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.

Implication of Fusarium graminearum primary metabolism in its resistance to benzimidazole fungicides as revealed by 1H NMR metabolomics.

Fungal metabolomics is a field of high potential but yet largely unexploited. Focusing on plant-pathogenic fungi, no metabolomics studies exist on their resistance to fungicides, which represents a major issue that the agrochemical and agricultural sectors are facing. Fungal infections cause quantitative, but also qualitative yield losses, especially in the case of mycotoxin-producing species. The aim of the study was to correlate metabolic changes in Fusarium graminearum strains' metabolomes with their carbendazim-resistant level and discover corresponding metabolites-biomarkers, with primary focus on its primary metabolism. For this purpose, comparative 1H NMR metabolomics was applied to a wild-type and four carbendazim-resistant Fusarium graminearum strains following or not exposure to the fungicide. Results showed an excellent discrimination between the strains based on their carbendazim-resistance following exposure to low concentration of the fungicide (2 mg L-1). Both genotype and fungicide treatments had a major impact on fungal metabolism. Among the signatory metabolites, a positive correlation was discovered between the content of F. graminearum strains in amino acids of the aromatic and pyruvate families, l-glutamate, l-proline, l-serine, pyroglutamate, and succinate and their carbendazim-resistance level. In contrary, their content in l-glutamine and l-threonine, had a negative correlation. Many of these metabolites play important roles in fungal physiology and responses to stresses. This work represents a proof-of-concept of the applicability of 1H NMR metabolomics for high-throughput screening of fungal mutations leading to fungicide resistance, and the study of its biochemical basis, focusing on the involvement of primary metabolism. Results could be further exploited in programs of resistance monitoring, genetic engineering, and crop protection for combating fungal resistance to fungicides.

Isolation and characterization of indigenous endophytic bacteria associated with leaves of switchgrass (Panicum virgatum L.) cultivars.

AIMS: To isolate and characterize indigenous bacterial endophytes from cultivars of switchgrass and study their antimicrobial and growth promoting potential. METHODS AND RESULTS: The diversity, molecular and biochemical characterizations of indigenous and culturable bacterial endophytes residing in leaves of switchgrass have not been studied previously. This study describes the characterization of 31 bacterial endophytes from three switchgrass cutlivars: Cave-in Rock, Blue Jacket and Tecumseh. Molecular and phylogenetic analysis based on the 16S rRNA sequence grouped the endophytes into eight different taxa that shared high homology of 98-99% with other known sequences. Bacterial endophytes were identified as Microbacterium testaceum, Curtobacterium flaccumfaciens, Bacillus subtilis and Bacillus pumilus, Pseudomonas fluorescens, Sphingomonas parapaucimobilis, Serratia sp. and Pantoea ananatis. Some endophytes were detected in switchgrass seeds and in plants that originated from seeds collected a year earlier, confirming vertical transmission to the next generation of the host. Selected endophytes produced cellulases and were capable of solubilizing inorganic phosphorus. Analysis of cell-free culture filtrate of selected strains using direct infusion orbitrap mass spectrometry confirmed the presence of several well-characterized lipopeptide toxins and phytohormones. Re-inoculation of the roots of switchgrass seedlings with endophytes singly or combined confirmed their migration to the upper aerial parts of the plant. CONCLUSIONS: Our findings suggest that switchgrass leaves harbour a diversity of bacterial endophytes, some of which could potentially be applied as growth promoting bacteria. SIGNIFICANCE AND IMPACT OF THE STUDY: This is the first report on the characterization of indigenous bacterial endophytes and their potential use as biofertilizers.

Fusarium graminearum1H NMR metabolomics.

Raw 1H NMR spectra of Fusarium graminearum hyphae can be found at the website of the pesticide metabolomics group (PMG) of the Agricultural University of Athens at the address: http://www.aua.gr/pesticide-metabolomicsgroup/Resources/Fusarium_graminearum_NMR_spectra.html, accession number PMG-01-17. The data set support the research article "Implication of Fusarium graminearum Primary Metabolism in its Resistance to Benzimidazole Fungicides as revealed by 1H NMR Metabolomics" [1].

Physiological and ultrastructural changes in "green islands" on Avena sterilis leaves caused by (8R,16R)-(-)-pyrenophorin.

The biochemical and ultrastructural changes in "green islands" (GIs) on detached Avena sterilis leaves caused by the macrodiolide (8R,16R)-(-)-pyrenophorin in the dark were examined. In the absence of light, leaf segments retained their photosynthetic pigments for 96 h after treatment with (8R,16R)-(-)-pyrenophorin (70 muM), whereas in the untreated leaves complete senescence, loss of photosynthetic pigments and cell disorganization were observed 72 h after detachment. Proteolytic enzyme activity in treated tissues with pyrenophorin remained at low levels for 96 h after treatment and protein dissipation was lower in the treated than in the untreated. Although tissues in "GIs" seem macroscopically healthy, electron microscopy observations revealed structurally disorganized cells filled with granular, electron-dense material. Chloroplasts were severely damaged and contained a large number of plastoglobuli. Similar ultrastructural changes were also observed in A. sterilis tissues treated with the phytotoxin under illumination, indicating a mechanism operating both under illumination and in the dark.

Comparative toxicity of the phytotoxins (8R,16R)-(-)-pyrenophorin and (5S,8R,13S,16R)-(-)-pyrenophorol on aquatic organisms.

The acute toxicities of the fungal phytotoxins (8R,16R)-(-)-pyrenophorin and (5S,8R,13S,16R)-(-)-pyrenophorol on Vibrio fischeri, Oscillatoria perornata, Pseudokirchneriella subcapitata, Lemna minor and Artemia fransiscana were evaluated. (8R,16R)-(-)-pyrenophorin was more toxic than (5S,8R,13S,16R)-(-)-pyrenophorol to V. fischeri, O. perornata, L. minor and A. fransiscana. The highest acute toxicity of (8R,16R)-(-)-pyrenophorin was exhibited on V. fischeri (5 min median effective concentration of 3.57 M 10(-5)) whereas the corresponding value for (5S,8R,13S,16R)-(-)-pyrenophorol was 801 M 10(-5). P. subcapitata exhibited a lack of sensitivity (median inhibitory concentration of >10 M 10(-5)) to both phytotoxins.