin, were synthesized as authentic standards. The stereochemistry of each amino acid was elucidated through a combination of the advanced Marfey method and chemical manipulation. Epichlicin showed inhibitory activity toward the spore germination of Cladosporium phlei, a pathogenic fungus of the timothy plant at an IC50 value of 22 nM.